sirolimus and Disease-Models--Animal

Autophagy is an explicit cellular process to deliver dissimilar cytoplasmic misfolded proteins, lipids and damaged organelles to the lysosomes for degradation and elimination. The mechanistic target of rapamycin (mTOR) is the main negative regulator of autophagy. The mTOR pathway is involved in regulating neurogenesis, synaptic plasticity, neuronal development and excitability. Exaggerated mTOR activity is associated with the development of temporal lobe epilepsy, genetic and acquired epilepsy, and experimental epilepsy. In particular, mTOR complex 1 (mTORC1) is mainly involved in epileptogenesis. The investigation of autophagy's involvement in epilepsy has recently been conducted, focusing on the critical role of rapamycin, an autophagy inducer, in reducing the severity of induced seizures in animal model studies. The induction of autophagy could be an innovative therapeutic strategy in managing epilepsy. Despite the protective role of autophagy against epileptogenesis and epilepsy, its role in status epilepticus (SE) is perplexing and might be beneficial or detrimental. Therefore, the present review aims to revise the possible role of autophagy in epilepsy.

Topics: Animals; Autophagy; Disease Models, Animal; Epilepsy; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

This article recapitulates the evidence on the role of mammalian targets of rapamycin (mTOR) complex pathways in multiple sclerosis (MS). Key biological processes that intersect with mTOR signaling cascades include autophagy, inflammasome activation, innate (e.g., microglial) and adaptive (B and T cell) immune responses, and axonal and neuronal toxicity/degeneration. There is robust evidence that mTOR inhibitors, such as rapamycin, ameliorate the clinical course of the animal model of MS, experimental autoimmune encephalomyelitis (EAE). New, evolving data unravel mechanisms underlying the therapeutic effect on EAE, which include balance among T-effector and T-regulatory cells, and mTOR effects on myeloid cell function, polarization, and antigen presentation, with relevance to MS pathogenesis. Radiologic and preliminary clinical data from a phase 2 randomized, controlled trial of temsirolimus (a rapamycin analogue) in MS show moderate efficacy, with significant adverse effects. Large clinical trials of indirect mTOR inhibitors (metformin) in MS are lacking; however, a smaller prospective, non-randomized study shows some potentially promising radiological results in combination with ex vivo beneficial effects on immune cells that might warrant further investigation. Importantly, the study of mTOR pathway contributions to autoimmune inflammatory demyelination and multiple sclerosis illustrates the difficulties in the clinical application of animal model results. Nevertheless, it is not inconceivable that targeting metabolism in the future with cell-selective mTOR inhibitors (compared to the broad inhibitors tried to date) could be developed to improve efficacy and reduce side effects.

Topics: Animals; Clinical Trials, Phase II as Topic; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Humans; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Prospective Studies; Randomized Controlled Trials as Topic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The coronavirus disease 2019 (COVID-19) has affected approximately 2 million individuals worldwide; however, data regarding fatal cases have been limited.. To report the clinical features of 162 fatal cases of COVID-19 from 5 hospitals in Wuhan between December 30, 2019 and March 12, 2020.. The demographic data, signs and symptoms, clinical course, comorbidities, laboratory findings, computed tomographic (CT) scans, treatments, and complications of the patients with fatal cases were retrieved from electronic medical records.. Young patients with moderate COVID-19 without comorbidity at admission could also develop fatal outcomes. The in-hospital survival time of the fatal cases was similar among the hospitals of different levels in Wuhan.

Topics: Adolescent; Adult; Animals; Asthma; Atrial Fibrillation; Autoantibodies; Biomarkers; Breast Neoplasms; Child; Conjunctivitis, Allergic; Cornea; COVID-19; Cyclosporine; Cytokines; Death, Sudden, Cardiac; Defibrillators, Implantable; Diet; Disease Models, Animal; Docetaxel; Double-Blind Method; Dry Eye Syndromes; Educational Status; Emulsions; Female; Fluorescein Angiography; Fluoresceins; Focus Groups; Heart Failure; Hemothorax; Humans; Inflammation; Keratoconus; Male; Meibomian Glands; Mice; Middle Aged; Multiple Sclerosis; Myocardial Infarction; Myocardium; Nerve Fibers; Nigeria; Obesity; Overweight; Pandemics; Primary Prevention; Prospective Studies; Qualitative Research; Registries; Retinal Ganglion Cells; Retinal Vessels; Schools; Sirolimus; Tertiary Care Centers; Th1 Cells; Th2 Cells; Tomography, Optical Coherence; Troponin I; Tumor Necrosis Factor-alpha; United States; Ventricular Remodeling

Epilepsy is a syndrome specified by frequent seizures and is one of the most prevalent neurological conditions, and that one-third of people of epilepsy are resistant to available drugs. Surgery is supposed to be the main treatment for the remedy of multiple drug-resistant epilepsy, but it is a drastic procedure. Advancement in genomic technologies indicates that gene therapy can make such surgery unnecessary. The considerable number of new studies show the significance of mutation in mammalian target of rapamycin pathway, NMDA receptors, GABA receptors, potassium channels and G-protein coupled receptors. Illustration of the meticulous drug in epilepsy targeting new expression of mutations in SCN8A, GRIN2A, GRIN2D and KCNT1 are conferred. Various methods are utilized to express a gene in a precise area of the brain; Transplantation of cells in an ex vivo approach (fetal cells, fibroblasts, immortalized cells), nonviral vector delivery and viral vector delivery like retrovirus, herpes simplex virus adenovirus and adeno-related virus. Gene therapy has thus been explored to generate anti-epileptogenic, anti-seizure and disease-modifying effects. Specific targeting of the epileptogenic region is facilitated by gene therapy, hence sparing the adjacent healthy tissue and decreasing the adverse effects that frequently go hand in hand with antiepileptic medication.

Topics: Animals; Brain; Disease Models, Animal; Epilepsy; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Metabolic Networks and Pathways; Mutation; Precision Medicine; Seizures; Sirolimus

The ε4 allele of Apolipoprotein (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), the most common form of dementia. Cognitively normal APOE4 carriers have developed amyloid beta (Aβ) plaques and cerebrovascular, metabolic and structural deficits decades before showing the cognitive impairment. Interventions that can inhibit Aβ retention and restore the brain functions to normal would be critical to prevent AD for the asymptomatic APOE4 carriers. A major goal of the study was to identify the potential usefulness of rapamycin (Rapa), a pharmacological intervention for extending longevity, for preventing AD in the mice that express human APOE4 gene and overexpress Aβ (the E4FAD mice). Another goal of the study was to identify the potential pharmacogenetic differences in response to rapamycin between the E4FAD and E3FAD mice, the mice with human APOE ε3 allele. We used multi-modal MRI to measure in vivo cerebral blood flow (CBF), neurotransmitter levels, white matter integrity, water content, cerebrovascular reactivity (CVR) and somatosensory response; used behavioral assessments to determine cognitive function; used biochemistry assays to determine Aβ retention and blood-brain barrier (BBB) functions; and used metabolomics to identify brain metabolic changes. We found that in the E4FAD mice, rapamycin normalized bodyweight, restored CBF (especially in female), BBB activity for Aβ transport, neurotransmitter levels, neuronal integrity and free fatty acid level, and reduced Aβ retention, which were not observe in the E3FAD-Rapa mice. In contrast, E3FAD-Rapa mice had lower CVR responses, lower anxiety and reduced glycolysis in the brain, which were not seen in the E4FAD-Rapa mice. Further, rapamycin appeared to normalize lipid-associated metabolism in the E4FAD mice, while slowed overall glucose-associated metabolism in the E3FAD mice. Finally, rapamycin enhanced overall water content, water diffusion in white matter, and spatial memory in both E3FAD and E4FAD mice, but did not impact the somatosensory responses under hindpaw stimulation. Our findings indicated that rapamycin was able to restore brain functions and reduce AD risk for young, asymptomatic E4FAD mice, and there were pharmacogenetic differences between the E3FAD and E4FAD mice. As the multi-modal MRI methods used in the study are readily to be used in humans and rapamycin is FDA-approved, our results may pave a way for future clinical testing of the pharmacogenetic response

Topics: Alzheimer Disease; Animals; Apolipoprotein E4; Apolipoproteins E; Blood-Brain Barrier; Cognition; Cognitive Dysfunction; Disease Models, Animal; Genotype; Mice; Mice, Transgenic; Pharmacogenetics; Plaque, Amyloid; Sirolimus

Amplifying endogenous neuroprotective mechanisms is a promising avenue for stroke therapy. One target is mammalian target of rapamycin (mTOR), a serine/threonine kinase regulating cell proliferation, cell survival, protein synthesis, and autophagy. Animal studies investigating the effect of rapamycin on mTOR inhibition following cerebral ischemia have shown conflicting results.. To conduct a systematic review and meta-analysis evaluating the effectiveness of rapamycin in reducing infarct volume in animal models of ischemic stroke.. Our search identified 328 publications. Seventeen publications met inclusion criteria (52 comparisons: 30 reported infarct size and 22 reported neurobehavioral score). Study quality was modest (median 4 of 9) with no evidence of publication bias. The point estimate for the effect of rapamycin was a 21.6% (95% CI, 7.6%-35.7% p < 0.01) improvement in infarct volume and 30.5% (95% CI 17.2%-43.8%, p < 0.0001) improvement in neuroscores. Effect sizes were greatest in studies using lower doses of rapamycin.. Low-dose rapamycin treatment may be an effective therapeutic option for stroke. Modest study quality means there is a potential risk of bias. We recommend further high-quality preclinical studies on rapamycin in stroke before progressing to clinical trials.

Topics: Animals; Brain Ischemia; Cerebellum; Disease Models, Animal; Humans; Immunosuppressive Agents; Neuroprotective Agents; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Choroid; Cornea; Diabetic Retinopathy; Disease Models, Animal; Endothelial Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Retina; Retinal Neovascularization; Retinal Vessels; Signal Transduction; Sirolimus; Vascular Endothelial Growth Factor A

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Ageing is the organisms increased susceptibility to death, which is linked to accumulated damage in the cells and tissues. Ageing is a complex process regulated by crosstalk of various pathways in the cells. Ageing is highly regulated by the Target of Rapamycin (TOR) pathway activity. TOR is an evolutionary conserved key protein kinase in the TOR pathway that regulates growth, proliferation and cell metabolism in response to nutrients, growth factors and stress. Comparing the ageing process in invertebrate model organisms with relatively short lifespan with mammals provides valuable information about the molecular mechanisms underlying the ageing process faster than mammal systems. Inhibition of the TOR pathway activity via either genetic manipulation or rapamycin increases lifespan profoundly in most invertebrate model organisms. This contribution will review the recent findings in invertebrates concerning the TOR pathway and effects of TOR inhibition by rapamycin on lifespan. Besides some contradictory results, the majority points out that rapamycin induces longevity. This suggests that administration of rapamycin in invertebrates is a promising tool for pursuing the scientific puzzle of lifespan prolongation.

Topics: Aging; Animals; Caenorhabditis elegans; Cell Proliferation; Disease Models, Animal; Drosophila melanogaster; Female; Humans; Immunosuppressive Agents; Longevity; Male; Mice; Podospora; Saccharomyces cerevisiae; Schizosaccharomyces; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The discovery that rapamycin increases lifespan in mice and restores/delays many aging phenotypes has led to the speculation that rapamycin has 'anti-aging' properties. The major question discussed in this review is whether a manipulation that has anti-aging properties can alter the onset and/or progression of Alzheimer's disease, a disease in which age is the major risk factor. Rapamycin has been shown to prevent (and possibly restore in some cases) the deficit in memory observed in the mouse model of Alzheimer's disease (AD-Tg) as well as reduce Aβ and tau aggregation, restore cerebral blood flow and vascularization, and reduce microglia activation. All of these parameters are widely recognized as symptoms central to the development of AD. Furthermore, rapamycin has also been shown to improve memory and reduce anxiety and depression in several other mouse models that show cognitive deficits as well as in 'normal' mice. The current research shows the feasibility of using pharmacological agents that increase lifespan, such as those identified by the National Institute on Aging Intervention Testing Program, to treat Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Autophagy; Behavior, Animal; Cerebrovascular Circulation; Cognition Disorders; Disease Models, Animal; Longevity; Memory Disorders; Mice; Neurofibrillary Tangles; Plaque, Amyloid; Sirolimus; TOR Serine-Threonine Kinases; Vasodilator Agents

When subjected to pressure overload, the ventricular myocardium shifts from fatty acids to glucose as its main source for energy provision and frequently increases its mass. Here, we review the evidence in support of the concept that metabolic remodeling, measured as an increased myocardial glucose uptake using dynamic positron emission tomography (PET) with the glucose analogue 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG), precedes the onset of left ventricular hypertrophy (LVH) and heart failure. Consistent with this, early intervention with propranolol, which attenuates glucose uptake, prevents the maladaptive metabolic response and preserves cardiac function in vivo. We also review ex vivo studies suggesting a link between dysregulated myocardial glucose metabolism, intracellular accumulation of glucose 6-phosphate (G6P) and contractile dysfunction of the heart. G6P levels correlate with activation of mTOR (mechanistic target of rapamycin) and endoplasmic reticulum stress. This sequence of events could be prevented by pretreatment with rapamycin (mTOR inhibition) or metformin (enzyme 5'-AMP-activated protein kinase activation). In conclusion, we propose that metabolic imaging with FDG PET may provide a novel approach to guide the treatment of patients with hypertension-induced LVH.

Topics: 3-O-Methylglucose; Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Fatty Acids; Glucose-6-Phosphate; Heart Failure; Heart Ventricles; Humans; Hypertension; Hypertrophy, Left Ventricular; Hypoglycemic Agents; Metformin; Mice; Myocardium; Positron-Emission Tomography; Rats; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Function, Left

Topics: Animals; Child Development Disorders, Pervasive; Disease Models, Animal; Eukaryotic Initiation Factors; Fragile X Mental Retardation Protein; Mice; Nerve Tissue Proteins; Neurofibromin 1; Oxytocin; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The development of new drugs for the treatment of epilepsy is a major challenge for modern neurology and its first steps demand basic research. Preclinical studies on animal models of epilepsy are mainly based on the analysis of brain electrical activity to detect seizures, when they are not just limited to behavioral tests like the Racine scale.. In the present review, we discuss the importance of using time-locked video and EEG recordings (Video-EEG) coupled with behavioral tests as tools to monitor and analyze the effects of anti-epileptic drugs in pre-clinical research. Particularly, we focus on the utility of a multimodal approach based on EEG/behavioral analysis to study the beneficial effects of chronic rapamycin treatment as a potential anti-epileptogenic therapy for a broad spectrum of epilepsy, including both genetic (as in tuberous sclerosis complex) and acquired diseases.. Changes and synchronization of neuronal activity of different areas have been correlated with specific behavior in both physiological and pathological conditions. In the epileptic brain, during a seizure there is an abnormal activation of many cells all at once, altering different networks.. A multimodal approach based on video, EEG analysis and behavioral tests would be the best option in preclinical studies of epilepsy.

Topics: Animals; Anticonvulsants; Brain; Disease Models, Animal; Electroencephalography; Epilepsy; Humans; Outcome Assessment, Health Care; Reproducibility of Results; Sirolimus; Videotape Recording

The pathogenesis of neurodegenerative diseases involves altered activity of proteolytic systems and accumulation of protein aggregates. Autophagy is an intracellular process in which damaged organelles and long-lived proteins are degraded and recycled for maintaining normal cellular homeostasis. Disruption of autophagic activity in neurons leads to modify the cellular homeostasis, causing deficient elimination of abnormal and toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons to clear abnormal protein aggregates and survive. This review aims to give an overview of some of the main modulators of autophagy that are currently being studied as possible alternatives in the search of therapies that slow the progression of neurodegenerative diseases, which are incurable to date.

Topics: Animals; Autophagy; Disease Models, Animal; Food; Humans; Isothiocyanates; Lithium; Neurodegenerative Diseases; Resveratrol; Sirolimus; Spermidine; Stilbenes; Sulfoxides; Trehalose; Valproic Acid

With the global aging population, Alzheimer's disease, Parkinson's disease and mild cognition impairment are increasing in prevalence. The success of rapamycin as an agent to extend lifespan in various organisms, including mice, brings hope that chronic mTOR inhibition could also refrain age-related neurodegeneration. Here we review the evidence suggesting that mTOR inhibition - mainly with rapamycin - is a valid intervention to delay age-related neurodegeneration. We discuss the potential mechanisms by which rapamycin may facilitate neurodegeneration prevention or restoration of cognitive function. We also discuss the known side effects of rapamycin and provide evidence to alleviate exaggerated concerns regarding its wider clinical use. We explore the small molecule alternatives to rapamycin and propose future directions for their development, mainly by exploring the possibility of targeting the downstream effectors of mTOR: S6K1 and especially S6K2. Finally, we discuss the strengths and weaknesses of the models used to determine intervention efficacy for neurodegeneration. We address the difficulties of interpreting data using the common way of investigating the efficacy of interventions to delay/prevent neurodegeneration by observing animal behavior while these animals are under treatment. We propose an experimental design that should isolate the variable of aging in the experimental design and resolve the ambiguity present in recent literature.

Topics: Aging; Animals; Cognition; Disease Models, Animal; Humans; Mice; Molecular Targeted Therapy; Neurodegenerative Diseases; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

The success of solid-organ transplantation was made possible by recognizing that destruction of the graft is caused by an alloimmune-mediated process. For the past decade, immunosuppressive protocols have used a combination of drugs that significantly decreased the rate of acute organ rejection. Despite advances in surgical and medical care of recipients of solid-organ transplants, long-term graft survival and patient survival have not improved during the past 2 decades. Current immunosuppression protocols include a combination of calcineurin inhibitors, such as tacrolimus, and antiproliferative agents (most commonly mycophenolate mofetil), with or without different dosing regimens of corticosteroids. Mammalian target of rapamycin inhibitors were introduced to be used in combination with cyclosporine-based therapy, but they did not gain much acceptance because of their adverse event profile. Belatacept, a costimulatory inhibitor, is currently being studied in different regimens in an effort to replace the use of calcineurin inhibitors to induce tolerance and to improve long-term outcomes. Induction therapy is now being used in more than 90% of kidney transplants and more than 50% cases of other solid-organ transplantation such as lung, heart, and intestinal transplants. As a result of these combination immunosuppressive (IS) therapy protocols, not only the incidence but also the intensity of episodes of acute rejection have decreased markedly, and at present 1-year graft and patient survival is almost 98% for kidney transplant recipients and approximately greater than 80% for heart and lung transplants. Evolving concepts include the use of donor-derived bone marrow mesenchymal cells to induce tolerance, to minimize the use of maintenance IS agents, and to prevent the development of adverse events associated with long-term use of maintenance IS therapy.

Topics: Abatacept; Alemtuzumab; Animals; Antibodies, Monoclonal, Humanized; Azathioprine; Bone Marrow Transplantation; Calcineurin Inhibitors; Cyclosporine; Disease Models, Animal; Everolimus; Heart Transplantation; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney Transplantation; Lung Transplantation; Mycophenolic Acid; Piperidines; Pyrimidines; Pyrroles; Randomized Controlled Trials as Topic; Sirolimus; Tacrolimus

Autophagy is a catabolic process that has been shown to have a role in many cellular processes including the removal of excessive or damaged proteins and protein aggregates. The salivary glands play a critical role in oral health, and their secretory capacity may be critically intertwined with the autophagic process. This review describes the role of autophagy activation in normal salivary gland homeostasis and during the glandular stress responses of therapeutic radiation, ductal ligation, autoimmunity, and salivary gland adenoid cystic carcinoma.

Topics: Animals; Autoimmune Diseases; Autophagy; Carcinoma, Adenoid Cystic; Disease Models, Animal; Homeostasis; Humans; Ligation; Salivary Gland Neoplasms; Salivary Glands; Sirolimus; Stress, Physiological

Glycogen storage disease (GSD) consists of more than 10 discrete conditions for which the biochemical and genetic bases have been determined, and new therapies have been under development for several of these conditions. Gene therapy research has generated proof-of-concept for GSD types I (von Gierke disease) and II (Pompe disease). Key features of these gene therapy strategies include the choice of vector and regulatory cassette, and recently adeno-associated virus (AAV) vectors containing tissue-specific promoters have achieved a high degree of efficacy. Efficacy of gene therapy for Pompe disease depend upon the induction of immune tolerance to the therapeutic enzyme. Efficacy of von Gierke disease is transient, waning gradually over the months following vector administration. Small molecule therapies have been evaluated with the goal of improving standard of care therapy or ameliorating the cellular abnormalities associated with specific GSDs. The receptor-mediated uptake of the therapeutic enzyme in Pompe disease was enhanced by administration of β2 agonists. Rapamycin reduced the liver fibrosis observed in GSD III. Further development of gene therapy could provide curative therapy for patients with GSD, if efficacy from preclinical research is observed in future clinical trials and these treatments become clinically available.

Topics: Animals; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Glycogen Storage Disease; Glycogen Storage Disease Type I; Glycogen Storage Disease Type II; Humans; Sirolimus; Small Molecule Libraries; Transgenes

The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiological and pathological conditions. Although many ASDs arise as a result of mutations in genes with multiple molecular functions, they appear to converge on common biological pathways that give rise to autism-relevant behaviors. Dysregulation of mTOR signaling has been identified as a phenotypic feature common to fragile X syndrome, tuberous sclerosis complex 1 and 2, neurofibromatosis 1, phosphatase and tensin homolog, and potentially Rett syndrome. Below are a summary of topics covered in a symposium that presents dysregulation of mTOR as a unifying theme in a subset of ASDs.

Topics: Animals; Autistic Disorder; Disease Models, Animal; Humans; Models, Biological; Signal Transduction; Sirolimus

An increasing amount of evidence suggests that the dysregulation of the Akt-mTOR (Akt-mammalian Target Of Rapamycin) signaling network is associated with intellectual disabilities, such as fragile X, tuberous sclerosis and Rett's syndrome. The Akt-mTOR pathway is involved in dendrite morphogenesis and synaptic plasticity, and it has been shown to modulate both glutamatergic and GABAergic synaptic transmission. We have recently shown that the AktmTOR pathway is hyperactive in the hippocampus of Ts1Cje mice, a model of Down's syndrome, leading to increased local dendritic translation that could interfere with synaptic plasticity. Rapamycin and rapalogs are specific inhibitors of mTOR, and some of these inhibitors are Food and Drug Administration-approved drugs. In this review, we discuss the molecular basis and consequences of Akt-mTOR hyperactivation in Down's syndrome, paying close attention to alterations in the molecular mechanisms underlying synaptic plasticity. We also analyze the pros and cons of using rapamycin/rapalogs for the treatment of the cognitive impairments associated with this condition.

Topics: Animals; Cognition Disorders; Disease Models, Animal; Down Syndrome; Humans; Immunosuppressive Agents; Mice; Oncogene Protein v-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) is aberrantly activated in many cancer types, and two rapamycin derivatives are currently approved by the Food and Drug Administration (FDA) of the United States for treating renal cell carcinoma. Mechanistically, mTOR is hyperactivated in human cancers either due to the genetic activation of its upstream activating signaling pathways or the genetic inactivation of its negative regulators. The tumor suppressor liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is involved in cell polarity, cell detachment and adhesion, tumor metastasis, and energetic stress response. A key role of LKB1 is to negatively regulate the activity of mTOR complex 1 (mTORC1). This review summarizes the molecular basis of this negative interaction and recent research progress in this area.

Topics: Adenocarcinoma; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Endometrial Neoplasms; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Colorectal cancer is the third leading cause of cancer death in Japan and the United States and is strongly associated with obesity, especially visceral obesity. Several metabolic mediators, such as adiponectin, have been suspected to play a role in obesity-related carcinogenesis. In a previous human study, the existence of a significant correlation between the number of human dysplastic aberrant crypt foci (ACF) and the visceral fat area was demonstrated, and also that of a significant inverse correlation between the number of dysplastic ACF and the plasma adiponectin level. Other studies have investigated the effect of adiponectin under the normal and high-fat diet conditions in a mouse model of azoxymethane-induced colon cancer. Enhanced formation of both ACF and tumors was observed in the adiponectin-deficient mice, as compared with that in the wild-type, under the high-fat diet condition but not under the normal diet condition. Furthermore, that the 5'-AMP-activated kinase/mammalian target of rapamycin pathway is involved in the promotion of colorectal carcinogenesis in adiponectin-deficient mice under the high-fat diet condition was shown. Therefore, that the 5'-AMP-activated kinase/mammalian target of rapamycin signaling pathway may play an important role in colorectal carcinogenesis was speculated. Metformin, a biguanide derivative widely used in the treatment of diabetes mellitus, has been shown to exert a suppressive effect on ACF formation in both mouse models and humans. Therefore, metformin might be a promising candidate as a safe drug for chemoprevention of colorectal carcinogenesis. Further studies with high evidence levels, such as randomized, controlled studies, are needed to clarify these relationships.

Topics: Aberrant Crypt Foci; Adiponectin; AMP-Activated Protein Kinases; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Colon; Colorectal Neoplasms; Diet, High-Fat; Disease Models, Animal; Epithelial Cells; Humans; Hypoglycemic Agents; Metformin; Mice; Obesity, Abdominal; Risk Factors; Signal Transduction; Sirolimus

Primary sclerosing cholangitis (PSC) is a chronic, cholestatic, idiopathic liver disease characterized by fibro-obliterative inflammation of the hepatic bile ducts. In a clinically significant proportion of patients, PSC progresses to cirrhosis, end-stage liver disease, and in some cases, cholangiocarcinoma. Despite clinical trials of nearly 20 different pharmacotherapies over several decades, safe and effective medical therapy, albeit critically needed, remains to be established. PSC is pathogenically complex, with genetic, immune, enteric microbial, environmental and other factors being potentially involved and, thus, not surprisingly, it manifests as a clinically heterogeneous disease with a relatively unpredictable course. It is likely that this complexity and clinical heterogeneity are responsible for the negative results of clinical trials, but novel insights about and approaches to PSC may shift this trend. The authors herein provide a review of previously tested pharmacologic agents, discuss emerging fundamental concepts and present viewpoints regarding how identifying therapies for PSC may evolve over the next several years.

Topics: Animals; Anti-Bacterial Agents; Cholangitis, Sclerosing; Disease Models, Animal; Endoscopy, Digestive System; Humans; Immunosuppressive Agents; Magnetic Resonance Imaging; Receptors, Lymphocyte Homing; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Transfer of human regulatory T cells (Tregs) has become an attractive therapeutic alternative to improve the long-term outcome in transplantation and thus reduce the side-effects of conventional immunosuppressive drugs. Here, we summarize the recent findings on human Treg subsets, their phenotype and in-vivo function.. In the last 2 years, it has become apparent that several Treg subsets exist that specifically regulate Th1-driven, Th2-driven, or Th17-driven immune responses; these subsets are very unstable and rapidly change their phenotype, for example, there is loss of Foxp3 expression upon extensive ex-vivo expansion and only the administration of rapamycin has been shown to be able to interfere reproducibly. New humanized mouse models incorporating human solid-organ grafts have been developed, which have been used to test the human Treg in-vivo function, and the first human Treg-cell products have been tested for safety and efficacy in stem cell transplantation.. With the recent findings, we have gained a better understanding of Treg heterogeneity, plasticity and function. Using the outcomes of clinical trials in stem cell transplantation, we have learned that adoptive therapy of Tregs is well tolerated and we are now awaiting the first result in solid-organ transplantation from the 'ONE Study'.

Topics: Animals; Disease Models, Animal; Forkhead Transcription Factors; Graft Rejection; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Immunotherapy, Adoptive; Organ Transplantation; Sirolimus; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells

Evidence is accumulating that autophagy occurs in advanced atherosclerotic plaques. Although there is an almost relentless discovery of molecules that are involved in autophagy, studies of selective autophagy induction or inhibition using knockout mice are just now beginning to reveal its biological significance. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading the damaged intracellular material. In this way, autophagy is protective and contributes to cellular recovery in an unfavorable environment. Pharmacological approaches have recently been developed to stabilize vulnerable, rupture-prone lesions through induction of autophagy. This approach has proven to be successful in short-term studies. However, how autophagy induction affects processes such as inflammation remains to be elucidated and is currently under investigation. This review highlights the possibilities for exploiting autophagy as a drug target for plaque stabilization.

Topics: Animals; Atherosclerosis; Autophagy; Disease Models, Animal; Everolimus; Humans; Immunosuppressive Agents; Mice; Plaque, Atherosclerotic; Rabbits; Sirolimus

Most current treatments for epilepsy are symptomatic therapies that suppress seizures but do not affect the underlying course or prognosis of epilepsy. The need for disease-modifying or "antiepileptogenic" treatments for epilepsy is widely recognized, but no such preventive therapies have yet been established for clinical use. A rational strategy for preventing epilepsy is to target primary signaling pathways that initially trigger the numerous downstream mechanisms mediating epileptogenesis. The mammalian target of rapamycin (mTOR) pathway represents a logical candidate, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. The importance of the mTOR pathway in epileptogenesis is best illustrated by tuberous sclerosis complex (TSC), one of the most common genetic causes of epilepsy. In mouse models of TSC, mTOR inhibitors prevent the development of epilepsy and underlying brain abnormalities associated with epileptogenesis. Accumulating evidence suggests that mTOR also participates in epileptogenesis due to a variety of other causes, including focal cortical dysplasia and acquired brain injuries, such as in animal models following status epilepticus or traumatic brain injury. Therefore, mTOR inhibition may represent a potential antiepileptogenic therapy for diverse types of epilepsy, including both genetic and acquired epilepsies.

Topics: Animals; Anticonvulsants; Brain Injuries; Cell Death; Cell Division; Cell Proliferation; Disease Models, Animal; Epilepsy; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Malformations of Cortical Development; Mice; Models, Genetic; Neuronal Plasticity; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis

Tuberous sclerosis complex (TSC) is a multiorgan genetic disease caused by mutations in the TSC1 or TSC2 genes. TSC has been recognized for many years as an important cause of severe neurological disease with patients suffering from epilepsy, developmental delay, autism, and psychiatric problems. During the last year, there have been enormous advances in basic and translational research pertaining to TSC.. In this review, I discuss the basic science findings that position the TSC1 and TSC2 genes as critical regulators of the mammalian target of rapamycin kinase within mammalian target of rapamycin complex 1. In addition, I will discuss the development of new animal models, translational data, and recent clinical trials using mammalian target of rapamycin complex 1 inhibitors such as rapamycin.. The past few years have seen spectacular advances that have energized TSC-related research and challenged existing symptomatic treatments. Although it remains to be seen whether use of mammalian target of rapamycin complex 1 inhibitors will revolutionize the care of patients with TSC, the application of basic and translational research towards a specific clinical disorder emphasizes the potential and promise of molecular medicine.

Topics: Animals; Clinical Trials as Topic; Disease Models, Animal; Epilepsy; Humans; Signal Transduction; Sirolimus; Transcription Factors; Tuberous Sclerosis

Infantile spasms are the classical seizure type of West syndrome. Infantile spasms often herald a dismal prognosis, due to the high probability to evolve into intractable forms of epilepsies with significant cognitive deficits, especially if not adequately treated. The current therapies-high doses of adrenocorticotropic hormone, steroids, or the gamma-aminobutyric acid (GABA) transaminase inhibitor vigabatrin--are often toxic and may not always be effective. The need to identify new therapies for spasms has led to the generation of a number of rodent models of infantile spasms. These include acute and chronic models of infantile spasms, with cryptogenic or symptomatic origin, many of which are based on specific etiologies. In this review, we summarize the clinical experience with treating infantile spasms and the main features of the new animal models of infantile spasms and discuss their utility in the preclinical development of new therapies for infantile spasms.

Topics: Adrenocorticotropic Hormone; Animals; Anticonvulsants; Disease Models, Animal; Humans; Infant; Mice; Rats; Receptors, GABA; Sirolimus; Spasms, Infantile; Vigabatrin

Left ventricular hypertrophy (LVH) contributes to elevated cardiac mortality with graft function in renal transplant recipients. Antihypertensive therapy, and especially angiotensin-converting enzyme (ACE) inhibitors, proved to be effective in regressing the LVH of renal transplant recipients, at least in part by interacting with immunosuppressive agents, thus raising the possibility that immunosuppressive therapy might affect changes in the left ventricular mass (LVM) of recipients. This review mainly focuses on the potential role of mammalian target of rapamycin (mTOR) inhibition to regress cardiac hypertrophy in both experimental models and in the clinical setting. We comment on the results of experimental studies conducted on animal models, which showed regression of cardiac hypertrophy by sirolimus (SRL). We also discuss clinical studies that show that conversion from calcineurin inhibitors to SRL is effective to achieve regression of LVH in both kidney and cardiac transplant recipients, mainly by reducing the true left ventricular wall hypertrophy.

Topics: Animals; Antihypertensive Agents; Calcineurin Inhibitors; Disease Models, Animal; Humans; Hypertrophy, Left Ventricular; Immunosuppressive Agents; Kidney Transplantation; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of countless cysts in both kidneys, which compress the cyst-free renal parenchyma, leading to a loss of renal function and the need for renal replacement therapy and/or kidney transplantation in ∼50% of affected patients. In animal models of experimental polycystic kidney disease, the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus effectively reduce cyst growth and loss of renal function. Furthermore, an analysis of renal transplant patients with ADPKD has shown that cystic kidney and liver volumes regress more on a sirolimus-based regimen than on a calcineurin inhibitor-based immunosuppressive regimen. Several prospective controlled clinical trials have been initiated to investigate whether mTOR inhibitors retard cyst growth and slow renal functional deterioration in patients with ADPKD. Study results are expected in 2010.

Topics: Animals; Disease Models, Animal; Everolimus; Humans; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

The formation of intra-neuronal mutant protein aggregates is a characteristic of several human neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders, including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins. Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical mTOR-independent pathway, which has multiple drug targets, involving links between Ca(2+)-calpain-G(salpha) and cAMP-Epac-PLC-epsilon-IP(3) signalling. Both pathways enhance the clearance of mutant huntingtin fragments and attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the expression of different autophagy genes is reduced, implicating that its effects are not mediated by autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for HD and related conditions.

Topics: Animals; Antibiotics, Antineoplastic; Autophagy; Disease Models, Animal; Drosophila; Gene Expression Regulation; Humans; Huntingtin Protein; Mutation; Nerve Tissue Proteins; Neurodegenerative Diseases; Nuclear Proteins; Peptides; Protein Biosynthesis; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder with variable phenotypic expression, due to a mutation in one of the two genes, TSC1 and TSC2, and a subsequent hyperactivation of the downstream mTOR pathway, resulting in increased cell growth and proliferation. The central nervous system is consistently involved in TSC, with 90% of individuals affected showing structural abnormalities, and almost all having some degree of CNS clinical manifestations, including seizures, cognitive impairment and behavioural problems. TSC is proving to be a particularly informative model for studying contemporary issues in developmental neurosciences. Recent advances in the neurobiology of TSC from molecular biology, molecular genetics, and animal model studies provide a better understanding of the pathogenesis of TSC-related neurological symptoms. Rapamycin normalizes the dysregulated mTOR pathway, and recent clinical trials have demonstrated its efficacy in various TSC manifestations, suggesting the possibility that rapamycin may have benefit in the treatment of TSC brain disease.

Topics: Animals; Behavior; Central Nervous System; Cognition; Disease Models, Animal; Humans; Mice; Rats; RNA, Messenger; Seizures; Sirolimus; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Topics: Animals; Antineoplastic Agents; Chemoprevention; Clinical Trials as Topic; Disease Models, Animal; Head and Neck Neoplasms; Humans; Mice; Sirolimus

Activation of mammalian target of rapamycin (mTOR) signaling occurs in a wide variety of human tumors and can lead to increased susceptibility to mTOR inhibitors. Temsirolimus, a novel analog of rapamycin, has shown promising preclinical and early clinical anti-tumor activity in various solid and hematologic tumor types, either alone or in combination with chemotherapy or other targeted agents. Randomized phase III trials have already demonstrated significant clinical benefits of treatment with single-agent temsirolimus in advanced renal cell carcinoma and relapsed and/or refractory mantle cell lymphoma. Other malignancies studied in the phase I and II trial settings include glioblastoma, breast cancer, endometrial cancer, non-Hodgkin lymphomas, and multiple myeloma. This article reviews a comprehensive collection of the clinical trial results reported to date for temsirolimus in various solid and hematologic malignancies, as well as current strategies being tested in ongoing trials. The findings with temsirolimus in multiple tumors provide a valuable framework for future development of temsirolimus and other mTOR inhibitors.

Topics: Animals; Antineoplastic Agents; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Hematologic Neoplasms; Humans; Neoplasms; Sirolimus

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the massive enlargement of both kidneys caused by numerous cysts. The cyst formation begins in utero and the continuous growth of cysts leads to compression and destruction of non-cystic renal parenchyma, so that finally a kidney replacement in the 5th to 6th decade of life is necessary. So far there is no therapy that halts disease progression. Animal data show that pharmacological inhibition of a central regulator of cell proliferation, the so-called mammalian target of rapamycin (mTOR), slows disease progression. This overview provides an insight into the disease and the specific mTOR inhibitor sirolimus, which is currently tested in clinical trials.

Topics: Adult; Animals; Disease Models, Animal; Disease Progression; Female; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Polycystic Kidney, Autosomal Dominant; Randomized Controlled Trials as Topic; Rats; Sirolimus

Topics: Animals; Calcium-Binding Proteins; Caroli Disease; Cholangiopancreatography, Magnetic Resonance; Cysts; Disease Models, Animal; Elasticity Imaging Techniques; Glucosidases; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Cirrhosis; Liver Diseases; Magnetic Resonance Imaging; Membrane Proteins; Molecular Biology; Molecular Chaperones; Octreotide; Peptides, Cyclic; Polycystic Kidney Diseases; Prevalence; RNA-Binding Proteins; Sirolimus; Somatostatin; Tomography, X-Ray Computed

Polymer-based sirolimus- (Cypher) and paclitaxel-eluting (Taxus) drug eluting stents have become the treatment of choice for patients with symptomatic coronary artery disease undergoing percutaneous coronary intervention (PCI). Although these stents reduce rates of restenosis compared with bare metal stents (BMS), late thrombosis, a life threatening complication, has emerged as a major safety concern. Our understanding of the pathophysiology of late DES thrombosis is derived from animal and human pathologic samples taken after implantation of these devices. These data indicate that both DES cause substantial impairment in arterial healing characterized by lack of complete reendothelialization and persistence of fibrin when compared with BMS. This delayed healing is the primary substrate underlying all cases of late DES thrombosis at autopsy. Several additional risk factors for late stent thrombosis such as penetration of necrotic core, malapposition, overlapping stent placement, excessive stent length, and bifurcation lesions represent additional barriers to healing and should be avoided if DES are to be used to minimize the risk of late thrombosis. Because the time course of complete healing with DES in man is unknown, the optimal duration of antiplatelet treatment remains to be determined.

Topics: Aged; Angioplasty, Balloon, Coronary; Animals; Biopsy, Needle; Coronary Angiography; Coronary Restenosis; Coronary Stenosis; Disease Models, Animal; Endothelium, Vascular; Humans; Immunohistochemistry; Middle Aged; Paclitaxel; Prognosis; Prosthesis Design; Rabbits; Risk Factors; Sensitivity and Specificity; Sirolimus; Stents; Time Factors; Wound Healing

Comparative preclinical histologic studies remain the most effective method for assessing the healing characteristics of vascular stents. The 2 most commonly used animal models to assess vascular responses to stent implantation are the porcine coronary artery and the rabbit iliac artery. Neither model alone is comparable to the human response to the implantation of a drug-eluting stent (DES). In the rabbit model at 28 days, the pathologies of the zotarolimus-eluting stent (ZES), the paclitaxel-eluting stent (PES), the sirolimus-eluting stent (SES), and a bare metal stent (BMS) were assessed. There was less inflammation with the ZES than with the SES or PES, and there were uncovered struts with the SES and PES but not with the ZES and BMS. In the pig model at 30, 90, and 180 days, the pathologies of the ZES, SES, and BMS were assessed. At 30 days, the thickness of neointima and the grade of inflammation were less with the SES than with the ZES and BMS, but at 90 and 180 days, the measures increased for the SES and were greater than those with the ZES and BMS, whereas the measures for the ZES and BMS did not change over time. In the rabbit model, the endothelialization of overlapping the SES, PES, and ZES was assessed. There was significantly greater endothelialization in the area above stent struts in the overlapping segment for the ZES than for the SES (p = 0.028). The level of endothelialization for the PES was less than that for the ZES, but the difference was not significant. Because arterial healing is multifactorial, it is extremely important that the next generation of DESs undergo preclinical testing in pig and rabbit models to examine endothelialization, inflammation, release kinetics, and neointimal reduction to establish the safety of these devices in humans.

Topics: Animals; Antineoplastic Agents; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Humans; Iliac Artery; Paclitaxel; Rabbits; Sirolimus; Swine; Time Factors; Tunica Intima

Despite the success of coronary stent implantations in the last decade, in-stent restenosis due to neointimal hyperplasia remains a problem to overcome. Neointimal hyperplasia is a vascular response to stent injury and mainly consists of proliferation of smooth muscle cells and deposition of extracellular matrix. Recently, local drug delivery has been advocated as a potential strategy to prevent in-stent restenosis. Unprecedented results have been obtained in early clinical studies on sirolimus-eluting and paclitaxel-eluting stents. Trials using various pharmaceutical coatings on different coronary stents are ongoing. More types of drug-eluting stents are expected on the market in the near future. Meanwhile, the evaluation of drug-eluting stents is entering the second phase in which the safety and efficacy in more complex lesion subsets and different clinical presentations are being investigated. Results including cost-benefit analyses are expected to have a tremendous impact on the practice of interventional cardiology in the next decade.

Topics: Animals; Anti-Bacterial Agents; Coronary Restenosis; Disease Models, Animal; Drug Delivery Systems; Drug-Eluting Stents; Humans; Paclitaxel; Prosthesis Design; Sirolimus; Tubulin Modulators

In 2001, the first human study with drug-eluting stents (DES) was published showing a nearly complete abolition of restenosis by using a sirolimus-eluting stent. This success was very encouraging to test new compounds in combination with the DES platform. Nevertheless, several other anti-restenotic compounds have been used in human clinical trials with disappointing outcomes. Little is known concerning potential adverse effects on vessel wall integrity and (re)healing, atherosclerotic lesion formation, progression, and plaque stability of these DES. Although efficacy and safety need to be determined clinically, preclinical testing of candidate drugs in well-defined animal models is extremely helpful to gain insight into the basic biological responses to candidate compounds. Here, we discuss and report an animal model which enables rapid screening of candidate drugs for DES on an atherosclerotic background. The results from drug testing using this novel model could help to quickly and cost-effectively establish the dose range of candidate drugs with reasonable potential for DES.

Topics: Animals; Atherosclerosis; Coronary Restenosis; Disease Models, Animal; Drug Delivery Systems; Drug Evaluation, Preclinical; Humans; Mice; Paclitaxel; Sirolimus; Stents

Drug-eluting stents are revolutionizing interventional cardiology. Sirolimus-eluting stents are in widespread clinical use, associated with well-documented remarkably low restenosis rates, and a number of other agents appear promising in clinical trials. These human studies have been preceded by numerous animal studies, foremost among them the pig coronary model of in-stent restenosis (ISR). The histologic response to porcine coronary stenting was described over a decade ago. Porcine stenting studies now provide examinations not only of histology, but also mechanisms of action, toxicity, and biocompatibility. This review therefore examines the current status of this porcine coronary model of ISR. Contemporary methods of pig coronary stenting are discussed. The morphometric, cellular, and molecular analyses of the responses to stent injury are then described. Finally, recent pig coronary drug-eluting stent studies are examined, with a discussion of their advantages, limitations, and possible future modifications.

Topics: Animals; Coronary Restenosis; Disease Models, Animal; Drug Delivery Systems; Immunosuppressive Agents; Sirolimus; Stents; Swine

Sirolimus (rapamycin), a macrolide antibiotic with known potent immunosuppressive properties, acts in the first phase (G1) of the cell cycle, blocking its further progression to the phase of DNA synthesis (S). In experimental models, rapamycin is effective in inhibiting smooth muscle cell proliferation and migration after vessel wall injury with balloon angioplasty. These results lead to the clinical application of sirolimus-eluting stents in 45 patients in Sao Paulo and Rotterdam (FIM Registry) and 238 patients in a randomized, European multicenter trial (RAVEL). These trials showed, by angiography and intravascular ultrasound, almost complete abolition of in-stent late hyperplasia up to one year after the procedure. In this review, we describe the experimental and clinical results of sirolimus-eluting stents including our experience of 26 stents implanted in 17 patients. In elective de novo lesions has shown remarkably clear lumens at follow-up angiography and intravascular ultrasound within the stented segments were observed with no lesion progression at the stent margins or thrombosis after a 2 month regimen of aspirin, and ticlopodine or clopidogrel. New large-scale ongoing clinical trials will investigate the efficacy of sirolimus-eluting stents in lesions that are traditionally associated with high restenosis rates after stent implantation, such as long lesions, bifurcations and instent restenosis.

Topics: Angina Pectoris; Angioplasty, Balloon, Coronary; Animals; Aspirin; Clopidogrel; Coronary Angiography; Coronary Disease; Coronary Restenosis; Disease Models, Animal; Drug Delivery Systems; Follow-Up Studies; Humans; Immunosuppressive Agents; Injections, Intramuscular; Multicenter Studies as Topic; Platelet Aggregation Inhibitors; Randomized Controlled Trials as Topic; Rats; Sirolimus; Stents; Swine; Thrombosis; Ticlopidine; Time Factors; Ultrasonography, Interventional

Topics: Animals; Aorta; Chronic Disease; Disease Models, Animal; Graft Rejection; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Muscle, Smooth, Vascular; Sirolimus; Vascular Diseases

Rapamycin (sirolimus) is a macrolide, related to cyclosporine with immunosuppressive properties and antiproliferative activity in various human tumor cells lines and tumor xenograft models. The cytosolic kinase mTOR which controls the initiation of the translation of messenger RNA is the main known target of rapamycin. During clinical studies, rapamycin given by oral route as immunosuppressant did not show dose-limited toxicity and only asymptomatic thrombopenia and hyperlipemia were observed. In murine models, best antitumoral activity was observed using parental routes. CCI-779, an analog formulated for intravenous use has antitumor activity without significant immunosuppressive property in mice and is currently in phase I trials in man.

Topics: Administration, Oral; Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Clinical Trials, Phase I as Topic; Disease Models, Animal; Humans; Immunosuppressive Agents; Injections, Intravenous; Mice; Neoplasm Transplantation; Protein Biosynthesis; Protein Kinase Inhibitors; RNA, Messenger; Sirolimus; Transplantation, Heterologous; Tumor Cells, Cultured

Topics: Animals; Autoimmune Diseases; Disease Models, Animal; Immunosuppressive Agents; Neoplasms, Experimental; Organ Transplantation; Polyenes; Sirolimus

In summary, many new modalities of immunosuppression after transplantation are being investigated (Fig. 1). These approaches include various new drugs or monoclonal antibodies that target different cell subsets, cellular activation pathways, cellular effector function or mediators (such as cytokines) of effector function, ligands that stabilize cellular interactions, or antimetabolites that preferentially affect lymphocytes (Tables 4 and 5). Because of the excellent early graft and patient survival results after liver transplantation under various current immunosuppressive protocols, future clinical trials using these various new modalities will require large numbers of patients to show statistically significant differences in graft or patient survival. Therefore, other criteria in addition to graft and patient survival must be analyzed to evaluate the importance of new immunosuppressive therapies. These criteria may include incidence of acute or chronic rejection, long-term graft function, incidence of infectious complications, length of hospitalization, drug toxicity, and patient tolerance and compliance with new therapies.

Topics: Alprostadil; Animals; Antibodies, Monoclonal; Biphenyl Compounds; Child; Disease Models, Animal; Dogs; Guanidines; Humans; Immunosuppressive Agents; Liver Transplantation; Mycophenolic Acid; Polyenes; Rats; Sirolimus; Tacrolimus

Topics: Animals; Anti-Bacterial Agents; Carrier Proteins; Cyclosporine; Dermatitis, Atopic; Dermatitis, Contact; Disease Models, Animal; Guinea Pigs; Immunosuppressive Agents; Mice; Polyenes; Receptors, Drug; Receptors, Immunologic; Sirolimus; Swine; Tacrolimus; Tacrolimus Binding Proteins

Primary osteosarcoma of the breast is a very rare malignancy that shares histological features with osteosarcoma. It is also highly sensitive to methionine restriction due to methionine addiction. We previously established a patient-derived orthotopic xenograft (PDOX) nude-mouse model derived from tumor tissue of a patient with primary mammary osteosarcoma. In the present study, we investigated the efficacy of oral-recombinant methioninase (o-rMETase), combined with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on a mammary osteosarcoma PDOX nude-mouse model.. The PDOX mouse model was established by surgically transplanting a specimen of primary osteosarcoma of the breast into the mammary gland of nude mice. Mice implanted with tumors were randomly divided into four groups: Control group, N=5; rapamycin-treated group, N=5; o-rMETase-treated group, N=5; and a group treated with the combination of o-rMETase and rapamycin, N=5. Mice were treated for 2 weeks after transplantation, and tumor volume was measured during the treatment period.. Treatment with the combination of rapamycin and o-rMETase eradicated the osteosarcoma of the breast compared to the untreated control (p=0.000008). o-rMETase alone did not significantly inhibit tumor growth, and rapamycin alone only partially inhibited the tumor (p=0.78 and p=0.018, respectively) compared to the untreated control. There was not a significant difference in mouse weight between the groups.. The combination of rapamycin and o-rMETase was highly effective against primary osteosarcoma of the breast in a PDOX model, suggesting a future clinical strategy for this rare cancer type that currently has no first-line treatment.

Topics: Animals; Bone Neoplasms; Carbon-Sulfur Lyases; Disease Models, Animal; Heterografts; Methionine; Mice; Mice, Nude; Osteosarcoma; Recombinant Proteins; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The coronavirus disease 2019 (COVID-19) has affected approximately 2 million individuals worldwide; however, data regarding fatal cases have been limited.. To report the clinical features of 162 fatal cases of COVID-19 from 5 hospitals in Wuhan between December 30, 2019 and March 12, 2020.. The demographic data, signs and symptoms, clinical course, comorbidities, laboratory findings, computed tomographic (CT) scans, treatments, and complications of the patients with fatal cases were retrieved from electronic medical records.. Young patients with moderate COVID-19 without comorbidity at admission could also develop fatal outcomes. The in-hospital survival time of the fatal cases was similar among the hospitals of different levels in Wuhan.

Topics: Adolescent; Adult; Animals; Asthma; Atrial Fibrillation; Autoantibodies; Biomarkers; Breast Neoplasms; Child; Conjunctivitis, Allergic; Cornea; COVID-19; Cyclosporine; Cytokines; Death, Sudden, Cardiac; Defibrillators, Implantable; Diet; Disease Models, Animal; Docetaxel; Double-Blind Method; Dry Eye Syndromes; Educational Status; Emulsions; Female; Fluorescein Angiography; Fluoresceins; Focus Groups; Heart Failure; Hemothorax; Humans; Inflammation; Keratoconus; Male; Meibomian Glands; Mice; Middle Aged; Multiple Sclerosis; Myocardial Infarction; Myocardium; Nerve Fibers; Nigeria; Obesity; Overweight; Pandemics; Primary Prevention; Prospective Studies; Qualitative Research; Registries; Retinal Ganglion Cells; Retinal Vessels; Schools; Sirolimus; Tertiary Care Centers; Th1 Cells; Th2 Cells; Tomography, Optical Coherence; Troponin I; Tumor Necrosis Factor-alpha; United States; Ventricular Remodeling

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (

Topics: Aged; Animals; B7-H1 Antigen; Cell Proliferation; Cystectomy; Disease Models, Animal; Female; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Phosphorylation; Postoperative Complications; Programmed Cell Death 1 Receptor; Ribosomal Protein S6; Sirolimus; T-Lymphocytes; Urinary Bladder; Urinary Bladder Neoplasms

CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.

Topics: Adult; Animals; Child; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Female; Heart Failure; HeLa Cells; Humans; Lipoma; Male; Mice; Molecular Targeted Therapy; Musculoskeletal Abnormalities; Nevus; Phenotype; Scoliosis; Sirolimus; Syndrome; Thiazoles; Vascular Malformations; Vascular Neoplasms

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Topics: Adolescent; Adult; Animals; Disease Models, Animal; Female; Human Umbilical Vein Endothelial Cells; Humans; Immunosuppressive Agents; Male; Mice; Mice, Nude; Middle Aged; Mutation, Missense; Pilot Projects; Proto-Oncogene Proteins c-akt; Receptor, TIE-2; Signal Transduction; Sirolimus; Vascular Malformations; Veins

Remodeling of the coronary arteries is a common feature in severe cases of Kawasaki disease (KD). This pathology is driven by the dysregulated proliferation of vascular fibroblasts, which can lead to coronary artery aneurysms, stenosis, and myocardial ischemia. We undertook this study to investigate whether inhibiting fibroblast proliferation might be an effective therapeutic strategy to prevent coronary artery remodeling in KD.. We used a murine model of KD (induced by the injection of the Candida albicans water-soluble complex [CAWS]) and analyzed patient samples to evaluate potential antifibrotic therapies for KD.. We identified the mechanistic target of rapamycin (mTOR) pathway as a potential therapeutic target in KD. The mTOR inhibitor rapamycin potently inhibited cardiac fibroblast proliferation in vitro, and vascular fibroblasts up-regulated mTOR kinase signaling in vivo in the CAWS mouse model of KD. We evaluated the in vivo efficacy of mTOR inhibition and found that the therapeutic administration of rapamycin reduced vascular fibrosis and intimal hyperplasia of the coronary arteries in CAWS-injected mice. Furthermore, the analysis of cardiac tissue from KD fatalities revealed that vascular fibroblasts localizing with inflamed coronary arteries up-regulate mTOR signaling, confirming that the mTOR pathway is active in human KD.. Our findings demonstrate that mTOR signaling contributes to coronary artery remodeling in KD, and that targeting this pathway offers a potential therapeutic strategy to prevent or restrict this pathology in high-risk KD patients.

Topics: Animals; Coronary Artery Disease; Coronary Vessels; Disease Models, Animal; Humans; Mice; Mucocutaneous Lymph Node Syndrome; Sirolimus; TOR Serine-Threonine Kinases

Resistance to immunotherapy and chemotherapy hinders the prognosis of pancreatic cancer(PC). We hypothesized that the combination of mTOR inhibitor sirolimus and gemcitabine would change the metabolic landscape of PC and enhance the anti-PD-L1 therapy.. In KPC mice, the following regimens were administered and tumor growth inhibition rates(TGI%) were calculated: sirolimus(S), PD-L1 antibody(P), gemcitabine(G), sirolimus + PD-L1 antibody(SP), sirolimus + gemcitabine(SG), PD-L1 + gemcitabine(PG) and sirolimus + PD-L1 antibody + gemcitabine(SPG). The metabolic changes of tumors were identified by LC-MS and subpopulations of immune cells were measured by flow cytometry. Sirolimus treated macrophages were co-cultured with PC cells in vitro, and the metabolic changes of macrophages and tumor cells as well as tumor cells' viability were detected.. The monotherapy of S, P and G didn't inhibit tumor growth significantly. The combination of SP, PG and SG didn't improve the TGI% significantly compared with monotherapy. However, the TGI% of SPG combination was higher than other groups. The proportion of CD68. mTOR inhibitor can change the immune microenvironment of PC via metabolic reprogramming, thus promoting the efficacy of PD-L1 blockade when combined with gemcitabine.

Topics: Animals; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Disease Models, Animal; Gemcitabine; Mice; Pancreatic Neoplasms; Sirolimus; TOR Serine-Threonine Kinases; Tumor Microenvironment

CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Forkhead Transcription Factors; Graft vs Host Disease; Humans; Mammals; Mice; Sirolimus; T-Lymphocytes, Regulatory; Tretinoin

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Topics: Allografts; Animals; Disease Models, Animal; Graft Rejection; Graft Survival; Heart Transplantation; Hematopoietic Stem Cell Transplantation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Sirolimus

Pyroptosis, an inflammasome-mediated mode of death, plays an important role in glaucoma. It has been shown that regulating the mTOR pathway can inhibit pyroptosis. Unfortunately, whether rapamycin (RAPA), a specific inhibitor of the mTOR pathway, can inhibit optic nerve crush (ONC)-induced pyroptosis to protect retinal ganglion cells (RGCs) has not been investigated. Our research aimed to confirm the effect of intravitreal injection of RAPA on RGCs. Furthermore, we used the ONC model to explore the underlying mechanisms. First, we observed that intravitreal injection of RAPA alleviated RGC damage induced by various types of injury. We then used the ONC model to further explore the potential mechanism of RAPA. Mechanistically, RAPA not only reduced the activation of glial cells in the retina but also inhibited retinal pyroptosis-induced expression of inflammatory factors such as nucleotide-binding oligomeric domain-like receptor 3 (NLRP3), apoptosis-associated speckle-like protein containing a CARD (ASC), N-terminal of gasdermin-D (GSDMD-N), IL-18 and IL-1β. Moreover, RAPA exerted protective effects on RGC axons, possibly by inhibiting glial activation and regulating the mTOR/ROCK pathway. Therefore, this study demonstrates a novel mechanism by which RAPA protects against glaucoma and provides further evidence for its application in preclinical studies.

Topics: Animals; Disease Models, Animal; Glaucoma; Humans; Neuroinflammatory Diseases; Optic Nerve; Optic Nerve Injuries; Retinal Ganglion Cells; Sirolimus; TOR Serine-Threonine Kinases

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA), which removes the O-GlcNAc modification from proteins, has been explored in mouse models of amyloid-beta and tau pathology. However, the O-GlcNAcylation-dependent link between gene expression and neurological behavior remains to be explored. Using chronic administration of Thiamet G (TG, an OGA inhibitor) in vivo, we used a protocol designed to relate behavior with the transcriptome and selected biochemical parameters from the cortex of individual animals. TG-treated mice showed improved working memory as measured using a Y-maze test. RNA sequencing analysis revealed 151 top differentially expressed genes with a Log2fold change >0.33 and adjusted p-value <0.05. Top TG-dependent upregulated genes were related to learning, cognition and behavior, while top downregulated genes were related to IL-17 signaling, inflammatory response and chemotaxis. Additional pathway analysis uncovered 3 pathways, involving gene expression including 14 cytochrome c oxidase subunits/regulatory components, chaperones or assembly factors, and 5 mTOR (mechanistic target of rapamycin) signaling factors. Multivariate Kendall correlation analyses of behavioral tests and the top TG-dependent differentially expressed genes revealed 91 statistically significant correlations in saline-treated mice and 70 statistically significant correlations in TG-treated mice. These analyses provide a network regulation landscape that is important in relating the transcriptome to behavior and the potential impact of the O-GlcNAC pathway.

Topics: Animals; Disease Models, Animal; Gene Expression; Mice; Protein Processing, Post-Translational; Signal Transduction; Sirolimus

FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice.. We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig.. We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process.. These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

Topics: Abatacept; Animals; Clinical Deterioration; CTLA-4 Antigen; Disease Models, Animal; Forkhead Transcription Factors; Humans; Immune System Diseases; Mice; Sirolimus; T-Lymphocytes, Regulatory

Topics: Animals; Autism Spectrum Disorder; Disease Models, Animal; Mice; Mice, Inbred C57BL; Nucleus Accumbens; Sirolimus; Social Behavior

Alkali burn-induced corneal injury often causes inflammation and neovascularization and leads to compromised vision. We previously reported that rapamycin ameliorated corneal injury after alkali burns by methylation modification. In this study, we aimed to investigate the rapamycin-medicated mechanism against corneal inflammation and neovascularization. Our data showed that alkali burn could induce a range of different inflammatory response, including a stark upregulation of pro-inflammatory factor expression and an increase in the infiltration of myeloperoxidase- and F4/80-positive cells from the corneal limbus to the central stroma. Rapamycin effectively downregulated the mRNA expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), toll-like receptor 4 (TLR4), nucleotide binding oligomerization domain-like receptors (NLR) family pyrin domain-containing 3 (NLRP3), and Caspase-1, and suppressed the infiltration of neutrophils and macrophages. Inflammation-related angiogenesis mediated by matrix metalloproteinase-2 (MMP-2) and rapamycin restrained this process by inhibiting the TNF-α upregulation in burned corneas of mice. Rapamycin also restrained corneal alkali burn-induced inflammation by regulating HIF-1α/VEGF-mediated angiogenesis and the serum cytokines TNF-α, IL-6, Interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The findings of this study indicated rapamycin may reduce inflammation-associated infiltration of inflammatory cells, shape the expression of cytokines, and balance the regulation of MMP-2 and HIF-1α-mediated inflammation and angiogenesis by suppressing mTOR activation in corneal wound healing induced by an alkali injury. It offered novel insights relevant for a potent drug for treating corneal alkali burn.

Topics: Alkalies; Animals; Burns, Chemical; Cornea; Corneal Injuries; Corneal Neovascularization; Cytokines; Disease Models, Animal; Eye Burns; Inflammation; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic; Sirolimus; Tumor Necrosis Factor-alpha

Mitochondrial diseases represent a spectrum of disorders caused by impaired mitochondrial function, ranging in severity from mortality during infancy to progressive adult-onset disease. Mitochondrial dysfunction is also recognized as a molecular hallmark of the biological ageing process. Rapamycin, a drug that increases lifespan and health during normative ageing, also increases survival and reduces neurological symptoms in a mouse model of the severe mitochondrial disease Leigh syndrome. The Ndufs4 knockout (Ndufs4

Topics: Acarbose; Animals; Disease Models, Animal; Electron Transport Complex I; Leigh Disease; Mice; Mitochondria; Mitochondrial Diseases; Sirolimus

Benign prostatic hyperplasia (BPH) is a condition that becomes more common with age and manifests itself primarily as the expansion of the prostate and surrounding tissues. However, to date, the etiology of BPH remains unclear. In this respect, we performed single-cell RNA sequencing of prostate transition zone tissues from elderly individuals with different prostate volumes to reveal their distinct tissue microenvironment. Ultimately, we demonstrated that a reduced Treg/CD4+ T-cell ratio in the large-volume prostate and a relatively activated immune microenvironment were present, characterized partially by increased expression levels of granzymes, which may promote vascular growth and profibrotic processes and further exacerbate BPH progression. Consistently, we observed that the prostate gland of patients taking immunosuppressive drugs usually remained at a smaller volume. Furthermore, in mouse models, we confirmed that both suppression of the immune system with rapamycin and induction of Treg proliferation with low doses of IL-2 therapy indeed prevented the progression of BPH. Taken together, our findings suggest that an activated immune microenvironment is necessary for prostate volume growth and that Tregs can reverse this immune activation state, thereby inhibiting the progression of BPH.

Topics: Animals; Disease Models, Animal; Humans; Interleukin-2; Male; Mice; Prostate; Prostatic Hyperplasia; Sirolimus

Genetic analysis in human patients has linked mutations in PIK3CA, the catalytic subunit of PI-3'Kinase, to sporadic incidences of vascular malformations.. We have developed a mouse model with inducible and endothelial-specific expression of PIK3CA. This approach allows us to successfully model the human disease in a mature and established vascular bed and track the development of vascular malformations. To validate the utility of this model, we applied a topical rapamycin ointment, as rapamycin is therapeutically beneficial to patients in clinical trials. We found that the induced ear lesions showed significant attenuation after treatment, which was easily quantified.. These data collectively provide evidence of a new model to study vascular malformations in adult tissues, which should be particularly useful in environments lacking specialized small-animal imaging facilities.

Topics: Adult; Animals; Catalytic Domain; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Humans; Mice; Sirolimus; Vascular Malformations

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the

Topics: Animals; Death, Sudden, Cardiac; Disease Models, Animal; Everolimus; Fibrosis; Humans; Mammals; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Myocarditis; Sarcoidosis; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Osteoarthritis (OA) is a prevalent joint disease that affects all the tissues within the joint and currently lacks disease-modifying treatments in clinical practice. Despite the potential of rapamycin for OA disease alleviation, its clinical application is hindered by the challenge of achieving therapeutic concentrations, which necessitates multiple injections per week. To address this issue, rapamycin was loaded into poly(lactic-co-glycolic acid) nanoparticles (RNPs), which are nontoxic, have a high encapsulation efficiency and exhibit sustained release properties for OA treatment. The RNPs were found to promote chondrogenic differentiation of ATDC5 cells and prevent senescence caused by oxidative stress in primary mouse articular chondrocytes. Moreover, RNPs were capable to alleviate metabolism homeostatic imbalance of primary mouse articular chondrocytes in both monolayer and 3D cultures under inflammatory or oxidative stress. In the mouse destabilization of the medial meniscus (DMM) model, intra-articular injection of RNPs effectively mitigated joint cartilage destruction, osteophyte formation, chondrocytes hypertrophy, synovial inflammation, and pain. Our study demonstrates the feasibility of using RNPs as a potential clinically translational therapy to prevent the progression of post-traumatic OA.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Mice; Nanoparticles; Osteoarthritis; Sirolimus

Intimal hyperplasia (IH) is a major cause of vascular restenosis after bypass surgery, which progresses as a series of processes from the acute to chronic stage in response to endothelial damage during bypass grafting. A strategic localized drug delivery system that reflects the pathophysiology of IH and minimizes systemic side effects is necessary. In this study, the sequential release of sirolimus, a mechanistic target of rapamycin (mTOR) inhibitor, and statin, an HMG-COA inhibitor, was realized as a silk fibroin-based microneedle device in vivo. The released sirolimus in the acute stage reduced neointima (NI) and vascular fibrosis through mTOR inhibition. Furthermore, rosuvastatin, which was continuously released from the acute to chronic stage, reduced vascular stiffness and apoptosis through the inactivation of Yes-associated protein (YAP). The sequential release of sirolimus and rosuvastatin confirmed the synergistic treatment effects on vascular inflammation, VSMC proliferation, and ECM degradation remodeling through the inhibition of transforming growth factor (TGF)-beta/NF-κB pathway. These results demonstrate the therapeutic effect on preventing restenosis with sufficient vascular elasticity and significantly reduced IH in response to endothelial damage. Therefore, this study suggests a promising strategy for treating coronary artery disease through localized drug delivery of customized drug combinations.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Fibroins; Humans; Hyperplasia; Rosuvastatin Calcium; Sirolimus; TOR Serine-Threonine Kinases

The role of cellular senescence in the development of asthma is not well known. We aimed to evaluate the susceptibility of mice with cellular senescence to asthma development and determine whether the mTOR pathway played an important role in this process. Cellular senescence was induced in mice by intranasal instillation of 2% cigarette smoke extract (CSE). Subsequently, a low dose (0.1 μg) of house dust mite (HDM) allergens, which cause no inflammation and airway hyperresponsiveness (AHR) in mice without cellular senescence, was administered intranasally. To evaluate the role of the mTOR pathway in this model, rapamycin (TORC1 inhibitor) was injected intraperitoneally before CSE instillation. CSE significantly increased senescence-associated β-gal activity in lung homogenate and S100A8/9+ p-mTOR+ population in lung cells. Moreover, S100A8/9+ or HMGB1+ populations in airway epithelial cells with p-mTOR activity increased remarkably. Rapamycin attenuated all changes. Subsequent administration of low-dose HDM allergen induced murine asthma characterized by increased AHR, serum HDM-specific immunoglobulin E, and eosinophilic airway inflammation; these asthma characteristics disappeared after rapamycin injection. In vitro experiments showed significant activation of bone marrow-derived cells cocultured with S100A9 or HMGB1 overexpressing MLE-12 cells treated with HDM allergen, compared to those treated with HDM allergen only. CSE increased the levels of senescence markers (S100A8/9 and HMGB1) in airway epithelial cells, making the mice susceptible to asthma development due to low-dose HDM allergens by activating dendritic cells. Because rapamycin significantly attenuated asthma characteristics, the mTOR pathway may be important in this murine model.

Topics: Allergens; Animals; Asthma; Cellular Senescence; Cigarette Smoking; Disease Models, Animal; HMGB1 Protein; Lung; Mice; Mice, Inbred BALB C; Pyroglyphidae; Sirolimus; TOR Serine-Threonine Kinases

Compelling evidence has described that the incidence of hypertension and left ventricular hypertrophy (LVH) in postmenopausal women is significantly increased worldwide. Our team's previous research identified that androgen was an underlying factor contributing to increased blood pressure and LVH in postmenopausal women. However, little is known about how androgens affect LVH in postmenopausal hypertensive women. The purpose of this study was to evaluate the role of mammalian rapamycin receptor (mTOR) signaling pathway in myocardial hypertrophy in androgen-induced postmenopausal hypertension and whether mTOR inhibitors can protect the myocardium from androgen-induced interference to prevent and treat cardiac hypertrophy. For that, ovariectomized (OVX) spontaneously hypertensive rats (SHR) aged 12 weeks were used to study the effects of testosterone (T 2.85 mg/kg/weekly i.m.) on blood pressure and myocardial tissue. On the basis of antihypertensive therapy (chlorthalidone 8 mg/kg/day ig), the improvement of blood pressure and myocardial hypertrophy in rats treated with different dose gradients of rapamycin (0.8 mg/kg/day vs 1.5 mg/kg/day vs 2 mg/kg/day i.p.) in OVX + estrogen (E 9.6 mg/kg/day, ig) + testosterone group was further evaluated. After testosterone intervention, the OVX female rats exhibited significant increments in the heart weight/tibial length (TL), area of cardiomyocytes and the mRNA expressions of ANP, β-myosin heavy chain and matrix metalloproteinase 9 accompanied by a significant reduction in the uterine weight/TL and tissue inhibitor of metalloproteinase 1. mTOR, ribosomal protein S6 kinase (S6K1), 4E-binding protein 1 (4EBP1) and eukaryotic translation initiation factor 4E in myocardial tissue of OVX + estrogen + testosterone group were expressed at higher levels than those of the other four groups. On the other hand, rapamycin abolished the effects of testosterone-induced cardiac hypertrophy, decreased the systolic and diastolic blood pressure of SHR, and inhibited the activation of mTOR/S6K1/4EBP1 signaling pathway in a concentration-dependent manner. Collectively, these data suggest that the mTOR/S6K1/4EBP1 pathway is an important therapeutic target for the prevention of LVH in postmenopausal hypertensive female rats with high testosterone levels. Our findings also support the standpoint that the mTOR inhibitor, rapamycin, can eliminate testosterone-induced cardiomyocyte hypertrophy.

Topics: Animals; Blood Pressure; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Hypertrophy, Left Ventricular; Intracellular Signaling Peptides and Proteins; MTOR Inhibitors; Myocardium; Ovariectomy; Rats, Inbred SHR; Rats, Wistar; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Testosterone; TOR Serine-Threonine Kinases

Genetic mitochondrial diseases are the most frequent cause of inherited metabolic disorders and one of the most prevalent causes of heritable neurological disease. Leigh syndrome is the most common clinical presentation of pediatric mitochondrial disease, typically appearing in the first few years of life, and involving severe multisystem pathologies. Clinical care for Leigh syndrome patients is difficult, complicated by the wide range of symptoms including characteristic progressive CNS lesion, metabolic sequelae, and epileptic seizures, which can be intractable to standard management. While no proven therapies yet exist for the underlying mitochondrial disease, a ketogenic diet has led to some reports of success in managing mitochondrial epilepsies, with ketosis reducing seizure risk and severity. The impact of ketosis on other aspects of disease progression in Leigh syndrome has not been studied, however, and a rigorous study of the impact of ketosis on seizures in mitochondrial disease is lacking. Conversely, preclinical efforts have identified the intracellular nutrient signaling regulator mTOR as a promising therapeutic target, with data suggesting the benefits are mediated by metabolic changes. mTOR inhibition alleviates epilepsies arising from defects in TSC, an mTOR regulator, but the therapeutic potential of mTOR inhibition in seizures related to primary mitochondrial dysfunction is unknown. Given that ketogenic diet is used clinically in the setting of mitochondrial disease, and mTOR inhibition is in clinical trials for intractable pediatric epilepsies of diverse causal origins, a direct experimental assessment of their effects is imperative. Here, we define the impact of dietary ketosis on survival and CNS disease in the Ndufs4(KO) mouse model of Leigh syndrome and the therapeutic potential of both dietary ketosis and mTOR inhibition on seizures in this model. These data provide timely insight into two important clinical interventions.

Topics: Animals; Diet, Ketogenic; Disease Models, Animal; Electron Transport Complex I; Leigh Disease; Mice; Mice, Knockout; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Sepsis often leads to multiple organ failure or even death and is a significant health problem that contributes to a heavy economic burden. The lung is the first organ to be affected by sepsis. Presently, there is no specific drug or method to treat sepsis and sepsis-induced acute lung injury (ALI). H2S, along with CO and NO, is a physiological gas that acts as a signaling molecule and plays an active role in fighting various lung infections. GYY4137 is a novel H2S donor that is stable in vivo and in vitro. However, particularly in the context of ferroptosis, GYY4137 affects cecal ligation and puncture (CLP)-induced ALI by a mechanism that is not understood. Ferroptosis is a new form of cell necrosis. The primary mechanism is the accumulation of cellular lipid ROS in an iron-dependent manner. The principal objective of this project was to investigate the effects of GYY4137 on ferroptosis and autophagy in a mouse model of sepsis-induced ALI. We divided the experimental mice randomly into 5 groups: (1) sham group; (2) CLP group; (3) CLP + DMSO group: (4) CLP + GYY4137 (25 mg/kg) group; and (5) CLP + GYY4137 (50 mg/kg) group. (6) CLP + Rapamycin (2.0 mg/Kg) group. (7) CLP + Chloroquine (80 mg/Kg) group. (8) the Chloroquine (80 mg/Kg) + GYY (50 mg/Kg) group. The findings showed that GYY4137 significantly protected against CLP-induced ALI by improving sepsis-induced lung histopathological changes, diminishing lung tissue damage, ameliorating oxidative stress, and attenuating the severity of lung injury in mice. In this study, we found that GYY4137 could alleviate septicemia-induced ferroptosis in ALI by increasing the expression of GPx4 and SLC7A11 in lung tissue after CLP. One unexpected finding was the extent to which the levels of ferritin and ferritin light chain increased after CLP, which may be a compensatory mechanism for storing abnormally increased iron. We also found that the expression of p-mTOR, P62, and Beclin1 was significantly increased and that LC3II/LC3I declined after LPS stimulation, but the effect was inhibited by treatment with GYY4137, indicating that GYY4137 could inhibit the activation of autophagy in sepsis-induced ALI by blocking mTOR signaling.

Topics: Acute Lung Injury; Animals; Autophagy; Cecum; Cell Line; Disease Models, Animal; Ferroptosis; Hydrogen Sulfide; Ligation; Lung; Male; Mice; Mice, Inbred C57BL; Morpholines; Multiple Organ Failure; Organothiophosphorus Compounds; RAW 264.7 Cells; Sepsis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

This study test was designed to investigate the possible modulatory effect of rapamycin combined with HO-3867 in monocrotaline(MCT)-induced pulmonary arterial hypertension in rats. We hypothesized that combined treatment with rapamycin and HO-3867 is superior to either alone in attenuating MCT-induced rat pulmonary arterial hypertension (PAH). Pulmonary arterial hypertension was induced by a single intraperitoneal injection of monocrotaline (60 mg/kg). 2 weeks later, rapamycin (2 mg/kg i.p.) and HO3867 (10 mg/kg i.h.) were administered daily, alone and in combination, for 2 weeks. Right ventricular systolic pressure, echocardiography were recorded and then rats were sacrificed. Histological analysis of pulmonary arteries medial wall thickness, right ventricular hypertrophy index (RVHI), the ratio of right ventricular to body weight, and collagen volume fraction (CVF) of right ventricular were performed. Moreover, the expression of t-STAT3, p-STAT3, t-Akt, p-Akt in lung and t-STAT3, p-STAT3, t-S6, p-S6 in right ventricular were examined. The result showed that combined treatment provided a considerable improvement toward maintaining hemodynamic changes, lung vascular remodeling as well as amending RV remodeling and function. Furthermore, Combined treatment can normalize the protein levels of two signal pathways in lung and heart tissue, where p-S6 or p-Akt significantly decreased compared to HO-3867 alone, or p-STAT3 significantly reduced compared to rapamycin alone. In conclusion, combined treatment with rapamycin and HO-3867 is superior to either alone in attenuating MCT-induced PAH in rats.

Topics: Animals; Disease Models, Animal; Hypertension, Pulmonary; Monocrotaline; Piperidones; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sirolimus

The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.

Topics: Animals; Disease Models, Animal; Epilepsies, Partial; Epilepsy; GTPase-Activating Proteins; Membrane Transport Proteins; Mice; Mice, Knockout; Mutation; Nitrogen; Seizures; Sirolimus; Telencephalon; Tumor Suppressor Proteins

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

Topics: Animals; Cadherins; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Disease Models, Animal; GTP-Binding Protein alpha Subunits, G12-G13; Mice; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Sirolimus; TOR Serine-Threonine Kinases

The Food and Drug Administration–approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

Topics: Animals; Disease Models, Animal; Hearing Loss, Sensorineural; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Knockout; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Signal Transduction; Sirolimus; Tumor Suppressor Protein p53

T cells are the current choice for many cell therapy applications. They are relatively easy to access, expand in culture, and genetically modify. Rapamycin-conditioning ex vivo reprograms T cells, increasing their memory properties and capacity for survival, while reducing inflammatory potential and the amount of preparative conditioning required for engraftment. Rapamycin-conditioned T cells have been tested in patients and deemed to be safe to administer in numerous settings, with reduced occurrence of infusion-related adverse events. We demonstrate that ex vivo lentivirus-modified, rapamycin-conditioned CD4

Topics: alpha-Galactosidase; Animals; Disease Models, Animal; Lentivirus; Lysosomal Storage Diseases; Lysosomes; Mice; Sirolimus; T-Lymphocytes

Peritoneal adhesion formation is a challenging postoperative complication. We aim to evaluate the effect of orally administered sirolimus, prednisolone, and their combination to prevent this entity.. Eighty female albino underwent intraperitoneal injection of 3 mL of 10% sterile talc solution to induce peritoneal adhesion, and were subsequently and randomly divided into four groups (each n = 20); including a control group; 1 mg/kg oral prednisolone daily in the morning; 0.1 mg/kg oral sirolimus daily; and a combination group which received both drugs, with the same dosage. On the 29th day, abdominal cavities were explored, and classification was done based on Nair classification.. All rats were healthy on the 29th day, in which exploration was performed. The rats in the control group had extensive intra-abdominal adhesions, while 17 (85%) rats in the control group had substantial adhesion; however, the prednisolone, sirolimus, and combination group had lesser adhesion formation. Also, 14 (70%) rats of prednisolone group, 13 (65%) of sirolimus group, and 16 (80%) of combination group had insubstantial adhesion. The decrease in the grade of peritoneal adhesion bands was highly significant in the combination group (P > 0.001).. The combination of sirolimus and prednisolone was effective for preventing peritoneal adhesions in rats.

Topics: Abdominal Cavity; Animals; Disease Models, Animal; Female; Peritoneal Diseases; Postoperative Complications; Prednisolone; Rats; Sirolimus; Tissue Adhesions

The purpose of this experiment is to find out the function of Vitamin D (VD) in airway inflammation in asthmatic guinea pigs by regulating mammalian target of rapamycin (mTOR)-mediated autophagy. A total of 40 male guinea pigs were randomly assigned into the Con group, the ovalbumin (OVA)-sensitized group, the VD group, the VD + dimethyl sulfoxide group, and the VD + rapamycin (mTOR inhibitor) group. Then, serum from all groups was harvested for the measurement of immunoglobulin E (IgE), interleukin (IL)-4, and IL-5 levels. Next, bronchoalveolar lavage fluid was collected for cell counting. Moreover, lung tissues were extracted to assess levels of p-mTOR and autophagy factors (LC3B, Beclin1, Atg5, and P62). Compared with the Con group, the OVA group showed elevated levels of IgE, IL-4, and IL-5, increased contents of eosinophils, neutrophil, and lymphocytes, and declined monocytes. And the VD group improved inflammatory reactions in the guinea pigs. Besides, the OVA group showed lower levels of p-mTOR and P62 and higher autophagy levels than the Con group, while the VD group had opposite results. Rapamycin annulled the suppressive role of VD to airway inflammation in asthmatic guinea pigs. VD might inhibit OVA-induced airway inflammation by inducing mTOR activation and downregulating autophagy in asthmatic guinea pigs.

Topics: Animals; Asthma; Autophagy; Disease Models, Animal; Female; Guinea Pigs; Immunoglobulin E; Inflammation; Interleukin-5; Lung; Male; Mammals; Ovalbumin; Sirolimus; TOR Serine-Threonine Kinases; Vitamin D

Balloon angioplasty, stent implantation, and application of an arterial clamp during surgery can induce artery injury such as elastin breaks and endothelium injury, but there is little research focused on the injury induced by these therapeutic manipulations. We established a simple and reproducible small animal aortic injury model and examined intramural injection as a potential therapeutic method to alleviate injury.. The abdominal aorta of male Sprague Dawley (SD) rats or C57BL/6 J mice was clamped sequentially throughout its length. Transforming growth factor β1 (TGFβ1), SB431542, lipopolysaccharide (LPS), Necrostatin-1 (Nec-1), rapamycin, or MHY1485 contained in Pluronic gel was injected intramurally at day 0 or day 7. Animals were fed with chow containing 0.25% beta-aminopropionitrile (BAPN) to evaluate the influence of BAPN. All samples were harvested and examined by immunohistochemistry and immunofluorescence.. The clamped rat aorta showed luminal dilation, elastin fiber breaks, neointimal hyperplasia, and dissection (days 0-90). Intramural injection of TGFβ1, rapamycin and Nec-1 showed a protective effect on the injured aorta, whereas SB431542, MHY1485 and LPS showed more severe wall damage. The aortic lumen in rats fed with BAPN was significantly larger than in control rats (day 7). Mouse aorta showed similar injury with neointimal hyperplasia and elastin fiber breaks.. The rodent arterial injury model is reproducible and may mimic early changes of arterial injury. The model accommodates intramural injection of different drugs that may show mechanisms of arterial injury. Although this is a preliminary animal model, the intramural injection method may have potential clinical application in the future.

Topics: Aminopropionitrile; Animals; Aorta, Abdominal; Disease Models, Animal; Elastin; Hyperplasia; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Neointima; Poloxamer; Rats; Rats, Sprague-Dawley; Sirolimus

Alzheimer's disease (AD) is associated with amyloid-β (Aβ) accumulation that might be hindered by autophagy. There are two ways to induce autophagy: through mTOR-dependent and mTOR-independent pathways (here, by means of rapamycin and trehalose, respectively). The aim of this study was to evaluate the contribution of these pathways and their combination to the treatment of experimental AD. Mice were injected bilaterally intracerebroventricularly with an Aβ fragment (25-35) to set up an AD model. Treatment with rapamycin (10 mg/kg, every other day), trehalose consumption with drinking water (2 mg/mL, ad libitum), or their combination started 2 days after the surgery and lasted for 2 weeks. Open-field, plus-maze, and passive avoidance tests were used for behavioral phenotyping. Neuronal density, Aβ accumulation, and the expression of autophagy marker LC3-II and neuroinflammatory marker IBA1 were measured in the frontal cortex and hippocampus. mRNA levels of autophagy genes (Atg8, Becn1, and Park2) were assessed in the hippocampus. Trehalose but not rapamycin caused pronounced prolonged autophagy induction and transcriptional activation of autophagy genes. Both drugs effectively prevented Aβ deposition and microglia activation. Autophagy inhibitor 3-methyladenine significantly attenuated autophagy activation and disturbed the effect of the inducers on Aβ load. The inducers substantially reversed behavioral and neuronal deficits in Aβ-injected mice. In many cases, the best outcomes were achieved with the combined treatment. Thus, trehalose alone or combined autophagy activation by the two inducers may be a promising treatment approach to AD-like neurodegeneration. Some aspects of interaction between mTOR-dependent and mTOR-independent pathways of autophagy are discussed.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autophagy; Disease Models, Animal; Mice; Mice, Transgenic; Sirolimus; Therapies, Investigational; TOR Serine-Threonine Kinases; Trehalose

The mechanistic target of rapamycin (mTOR) signaling pathway plays a major role in key cellular processes including metabolism and differentiation; however, the role of mTOR in microglia and its importance in Alzheimer's disease (AD) have remained largely uncharacterized. We report that selective loss of

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Disease Models, Animal; Female; Male; Membrane Glycoproteins; Mice; Mice, Transgenic; Microglia; Plaque, Amyloid; Receptors, Immunologic; Sirolimus; TOR Serine-Threonine Kinases

We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes.. Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized.. In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance.. In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Glioma; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Sirolimus

Crocin is a monomer of Chinese traditional herbs extracted from saffron, relieving depression-like behavior. However, its underlying mechanism of action remains unclear. Herein, we explored whether crocin's antidepressant effect depended on the mammalian target of the rapamycin (mTOR) signaling pathway. The model of PC12 cells injury was established by corticosterone, the changes in cell survival rate were tested by the CCK-8 method, and the changes in cellular morphology were observed under a fluorescence microscope. The depression model was established by chronic unpredictable mild stress (CUMS), and its antidepressant effect was estimated by open field test (OFT), forced swimming test (FST), and tail suspension test (TST). Western blot was used to monitor the protein expression. The results showed that crocin could effectively improve cell survival rate and cellular synaptic growth, alleviate the depressive behavior of CUMS mice, and promote the expression of BDNF, P-mTOR, P-ERK, and PSD95. However, when rapamycin was pretreated, the antidepressant effects of crocin were inhibited. In summary, crocin plays a significant antidepressant effect. After pretreatment with rapamycin, the anti-depression effect of crocin was significantly inhibited. It is suggested that the mechanism of the anti-depression effect of crocin may be related to the mTOR signaling pathway.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Brain-Derived Neurotrophic Factor; Carotenoids; Disease Models, Animal; Hippocampus; Mammals; Mice; Rats; Signal Transduction; Sirolimus; Stress, Psychological; TOR Serine-Threonine Kinases

To investigate the effects of everolimus, a mechanistic/mammalian target of rapamycin (mTOR) inhibitor, on tumor growth and immune response in a mouse model of breast cancer. Human hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) MC4-L2 cell line was used to establish a mouse model of breast cancer. The inhibitory effects of high (10 mg/kg) and low (5 mg/kg) doses of everolimus were investigated on tumor growth. Additionally, the frequency of CD4+Foxp3+ regulatory T cells (Tregs), CD8+Foxp3+ Tregs, and CD4+ and CD8+ T cells expressing cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was explored by flow cytometry in bone marrow, lymph nodes, and spleen. Our results showed that both 10 mg/kg and 5 mg/kg doses of everolimus efficiently inhibited tumor growth, resulting in reduced breast tumor volume. In addition, it was revealed that everolimus-treated mice induced a higher frequency of CD4+Foxp3+ Tregs, CD8+Foxp3+ Tregs, and CD4+Foxp3+CTLA-4+ Tregs as well as CD4+ and CD8+ T cells expressing CTLA-4 in their bone marrow, lymph nodes, and spleen compared with standard control (vehicle-treated) in a dose-dependent manner. Furthermore, we found that everolimus treatment with 10 mg/kg and 5 mg/kg increased the frequency of Helios+Foxp3+ Tregs in the bone marrow of treated mice compared with the control group. Our results indicate that treatment with everolimus not only inhibits tumor growth but also exerts an immunomodulatory effect by inducing Tregs in the lymphoid organs of breast cancer-bearing mice. The combination of therapy with other anti-cancer agents may negate immune suppression and improve the efficacy of mTOR-targeted breast cancer therapy.

Topics: Animals; Breast Neoplasms; CTLA-4 Antigen; Disease Models, Animal; Everolimus; Female; Forkhead Transcription Factors; Humans; Mammals; Mice; Sirolimus; TOR Serine-Threonine Kinases

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes, which encode proteins that negatively regulate mTOR complex 1 (mTORC1) signaling. Current treatment strategies focus on mTOR inhibition with rapamycin and its derivatives. While effective at improving some aspects of TSC, chronic rapamycin inhibits both mTORC1 and mTORC2 and is associated with systemic side-effects. It is currently unknown which mTOR complex is most relevant for TSC-related brain phenotypes. Here we used genetic strategies to selectively reduce neuronal mTORC1 or mTORC2 activity in mouse models of TSC. We find that reduction of the mTORC1 component Raptor, but not the mTORC2 component Rictor, rebalanced mTOR signaling in Tsc1 knock-out neurons. Raptor reduction was sufficient to improve several TSC-related phenotypes including neuronal hypertrophy, macrocephaly, impaired myelination, network hyperactivity, and premature mortality. Raptor downregulation represents a promising potential therapeutic intervention for the neurological manifestations of TSC.

Topics: Animals; Disease Models, Animal; Down-Regulation; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Neurons; Regulatory-Associated Protein of mTOR; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis

Childhood asthma is a common chronic childhood disease. Branched-chain amino acid transaminase 1 (BCAT1) was reported to be upregulated in chronic airway diseases, while its role in childhood asthma is unclear. Asthma mouse models were established in neonatal mice by 10 µg ovalbumin (OVA) intraperitoneal injection and 3% OVA inhalational challenge. In OVA-challenged mice, BCAT1 levels were upregulated. BCAT1 inhibitor alleviated airway structure and inflammation by suppressing IgE, OVA-specific IgE and inflammatory cytokine release and inflammatory cell infiltration. BCAT1 inhibitor alleviated airway remodeling by inhibiting goblet cell hyperplasia, mucus secretion and the expression of α-SMA and collagen I/III. The BCAT1 inhibitor prevented OVA-enhanced autophagy by decreasing Beclin-1, Atg5 and LC3I/II and increasing p65 levels. In IL-13-stimulated BEAS-2B cells, rapamycin promoted inflammatory cytokine release and autophagy after BCAT1 inhibitor administration. Our research revealed that BCAT1 was upregulated in neonatal asthmatic mice and that a BCAT1 inhibitor might restrain airway inflammation and remodeling by decreasing autophagy, which offered a novel mechanistic understanding of childhood asthma.

Topics: Airway Remodeling; Amino Acids, Branched-Chain; Animals; Asthma; Autophagy; Beclin-1; Bronchoalveolar Lavage Fluid; Collagen; Cytokines; Disease Models, Animal; Immunoglobulin E; Inflammation; Interleukin-13; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Sirolimus; Transaminases

We previously demonstrated that prenatal exposure to valproic acid (VPA), an environmental model of autism spectrum disorder (ASD), leads to a hyperexcitable phenotype associated with downregulation of inward-rectifying potassium currents in nucleus accumbens (NAc) medium spiny neurons (MSNs) of adolescent rats. Aberrant mTOR pathway function has been associated with autistic-like phenotypes in multiple animal models, including gestational exposure to VPA. The purpose of this work was to probe the involvement of the mTOR pathway in VPA-induced alterations of striatal excitability. Adolescent male Wistar rats prenatally exposed to VPA were treated acutely with the mTOR inhibitor rapamycin and used for behavioral tests, ex vivo brain slice electrophysiology, single-neuron morphometric analysis, synaptic protein quantification and gene expression analysis in the NAc. We report that postnatal rapamycin ameliorates the social deficit and reverts the abnormal excitability, but not the inward-rectifying potassium current defect, of accumbal MSNs. Synaptic transmission and neuronal morphology were largely unaffected by prenatal VPA exposure or postnatal rapamycin treatment. Transcriptome analysis revealed extensive deregulation of genes implied in neurodevelopmental disorders and ionic mechanisms exerted by prenatal VPA, which was partially reverted by postnatal rapamycin. The results of this work support the existence of antagonistic interaction between mTOR and VPA-induced pathways on social behavior, neurophysiological phenotype and gene expression profile, thus prompting further investigation of the mTOR pathway in the quest for specific therapeutic targets in ASD.

Topics: Animals; Autism Spectrum Disorder; Behavior, Animal; Disease Models, Animal; Female; Male; Neurons; Phenotype; Potassium; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases; Valproic Acid

After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ. Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ. Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autophagy; Disease Models, Animal; Hippocampus; Mammals; Mice; Mice, Inbred C57BL; Mice, Transgenic; Moxibustion; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

After long times of ongoing research, still there is no appropriate cure for Alzheimer's disease (AD). Recently, epigenetic alterations, particularly miRNA, have gotten attention in AD research. Among various miRNA, miR-34c has been addressed to be elevated in the brain of AD patients, however, its exact role and downstream mechanisms have not been elucidated yet. This study aimed to investigate the therapeutic potential of miR-34c antagomir on cognitive dysfunction induced by streptozocin (STZ), considering postsynaptic density protein 95 (PSD-95) and mammalian target of rapamycin expression (mTOR). Forty rats were cannulated intraventricularly under deep anesthesia using stereotaxic apparatus and divided into five groups: saline + saline, STZ + saline, STZ + miR-34c antagomir, STZ + lipofectamine, and STZ + scrambled, and received the related treatments for two weeks. At the end of the treatments, spatial memory and locomotor activity were assessed by Morris water maze (MWM), and open fields, respectively. Finally, PSD-95 and mTOR levels were measured by quantitative real-time PCR (qPCR) and western blotting on hippocampal samples. Results showed that miR-34c antagomir markedly ameliorated spatial learning and memory deficits induced by STZ, and significantly enhanced PSD-95 and mTOR levels in the hippocampus. In conclusion, miR-34c antagomir may be considered as a promising novel therapeutic target for AD patients.

Topics: Alzheimer Disease; Animals; Antagomirs; Disease Models, Animal; Disks Large Homolog 4 Protein; Hippocampus; Mammals; Maze Learning; Memory Disorders; MicroRNAs; Rats; Sirolimus; Streptozocin; TOR Serine-Threonine Kinases

In this study we aimed to reduce tau pathology, a hallmark of Alzheimer's Disease (AD), by activating mTOR-dependent autophagy in a transgenic mouse model of tauopathy by long-term dosing of animals with mTOR-inhibitors. Rapamycin treatment reduced the burden of hyperphosphorylated and aggregated pathological tau in the cerebral cortex only when applied to young mice, prior to the emergence of pathology. Conversely, PQR530 which exhibits better brain exposure and superior pharmacokinetic properties, reduced tau pathology even when the treatment started after the onset of pathology. Our results show that dosing animals twice per week with PQR530 resulted in intermittent, rather than sustained target engagement. Nevertheless, this pulse-like mTOR inhibition followed by longer intervals of re-activation was sufficient to reduce tau pathology in the cerebral cortex in P301S tau transgenic mice. This suggests that balanced therapeutic dosing of blood-brain-barrier permeable mTOR-inhibitors can result in a disease-modifying effect in AD and at the same time prevents toxic side effects due to prolonged over activation of autophagy.

Topics: Alzheimer Disease; Animals; Brain; Disease Models, Animal; Mice; Mice, Transgenic; Sirolimus; tau Proteins; TOR Serine-Threonine Kinases

Marfan syndrome (MFS) is a genetic disorder leading to medial aortic degeneration and life-limiting dissections. To date, there is no causal prevention or therapy. Rapamycin is a potent and selective inhibitor of the mechanistic target of rapamycin (mTOR) protein kinase, regulating cell growth and metabolism. The mgR/mgR mice represent an accepted MFS model for studying aortic pathologies to understand the underlying molecular pathomechanisms. This study investigated whether rapamycin inhibits the development of thoracic aortic aneurysms and dissections in mgR/mgR mice.. Isolated primary aortic smooth muscle cells (mAoSMCs) from mgR/mgR mice were used for in vitro studies. Two mg kg/BW rapamycin was injected intraperitoneally daily for two weeks, beginning at 7-8 weeks of age. Mice were sacrificed 30 days post-treatment. Histopathological and immunofluorescence analyses were performed using adequate tissue specimens and techniques. Animal survival was evaluated accompanied by periodic echocardiographic examinations of the aorta.. The protein level of the phosphorylated ribosomal protein S6 (p-RPS6), a downstream target of mTOR, was significantly increased in the aortic tissue of mgR/mgR mice. In mAoSMCs isolated from these animals, expression of mTOR, p-RPS6, tumour necrosis factor α, matrix metalloproteinase-2 and -9 was significantly suppressed by rapamycin, demonstrating its anti-inflammatory capacity. Short-term rapamycin treatment of Marfan mice was associated with delayed aneurysm formation, medial aortic elastolysis and improved survival.. Short-term rapamycin-mediated mTOR inhibition significantly reduces aortic aneurysm formation and thus increases survival in mgR/mgR mice. Our results may offer the first causal treatment option to prevent aortic complications in MFS patients.

Topics: Animals; Aortic Aneurysm; Disease Models, Animal; Fibrillin-1; Longevity; Marfan Syndrome; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Ribosomal Protein S6; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

The global burden of chronic kidney disease is increasing, and the majority of these diseases are progressive. Special site-targeted drugs are emerging as alternatives to traditional drugs. Oligonucleotides (ODNs) have been proposed as effective therapeutic tools in specific molecular target therapies for several diseases. We designed ring-type non-coding RNAs (ncRNAs), also called mTOR ODNs to suppress mammalian target rapamycin (mTOR) translation. mTOR signaling is associated with excessive cell proliferation and fibrogenesis. In this study, we examined the effects of mTOR suppression on chronic renal injury. To explore the regulation of fibrosis and inflammation in unilateral ureteral obstruction (UUO)-induced injury, we injected synthesized ODNs via the tail vein of mice. The expression of inflammatory-related markers (interleukin-1β, tumor necrosis factor-α), and that of fibrosis (α-smooth muscle actin, fibronectin), was decreased by synthetic ODNs. Additionally, ODN administration inhibited the expression of autophagy-related markers, microtubule-associated protein light chain 3, Beclin1, and autophagy-related gene 5-12. We confirmed that ring-type ODNs inhibited fibrosis, inflammation, and autophagy in a UUO mouse model. These results suggest that mTOR may be involved in the regulation of autophagy and fibrosis and that regulating mTOR signaling may be a therapeutic strategy against chronic renal injury.

Topics: Actins; Animals; Autophagy; Beclin-1; Disease Models, Animal; Fibronectins; Fibrosis; Inflammation; Interleukin-1beta; Kidney; Mammals; Mice; Microtubule-Associated Proteins; Oligonucleotides; Renal Insufficiency, Chronic; RNA, Untranslated; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Ureteral Obstruction

As expected, Rapa/Lps and 5-FU significantly suppressed tumor formation, decreased the number of tumors, and tumor load both in two mouse models, and had no influence on mouse weight. Mechanically, the anti-tumor effect of the drug also was associated in inhibiting angiogenesis and proliferation. Furthermore, we found that Rapa/Lps obviously inhibited HUVECs tube formation and migration.. Altogether, we revealed the Rapa/Lps synergism with 5-FU decreased colon and small intestinal tumorigenesis in AOM/DSS-treated and APC

Topics: Animals; Azoxymethane; Colitis; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Fluorouracil; Lipopolysaccharides; Liposomes; Mice; Mice, Inbred C57BL; Sirolimus

Severe malnutrition accounts for half-a-million deaths annually in children under the age of five. Despite improved WHO guidelines, inpatient mortality remains high and is associated with metabolic dysfunction. Previous studies suggest a correlation between hepatic metabolic dysfunction and impaired autophagy. We aimed to determine the role of mTORC1 inhibition in a murine model of malnutrition-induced hepatic dysfunction. Wild type weanling C57/B6 mice were fed a 18 or 1% protein diet for two weeks. A third low-protein group received daily rapamycin injections, an mTORC1 inhibitor. Hepatic metabolic function was assessed by histology, immunofluorescence, gene expression, metabolomics and protein levels. Low protein-fed mice manifested characteristics of severe malnutrition, including weight loss, hypoalbuminemia, hypoglycemia, hepatic steatosis and cholestasis. Low protein-fed mice had fewer mitochondria and showed signs of impaired mitochondrial function. Rapamycin prevented hepatic steatosis, restored ATP levels and fasted plasma glucose levels compared to untreated mice. This correlated with increased content of LC3-II, and decreased content mitochondrial damage marker, PINK1. We demonstrate that hepatic steatosis and disturbed mitochondrial function in a murine model of severe malnutrition can be partially prevented through inhibition of mTORC1. These findings suggest that stimulation of autophagy could be a novel approach to improve metabolic function in severely malnourished children.

Topics: Animals; Disease Models, Animal; Fatty Liver; Malnutrition; Mechanistic Target of Rapamycin Complex 1; Mice; Sirolimus; TOR Serine-Threonine Kinases

We developed a preclinical model of myocardial ischemia/reperfusion (I/R) injury in conscious diabetic rabbits to identify an early pharmacological intervention for patients with diabetes and acute myocardial infarction (AMI). Here, we describe a reproducible protocol for induction of diabetes with subsequent manifestation of myocardial I/R injury in conscious rabbits to mimic the real-life scenario observed in clinical settings. Further, we demonstrate the efficacy of rapamycin at the onset of reperfusion to limit the adverse effect of AMI. For complete details on the use and execution of this protocol, please refer to Samidurai et al. (2020).

Topics: Alloxan; Animals; Apoptosis; Balloon Occlusion; Cardiotonic Agents; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Electrocardiography; Male; Myocardial Reperfusion Injury; Rabbits; Sirolimus; Troponin I

Tuberculosis (TB) treatment regimens are lengthy, causing non-adherence to treatment. Inadequate treatment can lead to relapse and the development of drug resistance TB. Furthermore, patients often exhibit residual lung damage even after cure, increasing the risk for relapse and development of other chronic respiratory illnesses. Host-directed therapeutics are emerging as an attractive means to augment the success of TB treatment. In this study, we used C3HeB/FeJ mice as an experimental model to investigate the potential role of rapamycin, a mammalian target of rapamycin inhibitor, as an adjunctive therapy candidate during the treatment of Mycobacterium tuberculosis infection with moxifloxacin. We report that administration of rapamycin with or without moxifloxacin reduced infection-induced lung inflammation, and the number and size of caseating necrotic granulomas. Results from this study strengthen the potential use of rapamycin and its analogs as adjunct TB therapy, and importantly underscore the utility of the C3HeB/FeJ mouse model as a preclinical tool for evaluating host-directed therapy candidates for the treatment of TB.

Topics: Animals; B-Lymphocytes; Cell Aggregation; Disease Models, Animal; Female; Lung; Mice; Moxifloxacin; Mycobacterium tuberculosis; Necrosis; Neutrophil Infiltration; Polymethacrylic Acids; Sirolimus; Tuberculosis

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Topics: Adenine; Animals; Autophagy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Transgenic; MTOR Inhibitors; Neuroinflammatory Diseases; Neuroprotective Agents; Parkinson Disease; Parkinson Disease, Secondary; Signal Transduction; Sirolimus; Substantia Nigra; TOR Serine-Threonine Kinases; Trehalose

Autophagy is a self-phagocytic and highly evolutionarily conserved intracellular lysosomal catabolic system, which plays a vital role in a variety of trauma models, including skin wound healing (SWH). However, the roles and potential mechanisms of autophagy in SWH are still controversial. We firstly investigated the role of autophagy in SWH-induced wound closure rate, inflammatory response, and histopathology, utilizing an inhibitor of autophagy 3-methyladenine (3-MA) and its agonist rapamycin (RAP). As expected, we found 3-MA treatment remarkably increased the wound closure rate, combated inflammation response, and mitigated histopathological changes, while RAP delivery aggravated SWH-induced pathological damage. To further exploit the underlying mechanism of autophagy regulating inflammation, the specific inhibitors of yes-associated protein (YAP), Verteporfin, and Anti-IL-33 were applied. Herein, treating with 3-MA markedly suppressed the expression of tumor necrosis factor-α (TNF-α), IL-1β, and IL-6, promoted that of IL-10, IL-33, and ST2, while RAP administration reverted SWH-induced the up-regulation of these inflammatory cytokines mentioned above. Importantly, Verteporfin administration not only down-regulated the expression levels of YAP, TNF-α, and IL-6 but also up-regulated that of IL-33 and IL-10. Unexpectedly, 3-MA or RAP retreatment did not have any impact on the changes in IL-33 among these inflammatory indicators. Furthermore, elevated expression of IL-33 promoted wound closure and alleviated the pathological damage, whereas, its antagonist Anti-IL-33 treatment overtly reversed the above-mentioned effects of IL-33. Moreover, 3-MA in combination with anti-IL-33 treatment reversed the role of 3-MA alone in mitigated pathological changes, but they failed to revert the effect of anti-IL-33 alone on worsening pathological damage. In sum, emerging data support the novel contribution of the YAP/IL-33 pathway in autophagy inhibition against SWH-induced pathological damage, and highlight that the autophagy/YAP/IL-33 signal axis is expected to become a new therapeutic target for SWH.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; Disease Models, Animal; Inflammation; Interleukin-33; Male; Mice; Mice, Inbred ICR; Signal Transduction; Sirolimus; Skin; Wound Healing; YAP-Signaling Proteins

Autophagy has been demonstrated to have a beneficial effect on diabetic nephropathy (DN). Rapamycin, an inhibitor of mTOR, was shown to stimulate β-cell autophagy. However, its effects on preventing or ameliorating DN is unclear, and its effects are worth studying. As fasting is now an attractive protective strategy, we aim to compare its effect to rapamycin effects on pancreatic and renal cells. Twenty-eight adult male Wistar Albino rats were randomly divided into four groups, using streptozotocin (STZ) to induce diabetes mellitus (DM). Autophagy was induced by two ways; rapamycin or fasting. The extent of autophagy and apoptosis were investigated by measuring the level of LC3B and p53 proteins, respectively, in pancreatic and kidney tissues using Western blotting (WB) technique and imaging the renal cells under transmission electron microscope. The efflux transporter P-glycoprotein was quantified by WB as well. Rapamycin-induced autophagy occurred concurrently with apoptosis. On the other hand, fasting supported P-glycoprotein recovery and renal cell survival together with disabling β-cells apoptosis. In conclusion, this study provides a potential link between rapamycin or fasting for the cross-regulation of apoptosis and autophagy in the setting of cell stress as DN. Unlike rapamycin, fasting enhanced the active expression of ABCB1 efflux protein, providing insights on the potential ameliorative effects of fasting in DN that require further elucidation.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autophagosomes; Autophagy; Biomarkers; Cell Survival; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Disease Susceptibility; Fasting; Kidney Function Tests; Liver Function Tests; Oxidative Stress; Rats; Sirolimus

Heart failure, which is characterized by cardiac remodelling, is one of the most common chronic diseases in the aged. Stimulator of interferon genes (STING) acts as an indispensable molecule modulating immune response and inflammation in many diseases. However, the effects of STING on cardiomyopathy, especially cardiac remodelling are still largely unknown. This study was designed to investigate whether STING could affect cardiac remodelling and to explore the potential mechanisms.. In vivo, aortic binding (AB) surgery was performed to construct the mice model of cardiac remodelling. A DNA microinjection system was used to trigger STING overexpression in mice. The STING mRNA and protein expression levels in mice heart were measured, and the cardiac hypertrophy, fibrosis, inflammation and cardiac function were also evaluated. In vitro, cardiomyocytes stimulated by Ang II and cardiac fibroblasts stimulated by TGF-β to performed to further study effects of STING on cardiac hypertrophy and fibroblast. In terms of mechanisms, the level of autophagy was detected in mice challenged with AB. Rapamycin, a canonical autophagy inducer, intraperitoneal injected into mice to study possible potential pathway.. In vivo, the STING mRNA and protein expression levels in mice heart challenged with AB for 6 weeks were significantly increased. STING overexpression significantly mitigated cardiac hypertrophy, fibrosis and inflammation, apart from improving cardiac function. In vitro, experiments further disclosed that STING overexpression in cardiomyocytes induced by Ang II significantly inhibited the level of cardiomyocyte cross-section area and the ANP mRNA. Meanwhile, TGF-β-induced the increase of α-SMA content and collagen synthesis in cardiac fibroblasts could be also blocked by STING overexpression. In terms of mechanisms, mice challenged with AB showed higher level of autophagy compared with the normal mice. However, STING overexpression could reverse the activation of autophagy triggered by AB. Rapamycin, a canonical autophagy inducer, offset the cardioprotective effects of STING in mice challenged with AB. Finally, further experiments unveiled that STING may inhibit autophagy by phosphorylating ULK1 on serine757.. STING may prevent cardiac remodelling induced by pressure overload by inhibiting autophagy, which could be a promising therapeutic target in heart failure. Video Abstract.

Topics: Angiotensin II; Animals; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Cardiomegaly; Disease Models, Animal; Gene Expression Regulation; Heart Failure; Humans; Membrane Proteins; Mice; Myocytes, Cardiac; Protective Agents; Signal Transduction; Sirolimus

Allograft-specific regulatory T cells (T

Topics: Allografts; Animals; Benzylamines; Biomarkers; Cyclams; Dendritic Cells; Disease Models, Animal; Drug Synergism; Graft Rejection; Graft Survival; Heart Transplantation; Immunomodulation; Mice; Prognosis; Receptors, CXCR4; Sirolimus; T-Lymphocytes, Regulatory; Transplantation Immunology; Treatment Outcome

To expound the roles of mTOR and NF-kB signaling pathway in intermittent hypoxia (IH)-induced damage of hippocampal neurons.. For rat experiments, mTOR inhibitor (Rapamycin, Rapa) and NF-κB signaling inhibitor (ammonium pyrrolidine dithiocarbamate, PDTC) were applied to inhibit mTOR and NF-κB signaling, respectively. For neuron experiments, hippocampal neurons from rat were successfully cultured. Different concentrations of Rapa and PDTC were added to the cultured hippocampal neurons. Rat or primary hippocampal neurons were exposed to normoxic or IH conditions after administration of Rapa and PDTC. The effects of Rapa and PDTC administration on learning and memory ability of rats and hippocampal injury after IH exposure were assayed by Morris water maze and H&E staining. Electron microscope was utilized to examine primary hippocampal neuron ultrastructure changes after IH exposure and Rapa or PDTC administration. The expressions of NF-κB-p65, IκBα, IKKβ, BDNF, TNF-α, IL-1β, PSD-95 and SYN in hippocampal neurons were examined.. Compared with normal control rats or neurons, IH-treated group had elevated expression levels of NF-kB, TNF-α and IL-1β and suppressed expression level of BDNF, PSD-95 and SYN. These results were reversed upon pre-treatment with Rapa and PDTC. Furthermore, IκBα and IKKβ expressions were down-regulated in IH group. No notable difference was manifested in PDTC pre-treatment group, while a prominent increase was shown after Rapa pre-administration.. The administration of PDTC and Rapa could prevent IH-induced hippocampal neuron impairment, indicating that inhibition of the mTOR and NF-κB pathway may likely act as a therapeutic target for obstructive sleep apnea.

Topics: Animals; Antioxidants; Cells, Cultured; Disease Models, Animal; Hippocampus; Hypoxia; Male; Neurons; NF-kappa B; Protein Kinase Inhibitors; Pyrrolidines; Rats; Rats, Wistar; Signal Transduction; Sirolimus; Sleep Apnea, Obstructive; Thiocarbamates; TOR Serine-Threonine Kinases

Focal cortical dysplasia (FCD) is a major cause for drug-resistant epilepsies. The molecular and cellular mechanisms of epileptogenesis in FCD are still poorly understood. Some studies have suggested that deficiencies of γ-aminobutyric acid (GABA) system may play an important role in type II FCD, but it remains controversial. In order to examine whether and how GABAergic interneurons and synaptic function are affected, we generated a somatic mTOR hyperactivation-based mouse model of type II FCD by in utero electroporation, quantified densities of interneurons in the malformed cortices, and recorded miniature inhibitory postsynaptic currents in dysmorphic neurons. We detected 20-25% reduction of GABAergic interneurons within malformed cortices, independent of cortical regions and cell subtypes but proportionate to the decrease of global neuron counts. GABAergic synaptic transmission from interneurons to mTOR hyperactivated dysmorphic neurons was dramatically disrupted, outweighing the decrease of interneuron counts. Postnatal mTOR inhibition partially rescued these alterations of GABAergic system. We also quantified the expression of GABA

Topics: Animals; Animals, Newborn; Disease Models, Animal; GABAergic Neurons; Gene Expression Profiling; Gene Expression Regulation; Inhibitory Postsynaptic Potentials; Interneurons; Malformations of Cortical Development; Mice, Transgenic; Neocortex; Sirolimus; Synaptic Transmission; TOR Serine-Threonine Kinases

Astaxanthin (Ast) is an effective neuroprotective and antioxidant compound used to treat Alzheimer's disease (AD); however, the underlying in vivo molecular mechanisms remain unknown. In this study, we report that Ast can activate the mammalian target of rapamycin (mTOR) pathway in the 8-month-old APP/PS1 transgenic mouse model of AD. Our results suggest that Ast could ameliorate the cognitive defects in APP/PS1 mice by activating the mTOR pathway. Moreover, mTOR activation perturbed the mitochondrial dynamics, increased the synaptic plasticity after 21 days of treatment with Ast (10 mg/kg/day), and increased the expression of Aβ-degrading enzymes, mitochondrial fusion, and synapse-associated proteins and decreased the expression of mitochondrial fission proteins. Intraperitoneal injection of the mTOR inhibitor, rapamycin, abolished the effects of Ast. In conclusion, Ast activates the mTOR pathway, which is necessary for mitochondrial dynamics and synaptic plasticity, leading to improved learning and memory. Our results support the use of Ast for the treatment of cognitive deficits. Graphical abstract In summary, Ast ameliorates cognitive deficits via facilitating the mTOR-dependent mitochondrial dynamics and synaptic damage, and reducing Aβ accumulation. This model supports the use of Ast for the treatment of cognitive deficits.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cognition; Disease Models, Animal; Mice; Mice, Transgenic; Presenilin-1; Sirolimus; TOR Serine-Threonine Kinases; Xanthophylls

Atherosclerosis arising from the pro-inflammatory conditions associated with chronic kidney disease (CKD) increases major cardiovascular morbidity and mortality. Rapamycin (RAPA) is known to inhibit atherosclerosis under CKD and non-CKD conditions, but it can cause dyslipidemia; thus, the co-application of lipid-lowering agents is recommended. Atorvastatin (ATV) has been widely used to reduce serum lipids levels, but its synergistic effect with RAPA in CKD remains unclear. Here, we analyzed the effect of their combined treatment on atherosclerosis stimulated by CKD in apolipoprotein E-deficient (ApoE-/-) mice. Oil Red O staining revealed that treatment with RAPA and RAPA＋ ATV, but not ATV alone, significantly decreased the atherosclerotic lesions in the aorta and aortic sinus, compared to those seen in the control (CKD) group. The co-administration of RAPA and ATV improved the serum lipid profile and raised the expression levels of proteins involved in reverse cholesterol transport (LXRα, CYP7A1, ABCG1, PPARγ, ApoA1) in the liver. The CKD group showed increased levels of various genes encoding atherosclerosispromoting cytokines in the spleen (Tnf-α, Il-6 and Il-1β) and aorta (Tnf-α and Il-4), and these increases were attenuated by RAPA treatment. ATV and RAPA＋ATV decreased the levels of Tnf-α and Il-1β in the spleen, but not in the aorta. Together, these results indicate that, in CKD-induced ApoE-/- mice, RAPA significantly reduces the development of atherosclerosis by regulating the expression of inflammatory cytokines and the co-application of ATV improves lipid metabolism. [BMB Reports 2021; 54(3): 170-175].

Topics: Animals; Apolipoproteins E; Atherosclerosis; Atorvastatin; Cytokines; Disease Models, Animal; Female; Lipid Metabolism; Mice; Mice, Knockout; Renal Insufficiency, Chronic; Sirolimus

Chronic prostatitis or chronic pelvic pain syndrome (CP/CPPS) is a disease with an unclear pathogenesis. Recent studies have reported that regulatory T (Treg) cells might be involved in the development of CP/CPPS. In this study we aimed to examine the functional role of Treg cells and explore the possible regulatory mechanism of Treg cells in CP/CPPS.. An experimental autoimmune prostatitis (EAP) mouse model was constructed; the numbers and functions of Treg cells in the EAP and control groups were tested. Then, cell differentiation experiments were conducted to evaluate the regulatory effect of autophagy on Treg cell differentiation. Furthermore, autologous CD4. We found that the number and function of Treg cells in the EAP group were diminished compared to those in the control group. Meanwhile, the tolerance of pain in EAP mice had also decreased. Moreover, after using the autophagy activator rapamycin, the expression of the inflammatory cytokines interleukin-1β was decreased and the pain symptoms were alleviated. A mechanistic study found that autophagy activation promoted the differentiation of Treg and increased the suppressive functions of Treg cells, along with the elevated expression of GATA-3 and cytotoxic T lymphocyte antigen 4 (CTLA-4). Furthermore, in vivo administration of the autophagy activator rapamycin had similar effects on recovering the frequency and function of Treg cells as well as the expression of GATA-3 and CTLA-4.. The impaired frequency and function of Treg cells may contribute to the progression of CP/CPPS, and autophagy is a protective mechanism that promotes the differentiation of Treg cells and restores the suppressive functions of Treg cells. Autophagy may be a novel therapeutic option for patients with CP/CPPS.

Topics: Animals; Autoimmune Diseases; Autophagy; Chronic Disease; CTLA-4 Antigen; Disease Models, Animal; GATA3 Transcription Factor; Male; Mice; Mice, Inbred NOD; Pain Measurement; Prostatitis; Rats; Rats, Sprague-Dawley; Sirolimus; T-Lymphocytes, Regulatory; Up-Regulation

Long-time use of pharmacological immunosuppressive agents frequently leads to metabolic disorders. Most studies have focused on islet toxicity leading to posttransplantation diabetes mellitus. In contrast, the link between intestinal dysbiosis and immunosuppressive drug-induced metabolic disorders remains unclear.. We established a mouse model of metabolic abnormality via sirolimus treatment. Fecal microbiota was examined using 16S rRNA gene MiSeq sequencing. Intestinal barrier function was assessed using fluorescein isothiocyanate-dextran assay and mucus immunostaining. Systemic inflammation was determined using a multiplexed fluorescent bead-based immunoassay.. Sirolimus induced dyslipidemia and glucose intolerance in mice in a dose-dependent manner. Interestingly, the clinical-mimicking dose of sirolimus altered the intestinal microbiota community, which was characterized by the enrichment of Proteobacteria, depletion of Akkermansia, and potential function shifts to those involved in lipid metabolism and the immune system. In addition, the clinical-mimicking dose of sirolimus reduced the thickness of the intestinal mucosal layer, increased the intestinal permeability, and enriched the circulating pro-inflammatory factors, including interleukin (IL)-12, IL-6, monocyte chemotactic protein 1, granulocyte-macrophage colony stimulating factor, and IL-1β. Our results showed a close association between intestinal dysbiosis, intestinal barrier failure, systemic inflammation, and metabolic disorders. Furthermore, we demonstrated that oral intervention in the gut microbiota by Lactobacillus rhamnosus HN001 protected against intestinal dysbiosis, especially by depleting the lipopolysaccharide-producing Proteobacteria, and attenuated the sirolimus-induced systemic inflammation, dyslipidemia, and insulin resistance.. Our study demonstrated a potentially causative role of intestinal dysbiosis in sirolimus-induced metabolic disorders, which will provide a novel therapeutic target for transplant recipients.

Topics: Animals; Bacteria; Cytokines; Disease Models, Animal; Dysbiosis; Dyslipidemias; Feces; Gastrointestinal Microbiome; Inflammation Mediators; Insulin Resistance; Intestinal Mucosa; Lacticaseibacillus rhamnosus; Male; Metabolic Syndrome; Mice, Inbred C57BL; Probiotics; Sirolimus

We previously showed that lifelong rapamycin treatment of short-lived Apc. We asked, what effect would pretreatment of Apc. These data indicate that enteric rapamycin prevents or delays colon neoplasia in Apc

Topics: Adenomatous Polyposis Coli Protein; Animals; Carcinogenesis; Colon; Colonic Neoplasms; Disease Models, Animal; Female; Heterozygote; Humans; Intestinal Mucosa; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Sirolimus; Survival Analysis; Time Factors

Acute lung injury (ALI) is characterized by alveolar macrophage overactivation and uncontrolled pulmonary inflammation. Mitochondrial damage-associated molecular patterns (MTDs), one type of damage-associated molecular patterns (DAMPs) released from ruptured mitochondrial, can induce inflammation which participates in the pathogenesis of ALI. Despite the critical role of autophagy in inflammatory response, little is known about its function in MTDs-induced ALI. Herein we have studied how autophagy attenuates MTDs-induced ALI in vitro and in vivo.. Exogenous MTDs were injected into mice through tail vein injection or directly treated with cultured alveolar macrophage cell lines to construct MTDs-induced ALI models. Rapamycin and 3-MA were used to regulate autophagy in vivo and in vitro. The expressions of Caspase-1, IL-1β, and their precursor were measured. Inhibition the activation of NLRP3 inflammasome to discover the candidate targets and potential molecular pathways involved in autophagy mitigating the MTDs-induced ALI.. After treatment with MTDs the expression levels of inflammatory cytokines and NLRP3 inflammasome-associated proteins were gradually increased in vitro and in vivo. Most importantly, with autophagy enhanced by rapamycin, all the secretion of inflammation cytokine, the level of lung injury, and the expression level of NLRP3 inflammasome-associated proteins were greatly decreased in MTDs-induced mouse model. MTDs-induced inflammation and lung injury were alleviated by autophagy enhancement. Autophagy can function as an effective way to alleviate inflammation in MTDs-induced ALI by inhibiting NLRP3 inflammasome and may represent a therapeutic target in modulating MTDs-induced inflammatory response.

Topics: Acute Lung Injury; Adenine; Alarmins; Animals; Autophagy; Cytokines; Disease Models, Animal; Inflammasomes; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Pneumonia; Sirolimus

Alkali burn to the cornea is one of the most intractable injuries to the eye due to the opacity resulting from neovascularization (NV) and fibrosis. Numerous studies have focused on studying the effect of drugs on alkali-induced corneal injury in mouse, but fewer on the involvement of alkali-induced DNA methylation and the PI3K/AKT/mTOR signaling pathway in the mechanism of alkali-induced corneal injury. Thus, the aim of this study was to determine the involvement of DNA methyltransferase 3 B-madiated DNA methylation and PI3K/AKT/mTOR signaling modulation in the mechanism of alkali-induced corneal injury in a mouse model. To this end, we used bisulfite sequencing polymerase chain reaction and Western blot analysis, to study the effects of 5-aza-2'-deoxycytidine and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, which inhibit methyltransferase and PI3K respectively, on DNA methylation and expression of downstream effectors of PI3K related to corneal NV, including TSC1 and mTOR genes. The results showed that, after an intraperitoneal injection of rapamycin (2 mg/kg/day) for seven days, the alkali-induced opacity and NV were remarkably decreased mainly by suppressing the infiltration of immune cells into injured corneas, angiogenesis, VEGF expression and myofibroblasts differentiation; as well as by promoting corneal cell proliferation and PI3K/AKT/mTOR signaling. More significantly, these findings showed that epigenetic regulatory mechanisms by DNA methylation played a key role in corneal NV, including in corneal alkali burn-induced methylation modification and rapamycin-induced DNA demethylation which involved the regulation of the PI3K/AKT/mTOR signaling pathway at the protein level. The precise findings of morphological improvement and regulatory mechanisms are helpful to guide the use of rapamycin in the treatment of corneal angiogenesis induced by alkaline-burn.

Topics: Actins; Animals; Blotting, Western; Burns, Chemical; Chromones; Corneal Injuries; Disease Models, Animal; DNA Methylation; Eye Burns; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Immunosuppressive Agents; Male; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Sirolimus; Sodium Hydroxide; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Vascular Endothelial Growth Factor A

Sjögren's syndrome (SS) is an autoimmune disease associated with severe exocrinopathy, which is characterized by profound lymphocytic infiltration (dacryoadenitis) and loss of function of the tear-producing lacrimal glands (LGs). Systemic administration of Rapamycin (Rapa) significantly reduces LG inflammation in the male Nonobese Diabetic (NOD) model of SS-associated autoimmune dacryoadenitis. However, the systemic toxicity of this potent immunosuppressant limits its application. As an alternative, this paper reports an intra-LG delivery method using a depot formulation comprised of a thermoresponsive elastin-like polypeptide (ELP) and FKBP, the cognate receptor for Rapa (5FV). Depot formation was confirmed in excised whole LG using cleared tissue and observation by both laser-scanning confocal and lightsheet microscopy. The LG depot was evaluated for safety, efficacy, and intra-LG pharmacokinetics in the NOD mouse disease model. Intra-LG injection with the depot formulation (5FV) retained Rapa in the LG for a mean residence time (MRT) of 75.6 h compared to Rapa delivery complexed with a soluble carrier control (5FA), which had a MRT of 11.7 h in the LG. Compared to systemic delivery of Rapa every other day for 2 weeks (seven doses), a single intra-LG depot of Rapa representing 16-fold less total drug was sufficient to inhibit LG inflammation and improve tear production. This treatment modality further reduced markers of hyperglycemia and hyperlipidemia while showing no evidence of necrosis or fibrosis in the LG. This approach represents a potential new therapy for SS-related autoimmune dacryoadenitis, which may be adapted for local delivery at other sites of inflammation; furthermore, these findings reveal the utility of optical imaging for monitoring the disposition of locally administered therapeutics.

Topics: Animals; Dacryocystitis; Disease Models, Animal; Lacrimal Apparatus; Male; Mice; Mice, Inbred NOD; Sirolimus; Sjogren's Syndrome; Tears

This study aimed to explore the therapeutic effect and mechanism of rapamycin (RAPA) on systemic lupus erythematosus (SLE) in BALB/C mice induced by pristane. The mice were randomly divided into 5 groups (n = 6): control, model, saline, RAPA (1 mg/kg) and RAPA (2 mg/kg). All groups were injected with pristane except control. HE staining revealed 1 mg/kg and 2 mg/kg RAPA treatments obviously alleviated pathological changes in the kidney of SLE mice such as glomeruli enlargement, hyperplasia of mesangial cells, epithelial and endothelial cells, infiltration of inflammatory cells, and edema-like degeneration of renal tubules. Compared with control group, body weights and anti-ribosomal P-protein antibody (ARPA) level of the mice in model group and saline group decreased (P < 0.05), while immune complex deposition and levels of anti-dsDNA antibody, anti-smRNP antibody and urine protein in model group and saline group increased (P < 0.05). However, compared with model group and saline group, body weights of the mice in RAPA (1 mg/kg) group and RAPA (2 mg/kg) group increased (P < 0.05), while immune complex deposition and levels of anti-dsDNA antibody, anti-smRNP antibody, ARPA, and urine protein in RAPA (1 mg/kg) group and RAPA (2 mg/kg) group decreased (P < 0.05). Compared with control group, the proportion of dentritic cells (DC) in the kidney and peripheral blood decreased while the proportion of Th1, Th2 and Th17 cells in the spleen, kidney and peripheral blood increased in model group and saline group (P < 0.05). Compared with model group and saline group, 1 mg/kg and 2 mg/kg RAPA treatments boosted the proportion of DC in the kidney and peripheral blood, reduced the proportion of Th1 and Th17 cells in the spleen, kidney and peripheral blood, and lessened the proportion of Th2 cells in the kidney and peripheral blood (P < 0.05). In conclusion, RAPA alleviated renal damage in SLE mice through improving immune response and function.

Topics: Animals; Antibodies, Antinuclear; Antigen-Antibody Complex; Dendritic Cells; Disease Models, Animal; Female; Immunosuppressive Agents; Kidney; Lupus Erythematosus, Systemic; Lupus Nephritis; Mice, Inbred BALB C; Sirolimus; T-Lymphocytes, Helper-Inducer; Terpenes

Topics: Animals; Autoimmune Diseases; Chorioallantoic Membrane; Conjunctiva; Disease Models, Animal; Drug Compounding; Electroretinography; Erythrocytes; Eye Diseases; Immunosuppressive Agents; Intravitreal Injections; Liposomes; Male; Mice; Micronucleus Tests; Rabbits; Retina; Safety; Sirolimus

Autophagy is an essential cellular process for the degradation and recycling of cellular components, and its dysregulation has been linked to neuronal cell death and neurodegeneration. In glaucoma, the role of autophagy in retinal ganglion cell (RGC) survival remains contradictory. Moreover, the effects of autophagy modulation at different time-points on RGC survival in a glaucoma model have not been investigated. In this study, we assessed the time-dependent role of autophagy in RGC survival in a circumlimbal suture-induced ocular hypertensive (OHT) rat model. Intraocular pressure (IOP) elevation led to a gradual autophagy induction, which reached a maximum between 1 and 4 weeks after OHT induction. On the other hand, early autophagy was impaired between 1 and 3 days after circumlimbal suturing, indicated by increased p62 levels due to reduced autophagosomal turnover. The intravitreal administration of rapamycin at different time-points after the application of the circumlimbal suture indicated that autophagy induction early during OHT development had potent survival-promoting effects in RGCs. In conclusion, our findings suggest that the role of autophagy in RGCs during OHT development might differ in a time-dependent manner. Modulating autophagy at the appropriate time might serve as a potential therapeutic approach to enhance RGC survival in OHT.

Topics: Adenylate Kinase; Animals; Autophagy; Biomarkers; Cell Survival; Chronic Disease; Disease Models, Animal; Intravitreal Injections; Male; Microtubule-Associated Proteins; Ocular Hypertension; Phosphorylation; Rats, Sprague-Dawley; Retinal Ganglion Cells; Signal Transduction; Sirolimus; Sutures; Time Factors; TOR Serine-Threonine Kinases

Alloreactive memory cells play a critical role after a second transplant and are difficult to suppress. This study investigated the effect of an immunotherapeutic strategy that combines anti-OX40L, rapamycin (Rapa), and a low dose of IL-2 in a memory cell-based adoptive model. In this model, the median survival time (MST) of the grafts of the combined treatment group was significantly extended compared to that of the control group and other treatment groups. A similar effect was observed regarding a reduction in memory T cells (Tm) and inflammatory cytokines production. Also, the percentages of Foxp3

Topics: Allografts; Animals; CD4 Antigens; CD8 Antigens; Cell Differentiation; Disease Models, Animal; Dose-Response Relationship, Drug; Graft Rejection; Graft Survival; Heart Transplantation; Homeodomain Proteins; Humans; Immunologic Memory; Interleukin-2; Male; Memory T Cells; Mice; Mice, Knockout; OX40 Ligand; Recombinant Proteins; Sirolimus; Skin Transplantation; T-Lymphocytes, Regulatory; Transplantation, Homologous

Epilepsy is one of the most common neurological disorders, with individuals having an increased susceptibility of seizures in the first few years of life, making children at risk of developing a multitude of cognitive and behavioral comorbidities throughout development. The present study examined the role of PI3K/Akt/mTOR pathway activity and neuroinflammatory signaling in the development of autistic-like behavior following seizures in the neonatal period. Male and female C57BL/6J mice were administered 3 flurothyl seizures on postnatal (PD) 10, followed by administration of minocycline, the mTOR inhibitor rapamycin, or a combined treatment of both therapeutics. On PD12, isolation-induced ultrasonic vocalizations (USVs) of mice were examined to determine the impact of seizures and treatment on communicative behaviors, a component of the autistic-like phenotype. Seizures on PD10 increased the quantity of USVs in female mice and reduced the amount of complex call types emitted in males compared to controls. Inhibition of mTOR with rapamycin significantly reduced the quantity and duration of USVs in both sexes. Changes in USVs were associated with increases in mTOR and astrocyte levels in male mice, however, three PD10 seizures did not result in enhanced proinflammatory cytokine expression in either sex. Beyond inhibition of mTOR activity by rapamycin, both therapeutics did not demonstrate beneficial effects. These findings emphasize the importance of differences that may exist across preclinical seizure models, as three flurothyl seizures did not induce as drastic of changes in mTOR activity or inflammation as observed in other rodent models.

Topics: Animals; Convulsants; Disease Models, Animal; Epilepsy; Female; Flurothyl; Immunologic Factors; Male; Mice; Mice, Inbred C57BL; Minocycline; MTOR Inhibitors; Seizures; Sex Factors; Sirolimus; Vocalization, Animal

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK3

Topics: Animals; Animals, Newborn; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Endothelial Cells; Gain of Function Mutation; Hemangioma, Cavernous, Central Nervous System; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Loss of Function Mutation; Male; MAP Kinase Kinase Kinase 3; Mechanistic Target of Rapamycin Complex 1; Mice; Mutation; Neoplasms; Sirolimus; TOR Serine-Threonine Kinases

Polycystic ovary syndrome (PCOS) is a multifactorial endocrinopathy that affects reproduction and metabolism. Mammalian target of rapamycin (mTOR) has been shown to participate in female reproduction under physiological and pathological conditions. This study aimed to investigate the role of mTOR complex 1 (mTORC1) signaling in dehydroepiandrosterone (DHEA)-induced PCOS mice.. Female C57BL/6J mice were randomly assigned into three groups: control group, DHEA group, and DHEA + rapamycin group. All DHEA-treated mice were administered 6 mg/100 g DHEA for 21 consecutive days, and the DHEA + rapamycin group was intraperitoneally injected with 4 mg/kg rapamycin every other day for the last 14 days of the DHEA treatment. There was no obvious change in the expression of mTORC1 signaling in the ovaries of the control and DHEA groups. Rapamycin did not protect against DHEA-induced acyclicity and PCO morphology, but impeded follicle development and elevated serum testosterone levels in DHEA-induced mice, which was related with suppressed Hsd3b1, Cyp17a1, and Cyp19a1 expression. Moreover, rapamycin also exacerbated insulin resistance but relieved lipid metabolic disturbance in the short term.. Rapamycin exacerbated reproductive imbalance in DHEA-induced PCOS mice, which characterized by elevated testosterone levels and suppressed steroid synthesis. This underscores the need for new mTORC1-specific and tissue-specific mTOR-related drugs for reproductive disorders.

Topics: Animals; Dehydroepiandrosterone; Disease Models, Animal; Female; Humans; Mice; Polycystic Ovary Syndrome; Reproduction; Sirolimus; Testosterone

Autophagy serves a crucial role in the etiology of kidney diseases, including drug‑induced renal impairment, inherited kidney disease, diabetic nephropathy and aristolochic acid nephropathy (AAN) and is, therefore, a potential target for treatment. We previously demonstrated that rapamycin could attenuate AAN in mice; however, the underlying mechanism remains to be elucidated. Therefore, whether the renal protective effect of rapamycin (an autophagy activator) is related to autophagy in aristolochic acid (AA)‑treated mice was of particular interest. The pathophysiological roles of rapamycin were investigated in AA‑induced nephrotoxicity in mice and the mechanisms of rapamycin action were explored by evaluating the modulation of autophagy in rapamycin‑treated mice and cultured renal tubular epithelial cells. Supplementation with rapamycin reversed AA‑induced kidney injury in mice and improved AA‑induced autophagy damage

Topics: Animals; Apoptosis; Aristolochic Acids; Autophagy; Cell Line; Disease Models, Animal; Humans; Kidney Diseases; Male; Mice, Inbred C57BL; Protective Agents; Sirolimus; TOR Serine-Threonine Kinases

Alzheimer's disease (AD) is a chronic neurodegenerative disease, which is characterized by cognitive and synaptic plasticity damage. Rapamycin is an activator of autophagy/mitophagy, which plays an important role in identifying and degrading damaged mitochondria. The aim of this study was to investigate the effect of rapamycin on cognitive and synaptic plasticity defects induced by AD, and further explore if the underlying mechanism was associated with mitophagy. The results show that rapamycin increases Parkin-mediated mitophagy and promotes fusion of mitophagosome and lysosome in the APP/PS1 mouse hippocampus. Rapamycin enhances learning and memory viability, synaptic plasticity, and the expression of synapse-related proteins, impedes cytochrome C-mediated apoptosis, decreases oxidative status, and recovers mitochondrial function in APP/PS1 mice. The data suggest that rapamycin effectively alleviates AD-like behaviors and synaptic plasticity deficits in APP/PS1 mice, which is associated with enhanced mitophagy. Our findings possibly uncover an important function of mitophagy in eliminating damaged mitochondria to attenuate AD-associated pathology.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cognition; Disease Models, Animal; Hippocampus; Mice; Mice, Transgenic; Mitophagy; Neurodegenerative Diseases; Neuronal Plasticity; Sirolimus

Obesity has become a relevant problem in transplantation medicine with steadily increasing numbers of obese graft recipients. However, the effect of immunomodulatory drugs on transplant-related outcomes among obese patients are unknown. Therefore, we evaluated the impact of rapamycin on allograft rejection and alloimmune response in a murine model of diet-induced obesity and fully-mismatched skin transplantation. Rapamycin significantly delayed allograft rejection in obese recipient mice compared to treated lean mice (14.5 days vs. 10.7 days, p = 0.005). Treatment with rapamycin increased frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs), augmented the immunosuppressive activity of M-MDSCs on T cells through indoleamine 2,3-dioxygenase pathway and shifted CD4

Topics: Allografts; Animals; Biomarkers; Cytokines; Disease Models, Animal; Disease Susceptibility; Gene Expression; Graft Rejection; Immune Tolerance; Immunomodulation; Immunophenotyping; Immunosuppressive Agents; Mice; Myeloid-Derived Suppressor Cells; Obesity; Sirolimus; Skin Transplantation; T-Lymphocyte Subsets; Transplant Recipients

Down syndrome (DS) is the most frequent genetic cause of intellectual disability including hippocampal-dependent memory deficits. We have previously reported hippocampal mTOR (mammalian target of rapamycin) hyperactivation, and related plasticity as well as memory deficits in Ts1Cje mice, a DS experimental model. Here we characterize the proteome of hippocampal synaptoneurosomes (SNs) from these mice, and found a predicted alteration of synaptic plasticity pathways, including long term depression (LTD). Accordingly, mGluR-LTD (metabotropic Glutamate Receptor-LTD) is enhanced in the hippocampus of Ts1Cje mice and this is correlated with an increased proportion of a particular category of mushroom spines in hippocampal pyramidal neurons. Remarkably, prenatal treatment of these mice with rapamycin has a positive pharmacological effect on both phenotypes, supporting the therapeutic potential of rapamycin/rapalogs for DS intellectual disability.

Topics: Animals; Dendritic Spines; Disease Models, Animal; Down Syndrome; Fragile X Mental Retardation Protein; Hippocampus; Long-Term Synaptic Depression; Mice, Transgenic; Mitochondrial Proteins; Neuronal Plasticity; Proteomics; Pyramidal Cells; Receptors, Metabotropic Glutamate; Sirolimus; Synapses

Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy.. Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [. The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy.. Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Topics: Aged; Animals; Cardiomegaly; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Female; Humans; Male; Mice, Inbred C57BL; Middle Aged; MTOR Inhibitors; Myocytes, Cardiac; Protein Biosynthesis; Serine Endopeptidases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown.. To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart.. We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1. Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

Topics: Animals; Cardiomegaly; Cells, Cultured; Diet; Disease Models, Animal; Enzyme Activation; Gene Knockdown Techniques; Glucose; Glucose-6-Phosphatase; Isomerases; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocytes, Cardiac; Oxidation-Reduction; Phosphorylation; Signal Transduction; Sirolimus; Tuberous Sclerosis Complex 2 Protein

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagA

Topics: Animals; Disease Models, Animal; Fasting; Glucose; Homeostasis; Humans; Liver; Mechanistic Target of Rapamycin Complex 1; Mice; Monomeric GTP-Binding Proteins; Nutrients; Phenotype; PPAR alpha; Proteomics; Signal Transduction; Sirolimus; Transcription, Genetic; Tuberous Sclerosis Complex 1 Protein

The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation (MV) maintains gas exchange in patients with ARDS but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during MV impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during MV. Conversely, treatment with rapamycin at the time MV is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during MV. Our data demonstrate that, in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during MV exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.

Topics: Animals; Disease Models, Animal; Humans; Lung; Lung Compliance; Mechanistic Target of Rapamycin Complex 1; Mice; Pulmonary Surfactants; Respiration, Artificial; Respiratory Distress Syndrome; Sirolimus; Ventilator-Induced Lung Injury

Numerous studies have reported that epilepsy causes memory deficits. The present study was aimed at studying the effect of rapamycin against the memory deficiency of the pentylenetetrazole (PTZ)-kindled animal model of epilepsy. In the present experiment, we randomly chose thirty male rats from the species of Wistar and categorized them in groups of control and experiment (6 for each group). The groups of experiment received the injection of rapamycin (0.5, 1 and 2 mg/kg) intraperitoneally (i.p.) and the group of control received normal saline (0.9%) treatment. Through the PTZ's sub-threshold dose (35 mg kg

Topics: Animals; Brain; Disease Models, Animal; Kindling, Neurologic; Male; Memory; Memory Disorders; Neurons; Pentylenetetrazole; Rats; Rats, Wistar; Seizures; Sirolimus

Infantile spasms may evolve into persistent epilepsies including Lennox-Gastaut syndrome. We compared adult epilepsy outcomes in models of infantile spasms due to structural etiology (multiple-hit model) or focal cortical inflammation and determined the anti-epileptogenic effects of pulse-rapamycin, previously shown to stop spasms in multiple-hit rats.. Spasms were induced in 3-day-old male rats via right intracerebral doxorubicin/lipopolysaccharide (multiple-hit model) infusions. Controls and sham rats were used. Separate multiple-hit rats received pulse-rapamycin or vehicle intraperitoneally between postnatal days 4 and 6. In adult mice, video-EEG (electroencephalography) scoring for seizures and sleep and histology were done blinded to treatment.. Motor-type seizures developed in 66.7% of multiple-hit rats, usually from sleep, but were reduced in the pulse-rapamycin-treated group (20%, p = .043 vs multiple-hit) and rare in other groups (0-9.1%, p < .05 vs multiple-hit). Spike-and-wave bursts had a slower frequency in multiple-hit rats (5.4-5.8Hz) than in the other groups (7.6-8.3Hz) (p < .05); pulse rapamycin had no effect on the hourly spike-and-wave burst rates in adulthood. Rapamycin, however, reduced the time spent in slow-wave-sleep (17.2%), which was increased in multiple-hit rats (71.6%, p = .003). Sham rats spent more time in wakefulness (43.7%) compared to controls (30.6%, p = .043). Multiple-hit rats, with or without rapamycin treatment, had right more than left corticohippocampal, basal ganglia lesions. There was no macroscopic pathology in the other groups.. Structural corticohippocampal/basal ganglia lesions increase the risk for post-infantile spasms epilepsy, Lennox-Gastaut syndrome features, and sleep dysregulation. Pulse rapamycin treatment for infantile spasms has anti-epileptogenic effects, despite the structural lesions, and decreases the time spent in slow wave sleep.

Topics: Animals; Disease Models, Animal; Electroencephalography; Epilepsy; Lennox Gastaut Syndrome; Male; Mice; Rats; Seizures; Sirolimus; Spasm

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.

Topics: Acute Kidney Injury; Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Cell Line; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mice; Mice, Inbred C57BL; Mustard Compounds; Phenylpropionates; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Reperfusion Injury; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Traumatic brain injury (TBI) provokes primary and secondary damage on endothelium and brain parenchyma, leading neurons die rapidly by necrosis. The mammalian target of rapamycin signalling pathway (mTOR) manages numerous aspects of cellular growth, and it is up-regulated after moderate to severe traumatic brain injury (TBI). Currently, the significance of this increased signalling event for the recovery of brain function is unclear; therefore, we used two different selective inhibitors of mTOR activity to discover the functional role of mTOR inhibition in a mouse model of TBI performed by a controlled cortical impact injury (CCI). Treatment with KU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin as well-known inhibitor of mTOR, was performed 1 and 4 hours subsequent to TBI. Results proved that mTOR inhibitors, especially KU0063794, significantly improved cognitive and motor recovery after TBI, reducing lesion volumes. Also, treatment with mTOR inhibitors ameliorated the neuroinflammation associated with TBI, showing a diminished neuronal death and astrogliosis after trauma. Our findings propose that the involvement of selective mTORC1/2 inhibitor may represent a therapeutic strategy to improve recovery after brain trauma.

Topics: Animals; Brain Injuries, Traumatic; Cell Death; Cell Proliferation; Disease Models, Animal; Enzyme Inhibitors; Immunosuppressive Agents; Male; Mice; Morpholines; MTOR Inhibitors; Neuroinflammatory Diseases; Neurons; Pyrimidines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

This preliminary research determines whether a combination of reverse end-to-side neurorrhaphy and rapamycin treatment achieves a better functional outcome than a single application after prolonged peripheral nerve injury. We found that the tibial nerve function of the reverse end-to-side + rapamycin group recovered better, with a higher tibial function index value, higher amplitude recovery rate, and shorter latency delay rate (

Topics: Animals; Anti-Bacterial Agents; Combined Modality Therapy; Disease Models, Animal; Electromyography; Female; Muscle Denervation; Muscle, Skeletal; Nerve Regeneration; Neurosurgical Procedures; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; Sirolimus; Tibial Neuropathy

Rapamycin (RAPA), which was first described as an anti-fungal agent, is a potent immunosuppressant that suppresses tumors and inhibits the mTOR signaling pathway. Heterotopic ossification (HO) is abnormal bone formation outside the skeletal system (e.g., in muscles, tendons, articular capsules and other soft tissues), often due to trauma or injury. There are currently no drugs available to treat traumatic HO, largely due to limited understanding of the disease. In this study, we focused on the role of oxidative stress (OS) in the early stage of traumatic HO, and explored the underlying mechanism of traumatic HO by using RAPA to specifically inhibit the mTOR pathway, which is known to play a role in the pathogenesis of HO. To assess the effects of RAPA in traumatic HO, we used an NSE-BMP4 transgenic mouse model that develops ossification in response to traumatic injury and intramuscular injection of cardiotoxin to initiate injury. These mice were then treated with RAPA or vehicle intraperitoneally every other day for 2 weeks. Our results demonstrate that RAPA can inhibit HO through a number of different mechanisms. We show that OS and a strong inflammatory response contribute to the hypoxia associated with the early stages of HO, and that RAPA inhibits these responses. Furthermore, RAPA reduces the vascularization triggered by mTOR signaling that leads to HO formation. Therefore, we believe that RAPA could be an effective treatment for the early stages of HO.

Topics: Animals; Disease Models, Animal; Immunosuppressive Agents; Mice; Mice, Transgenic; Ossification, Heterotopic; Oxidative Stress; Sirolimus; TOR Serine-Threonine Kinases

The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). In this study, we investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, we found that Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. Our results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models. Our findings not only provide a novel insight into the mechanisms of Nrf2-antioxidant-autophagy signaling, but also provide potential targets for PD interventions.

Topics: Adenine; Animals; Animals, Genetically Modified; Antioxidants; Antiparkinson Agents; Astrocytes; Autophagy; Behavior, Animal; Dihydroxyphenylalanine; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Male; Motor Activity; Nerve Degeneration; NF-E2-Related Factor 2; Oleanolic Acid; Parkinsonian Disorders; Phenotype; Rats, Sprague-Dawley; Repressor Proteins; Rotenone; Signal Transduction; Sirolimus

Rapamycin is a type of immunosuppressive agent that acts through inhibition of mammalian target of rapamycin (mTOR). Hepatopulmonary syndrome (HPS) is a lethal complication in cirrhotic patients. It is characterized by hypoxia and increased intrapulmonary shunts, in which pulmonary inflammation and angiogenesis play important roles. The current study aimed to evaluate the effect of rapamycin on HPS using the experimental model of common bile duct ligation (CBDL)-induced cirrhosis in rats.. The rats received low-dose (0.5 mg/kg), high-dose (2 mg/kg) rapamycin, or vehicle from the 15th to the 28th day post CBDL. Then the mortality rate, hemodynamics, biochemistry parameters, arterial blood gas and plasma levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α were evaluated on the 28th day post CBDL. Pulmonary histopathological stains were performed, and protein expression was examined. In parallel groups, the intrapulmonary shunts of CBDL rats were measured.. Compared with the control, a high-dose rapamycin treatment decreased portal pressure and improved hypoxia in CBDL rats. It also reduced the plasma level of VEGF and TNF-α and decreased intrapulmonary shunts. Meanwhile, it ameliorated pulmonary inflammation and angiogenesis and downregulated the protein expression of mTOR, P70S6K, nuclear factor kappa B (NFκB), VEGF, and VEGF receptor 2. In contrast, low-dose rapamycin did not attenuate intrapulmonary shunts despite ameliorating portal hypertension.. High-dose rapamycin ameliorates HPS in cirrhotic rats as evidenced by the alleviated hypoxia and decreased intrapulmonary shunts. Downregulation of the mTOR/P70S6K, NFκB, and VEGF signaling pathways might play a key role.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Disease Models, Animal; Hepatopulmonary Syndrome; Liver Cirrhosis; Male; NF-kappa B; Rats; Rats, Sprague-Dawley; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Over the years, iron accumulation in specific brain regions has been observed in normal aging and related to the pathogenesis of neurodegenerative disorders. Many neurodegenerative diseases may involve cognitive dysfunction, and we have previously shown that neonatal iron overload induces permanent cognitive deficits in adult rats and exacerbates age-associated memory decline. Autophagy is a catabolic pathway involved in the removal of toxic protein aggregates, which are a hallmark of neurodegenerative events. In the present study, we investigated whether iron accumulation would interfere with autophagy and also sought to determine the effects of rapamycin-induced stimulation of autophagy in attenuating iron-related cognitive deficits. Male Wistar rats received a single daily oral dose of vehicle or iron carbonyl (30 mg/kg) at postnatal days 12-14. In adulthood, they received daily intraperitoneal injections of vehicle or rapamycin (0.25 mg/kg) for 14 days. Results showed that iron given in the neonatal period impaired inhibitory avoidance memory and induced a decrease in proteins critically involved in the autophagy pathway, Beclin-1 and LC3, in the hippocampus. Rapamycin in the adulthood reversed iron-induced memory deficits, decreased the ratio phospho-mTOR/total mTOR, and recovered LC3 II levels in iron-treated rats. Our results suggest that iron accumulation, as observed in neurodegenerative disorders, hinders autophagy, which might play a role in iron-induced neurotoxicity. Rapamycin, by inducing authophagy, was able to ameliorate iron-induced cognitive impairments. These findings support the use of rapamycin as a potential neuroprotective treatment against the cognitive decline associated to neurodegenerative disorders.

Topics: Animals; Autophagy; Cognitive Dysfunction; Disease Models, Animal; Female; Hippocampus; Iron; Iron Overload; Memory Disorders; Neurodegenerative Diseases; Rats, Wistar; Sirolimus

An impairment of the intestinal barrier function is one of the major characteristics of Crohn's disease (CD). This study aimed to evaluate the impact of autophagy induction by rapamycin on the intestinal epithelial barrier function in CD model mice.. IL-10 knockout (IL-10 KO) mice were used as the human CD models in this study. All the mice were randomly assigned into four groups, (a) wild-type (WT) group; (b) IL-10 KO group; (c) IL-10 KO + rapamycin group and (d) IL-10 KO + 3-methyladenine (3-MA), containing 6 mice in each group. The disease activity index (DAI), histology, pro-inflammatory cytokines and chemotactic factors in colon tissues, intestinal and colonic permeability, distributions and expressions of tight junction (TJ) proteins, epithelial apoptosis of mice in four groups were evaluated and compared.. Autophagy induction by rapamycin treatment ameliorated DAI and histological colitis, decreased pro-inflammatory cytokines (TNF-α, IFN-γ and IL-17) and chemotactic factors (CXCL-1 and CXCL-2), decreased intestinal and colonic permeability, improved the distribution and expression of TJ proteins in IL-10 KO mice.. Autophagy induction by rapamycin significantly improved intestinal barrier function and protected IL-10 KO mice from the experimental chronic colitis.

Topics: Animals; Apoptosis; Autophagy; Crohn Disease; Disease Models, Animal; Humans; Interleukin-10; Intestinal Mucosa; Mice; Mice, Knockout; Permeability; Sirolimus

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs - including in HHT patient blood outgrowth ECs - and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

Topics: Activin Receptors, Type II; Animals; Bone Morphogenetic Proteins; Disease Models, Animal; Endothelial Cells; Growth Differentiation Factor 2; Indoles; Mice; Mice, Knockout; Signal Transduction; Sirolimus; Smad1 Protein; Smad5 Protein; Smad8 Protein; Telangiectasia, Hereditary Hemorrhagic; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor Receptor-2

The beneficial role of Cystic fibrosis transmembrane conductance regulator (CFTR) was reported in acute lung injury (ALI), however, there was no direct evidence supporting the relationship between CFTR and cell autophagy in ALI. Here, this study is to analyze the protective role of CFTR on autophagy in lipopolysaccharide (LPS)-induced ALI mice and its special mechanism.. ALI mouse models were established by the stimulation of LPS. ALI mice were subjected to tail vein injection of Lv-CFTR, intraperitoneal injection of autophagy activator RAPA or tail vein injection of Lv-sh-HMGB1 before lung tissues and bronchoalveolar lavage fluid (BALF) were collected. The expression levels of CFTR, HMGB1, Beclin-1, p62, p-AKT, p-mTOR, and LC3-II/LC3-I ratio were estimated by qRT-PCR and Western blot. The lung edema in ALI mice was inspected by wet/dry weight (W/D) ratio. Hematoxylin and eosin (H&E) staining was utilized to observe pathological features of lung tissue. Immunofluorescence was applied to determine the expression intensity of LC-3. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activity and malondialdehyde (MDA) content were assayed, and inflammatory response in ALI mice was measured.. ALI mouse models were successfully induced by LPS, evidenced by an enhanced inflammatory response in lung tissues, heightened W/D ratio and cell autophagy markers. ALI mice had suppressed expression of CFTR, while injection of CFTR overexpression in ALI mice attenuated inflammation, autophagy, MPO activity and MDA content in addition to elevating SOD activity. Moreover, CFTR overexpression could increase the p-AKT, and p-mTOR. Overexpression of HMGB1 could reverse the expression pattern in mice injected with CFTR overexpression.. CFTR could inhibit cell autophagy by enhancing PI3K/AKT/mTOR signaling pathway, thereby playing a protective role in LPS-induced ALI in mice.

Topics: Acute Lung Injury; Animals; Autophagy; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; HMGB1 Protein; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Signal Transduction; Sirolimus

Deregulation of mTOR (mammalian target of rapamycin) signalling occurs in diabetes, which exacerbates injury following myocardial infarction (MI). We therefore investigated the infarct-limiting effect of chronic treatment with rapamycin (RAPA, mTOR inhibitor) in diabetic mice following myocardial ischaemia/reperfusion (I/R) injury and delineated the potential protective mechanism.. Adult male diabetic (db/db) or wild-type (WT) (C57) mice were treated with RAPA (0.25 mg/kg/day, intraperitoneal) or vehicle (5% DMSO) for 28 days. The hearts from treated mice were subjected to global I/R in Langendorff mode. Cardiomyocytes, isolated from treated mice, were subjected to simulated ischaemia/reoxygenation (SI/RO) to assess necrosis and apoptosis. Myocardial infarct size was increased in diabetic heart following I/R as compared to WT. Likewise, enhanced necrosis and apoptosis were observed in isolated cardiomyocytes of diabetic mice following SI/RO. Treatment with RAPA reduced infarct size as well as cardiomyocyte necrosis and apoptosis of diabetes and WT mice. RAPA increased STAT3 phosphorylation and miRNA-17/20a expression in diabetic hearts. In addition, RAPA restored AKT phosphorylation (target of mTORC2) but suppressed S6 phosphorylation (target of mTORC1) following I/R injury. RAPA-induced cardioprotection against I/R injury as well as the induction of miR-17/20a and AKT phosphorylation were abolished in cardiac-specific STAT3-deficient diabetic mice, without alteration of S6 phosphorylation. The infarct-limiting effect of RAPA was obliterated in cardiac-specific miRNA-17-92-deficient diabetic mice. The post-I/R restoration of phosphorylation of STAT3 and AKT with RAPA were also abolished in miRNA-17-92-deficient diabetic mice. Additionally, RAPA suppressed the pro-apoptotic prolyl hydroxylase (Egln3/PHD3), a target of miRNA-17/20a in diabetic hearts, which was abrogated in miRNA-17-92-deficient diabetic mice.. Induction of STAT3-miRNA-17-92 signalling axis plays a critical role in attenuating MI in RAPA-treated diabetic mice. Our study indicates that chronic treatment with RAPA might be a promising pharmacological intervention for attenuating MI and improving prognosis in diabetic patients.

Topics: Animals; Apoptosis; Diabetes Mellitus; Disease Models, Animal; Isolated Heart Preparation; Male; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

Spinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application.. A total of 75 female Sprague-Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application.. NT-3 was found to significantly promote the recovery of motor function by Basso-Beattie-Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin.. This study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

Topics: Animals; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Beclin-1; Cell Proliferation; Cell Survival; Disease Models, Animal; Female; Humans; Motor Activity; Nerve Tissue Proteins; Neurotrophin 3; Oligodendroglia; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Sirolimus; Spinal Cord Injuries; Spinal Nerves

Topics: Animals; Antibodies; Disease Models, Animal; Hippocampus; Humans; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Mice; Ribosomal Proteins; Sirolimus; Social Behavior

The chronic inflammatory microenvironment is characterized by the elevated level of reactive oxygen species (ROS). Here, it is hypothesized that developing an ROS-scavenging scaffold loaded with rapamycin (Rapa@Gel) may offer a new strategy for modulating the local inflammatory microenvironment to improve intervertebral disk tissue regeneration. The therapeutic scaffold consisting of ROS-degradable hydrogel can be injected into the injured degeneration site of intervertebral disk (IVD) and can release therapeutics in a programmed manner. The ROS scavenged by scaffold reduces the inflammatory responses. It is found that when rats are treated with Rapa@Gel, this results in an increase in the percentage of M2-like macrophages and a decrease in M1-like macrophages in the inflammatory environment, respectively. Regeneration of IVD is achieved by Rapa@Gel local treatment, due to the increased M2 macrophages and reduced inflammation. This strategy may be extended to the treatment of many other inflammatory diseases.

Topics: Animals; Biocompatible Materials; Cytokines; Disease Models, Animal; Drug Delivery Systems; Hydrogels; Intervertebral Disc Degeneration; Male; Mice; Mice, Inbred C57BL; Rats, Sprague-Dawley; RAW 264.7 Cells; Reactive Oxygen Species; Sirolimus

A hallmark of polycystic kidney diseases (PKDs) is aberrant proliferation, which leads to the formation and growth of renal cysts. Proliferation is mediated by cyclin-dependent kinases (Cdks), and the administration of roscovitine (a pan-Cdk inhibitor) attenuates renal cystic disease in juvenile cystic kidney (jck) mice. Cdk2 is a key regulator of cell proliferation, but its specific role in PKD remains unknown. The aim of this study was to test the hypothesis that Cdk2 deficiency reduces renal cyst growth in PKD. Three studies were undertaken: (i) a time course (days 28, 56, and 84) of cyclin and Cdk activity was examined in jck mice and compared with wild-type mice; (ii) the progression was compared in jck mice with or without Cdk2 ablation from birth; and (iii) the effect of sirolimus (an antiproliferative agent) on Cdk2 activity in jck mice was investigated. Renal disease in jck mice was characterized by diffuse tubular cyst growth, interstitial inflammation and fibrosis, and renal impairment, peaking on day 84. Renal cell proliferation peaked during earlier stages of disease (days 28-56), whereas the expression of Cdk2-cyclin partners (A and E) and Cdk1 and 2 activity, was maximal in the later stages of disease (days 56-84). Cdk2 ablation did not attenuate renal disease progression and was associated with persistent Cdk1 activity. In contrast, the postnatal treatment of jck mice with sirolimus reduced both Cdk2 and Cdk1 activity and reduced renal cyst growth. In conclusion, (i) the kinetics of Cdk2 and Cdk2-cyclin partners did not correlate with proliferation in jck mice; and (ii) the absence of Cdk2 did not alter renal cyst growth, most likely due to compensation by Cdk1. Taken together, these data suggest that Cdk2 is dispensable for the proliferation of cystic epithelial cells and progression of PKD.

Topics: Animals; Cell Proliferation; Cyclin-Dependent Kinase 2; Disease Models, Animal; Disease Progression; Female; Kidney; Male; Mice; Polycystic Kidney Diseases; Sirolimus

To evaluate the effect of autophagy inducers on damage caused by vital dye in adult human RPE (ARPE) cells and in a rat model.. ARPE-19 cells were exposed to ICG or BBG (0.05 mg/ml) with rapamycin (200 nM) or metformin (2 mM) for 30 min and treated with or without 20 μM chloroquine (CQ) to identify the protein levels of LC3 and SQSTM1 by immunoblotting. In vivo study was performed by injecting 10 μl 0.05% ICG and 0.25% BBG into the subretinal space of the rat eyes, and/or co-treated them with metformin and rapamycin. The retinas were used to determine autophagy with the LC3-II level and apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay.. In this study, both ICG and BBG inhibited autophagy flux in adult human retinal pigment epithelium cells (ARPE-19), whereas only ICG consistently reduced autophagy in the retina of rats. Moreover, rapamycin and metformin induced autophagic flux in ARPE-19 cells and increased the LC3-II level in retinal tissues exposed to vital dyes. Both ICG and BBG increased apoptosis in the retina of rats. However, both rapamycin and metformin induced autophagy and reduced the apoptosis caused by vital dyes.. Taken together, these results suggest that rapamycin and metformin may diminish vital dye-induced retinal damage in vivo through activation of autophagy.

Topics: Adult; Animals; Apoptosis; Cell Survival; Cells, Cultured; Coloring Agents; Disease Models, Animal; Humans; Hypoglycemic Agents; Immunosuppressive Agents; Metformin; Rats; Retinal Diseases; Retinal Pigment Epithelium; Sirolimus

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 μmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia-reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia-reperfusion injury.

Topics: Alveolar Epithelial Cells; Animals; Autophagy; Cell Hypoxia; Dimethyl Sulfoxide; Disease Models, Animal; Endoplasmic Reticulum Stress; Humans; Inflammation; Inflammation Mediators; Lung; Lung Injury; Male; Microtubule-Associated Proteins; NF-kappa B; Rats; Reperfusion Injury; Signal Transduction; Sirolimus

To clarify the complex mechanism underlying epileptogeneis, a novel animal model was generated.. In our previous research, we have generated a melanocyte-lineage mTOR hyperactivation mouse model (Mitf-M-Cre Tsc2 KO mice; cKO mice) to investigate mTOR pathway in melanogenesis regulation, markedly reduced skin pigmentation was observed. Very unexpectedly, spontaneous recurrent epilepsy was also developed in this mouse model.. Compared with control littermates, no change was found in either brain size or brain mass in cKO mice. Hematoxylin staining revealed no obvious aberrant histologic features in the whole brains of cKO mice. Histoimmunofluorescence staining and electron microscopy examination revealed markedly increased mTOR signaling and hyperproliferation of mitochondria in cKO mice, especially in the hippocampus. Furthermore, rapamycin treatment reversed these abnormalities.. This study suggests that our melanocyte-lineage mTOR hyperactivation mouse is a novel animal model of epilepsy, which may promote the progress of both epilepsy and neurophysiology research.

Topics: Animals; Brain; Cells, Cultured; Disease Models, Animal; Electroencephalography; Epilepsy; Hippocampus; Melanocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein

Amino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs.. An in vivo model of I/R injury was tested in control, mTOR. Mice treated with BCAAs had a significant reduction in infarct size as a percentage of the area at risk compared to controls (34.1 ± 3.9% vs. 44.7 ± 2.6%, P = 0.001), whereas mice treated with the mTOR inhibitor rapamycin were not protected by BCAA administration (42.2 ± 6.5%, vs. control, P = 0.015). This protection was not detected in our hetero knockout mice of mTOR. Western blot analysis revealed no change in AKT signaling whereas activation of mTOR was identified. Furthermore, BCAAs prevented swelling which was reversed by the addition of rapamycin. In myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA-induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001).. BCAA treatment exhibits a protective effect in myocardial I/R injury and that mTOR plays an important role in this preconditioning effect.

Topics: Amino Acids, Branched-Chain; Animals; Blotting, Western; Cardiotonic Agents; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin (RAPA) is a potent angiogenic inhibitor and the aim of this study is to identify the inhibitory effect of RAPA on retinal neovascularization (RNV) in experimental oxygen-induced retinopathy (OIR).. Forty-two 7-day-old C57BL/6 J mice were randomly divided into normoxia control group (14 mice), OIR group (14 mice), and rapamycin (RAPA) group. OIR model was induced in OIR and RAPA group. Vehicle and RAPA (2 mg/kg/d) was injected intraperitoneally daily from postnatal day 12 (P12) in OIR and RAPA groups, respectively. RNV was evaluated using fluorescence angiography and histopathology on P17. Non-perfused areas of retina were analyzed by Image-Pro plus 6.0 software. Retinal expression of cyclin D1 was detected both at mRNA and protein levels.. RAPA treatment significantly decreased RNV, non-perfused areas and number of endothelial cell nuclei breaking through the internal limiting membrane (ILM) in OIR mice. Moreover, RAPA decreased activation of cyclin D1 in retina caused by OIR.. RAPA can inhibit RNV by downregulating the expression of cyclin D1, which indicates its therapeutic potential in treating RNV-related diseases.

Topics: Animals; Animals, Newborn; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Oxygen; Retinal Neovascularization; RNA, Messenger; Sirolimus

Autophagy plays a crucial role in the pathological process of cardiovascular diseases. However, little is known about the pathological mechanism underlying autophagy regulation in dilated cardiomyopathy (DCM).. We explored whether up-regulating autophagy could improve cardiac function in mice with experimental DCM through the mTOR-4EBP1 pathway. Animal model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Both up- or down-regulation of autophagy were studied by administration of rapamycin or 3-MA in parallel. Morphology, Western blotting, and echocardiography were applied to confirm the pathological mechanisms.. Autophagy was activated and autophagosomes were significantly increased in the rapamycin group. The collagen volume fraction (CVF) was decreased in the rapamycin group compared with the DCM group (9.21 ± 0.82% vs 14.38 ± 1.24%, P < 0.01). The expression of p-mTOR and p-4EBP1 were significantly decreased in rapamycin-induced autophagy activation, while the levels were increased by down-regulating autophagy with 3-MA. In the rapamycin group, the LVEF and FS were significantly increased compared with the DCM group (54.12 ± 6.48% vs 45.29 ± 6.68%, P < 0.01; 26.89 ± 4.04% vs 22.17 ± 2.82%, P < 0.05). As the inhibitor of autophagy, 3-MA aggravated the progress of maladaptive cardiac remodeling and declined cardiac function in DCM mice.. The study indicated a possible mechanism for improving cardiac function in mice with experimental DCM by up-regulating autophagy via the mTOR-4EBP1 pathway, which could be a promising therapeutic strategy for DCM.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Cardiomyopathy, Dilated; Cell Cycle Proteins; Disease Models, Animal; Fibrosis; Male; Mice, Inbred BALB C; Myocytes, Cardiac; Recovery of Function; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Function, Left; Ventricular Remodeling

We hypothesized that the combination of a local stimulus for activating tumor-specific T cells and an anti-immunosuppressant would improve treatment of gliomas. Virally encoded IL15Rα-IL15 as the T-cell activating stimulus and a prostaglandin synthesis inhibitor as the anti-immunosuppressant were combined with adoptive transfer of tumor-specific T cells.. vvDD-IL15Rα-YFP and vMyx-IL15Rα-tdTr each infected and killed GL261 cells. IL15Rα-IL15-armed oncolytic poxviruses provide potent antitumor effects against brain tumors when combined with adoptive T-cell therapy, rapamycin, and celecoxib.

Topics: Animals; Brain Neoplasms; Celecoxib; Cell Line, Tumor; Combined Modality Therapy; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Drug Synergism; Female; Glioma; Immunosuppressive Agents; Immunotherapy; Immunotherapy, Adoptive; Interleukin-15; Male; Mice, Inbred C57BL; Myxoma virus; Oncolytic Virotherapy; Receptors, Interleukin-15; Sirolimus; Vaccinia virus

Topics: Acetylation; Adaptor Proteins, Signal Transducing; Animals; Atherosclerosis; Autophagy; Autophagy-Related Proteins; Cells, Cultured; Disease Models, Animal; Female; Hippo Signaling Pathway; Human Umbilical Vein Endothelial Cells; Humans; Mechanotransduction, Cellular; Mice, Knockout, ApoE; Plaque, Atherosclerotic; Protein Serine-Threonine Kinases; Proteolysis; Regional Blood Flow; Sirolimus; Sirtuin 1; Stress, Mechanical; Transcription Factors; YAP-Signaling Proteins

Penehyclidine hydrochloride (PHC) suppresses renal ischemia and reperfusion (I/R) injury (IRI); however, the underlying mechanism of action that achieves this function remains largely unknown. The present study aimed to investigate the potential role of autophagy in PHC‑induced suppression of renal IRI, as well as the involvement of cell proliferation and apoptosis. A rat IRI model and a cellular hypoxia/oxygenation (H/R) model were established; PHC, 3‑methyladenine (3‑MA) and rapamycin (Rapa) were administered to the IRI model rats prior to I/R induction and to H/R cells following reperfusion. Serum creatinine was measured using a biochemistry analyzer, whereas aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) expression levels were detected using ELISA kits. Renal tissue injury was evaluated by histological examination. In addition, microtubule‑associated protein light chain 3B (LC3B) expression, autophagosome formation, cell proliferation and apoptosis were detected in the cellular H/R model. The results demonstrated that I/R induced renal injury in IRI model rats, upregulated serum creatinine, ALAT and ASAT expression levels, and increased autophagic processes. In contrast, pretreatment with PHC or Rapa significantly prevented these I/R‑induced changes, whereas the administration of 3‑MA enhanced I/R‑induced injuries through suppressing autophagy. PHC and Rapa increased LC3B and Beclin‑1 expression levels, but decreased sequestome 1 (p62) expression in the cellular H/R model, whereas 3‑MA prevented these PHC‑induced changes. PHC and Rapa promoted proliferation and autophagy in the cellular H/R model; these effects were accompanied by increased expression levels of LC3B and Beclin‑1, and reduced p62 expression levels, whereas these levels were inhibited by 3‑MA. Furthermore, PHC and Rapa inhibited apoptosis in the cellular H/R model through increasing Bcl‑2 expression levels, and suppressing Bax and caspase‑3 expression levels; the opposite effect was induced by 3‑MA. In conclusion, PHC suppressed renal IRI through the induction of autophagy, which in turn promoted proliferation and suppressed apoptosis in renal cells.

Topics: Adenine; Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Autophagy; Beclin-1; Caspase 3; Cell Proliferation; Disease Models, Animal; Ischemia; Kidney; Male; Quinuclidines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Pulmonary vein obstruction (PVO) frequently occurs after repair of total anomalous pulmonary vein connection with progression of intimal hyperplasia from the anastomotic site toward upstream pulmonary veins (PVs). However, the understanding of mechanism in PVO progression is constrained by lack of data derived from a physiological model of the disease, and no prophylaxis has been established. We developed a new PVO animal model, investigated the mechanisms of PVO progression, and examined a new prophylactic strategy.. We developed a chronic PVO model using infant domestic pigs by cutting and resuturing the left lower PV followed by weekly hemodynamic parameter measurement and angiographic assessment of the anastomosed PV. Subsequently, we tested a novel therapeutic strategy with external application of rapamycin-eluting film to the anastomotic site.. We found the pig PVO model mimicked human PVO hemodynamically and histopathologically. This model exhibited increased expression levels of Ki-67 and phospho-mammalian target of rapamycin in smooth muscle-like cells at the anastomotic neointima. In addition, contractile to synthetic phenotypic transition; that is, dedifferentiation of smooth muscle cells and mammalian target of rapamycin pathway activation in the neointima of upstream PVs were observed. Rapamycin-eluting films externally applied around the anastomotic site inhibited the activation of mammalian target of rapamycin in the smooth muscle-like cells of neointima, and delayed PV anastomotic stenosis.. We demonstrate the evidence on dedifferentiation of smooth muscle-like cells and mammalian target of rapamycin pathway activation in the pathogenesis of PVO progression. Delivery of rapamycin to the anastomotic site from the external side delayed PV anastomotic stenosis, implicating a new therapeutic strategy to prevent PVO progression.

Topics: Angiography; Animals; Biomarkers; Disease Models, Animal; Disease Progression; Muscle, Smooth; Neointima; Pulmonary Veins; Pulmonary Veno-Occlusive Disease; Sirolimus; Swine; TOR Serine-Threonine Kinases; Vascular Remodeling

SophoraflavanoneG (SG), an important prenylated flavonoid isolated from Sophoraalopecuroides.L, is effective for many illnesses. The present study was designed to investigate whether the compound could reverse depressive-like symptoms and investigate its possible mechanisms. Chronic Unpredictable Mild Stress (CUMS) mice were treated with fluoxetine and SG. The immobility time in forced swimming test (FST) and tail suspension test (TST) were recorded. The levels of pro-inflammatory cytokines and neurotransmitters in the hippocampus were evaluated. Furthermore, the protein expressions of PI3K, AKT, mTOR, p70S6K, BDNF, and Trkb in hippocampus were detected. Rapamycin, the selective mTOR inhibitor, was used to estimate the potential mechanism. As a result, after 7 days of SG treatment, the immobility time in FST and TST was declined obviously. The levels of IL-6, IL-1β, and TNF-α in the hippocampus were significantly reduced, and the quantity of 5-HT and NE was raised considerably in SG-treated group compared with the CUMS-exposed group. Additionally, SG could up-regulate the expressions of PI3K, AKT, mTOR, 70S6K, BDNF, and Trkb. The blockade of mammalian target of rapamycin signaling blunted the antidepressant effect and reversed the up-regulation of BDNF expression caused by SG. These findings suggested that SG treatment alleviated depressive-like symptoms via mTOR-mediated BDNF/Trkb signaling.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Chronic Disease; Depression; Disease Models, Animal; Flavanones; Food Preferences; Hindlimb Suspension; Hippocampus; Male; Mice; Mice, Inbred BALB C; Signal Transduction; Sirolimus; Stress, Psychological; Sucrose; TOR Serine-Threonine Kinases

Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown.. Here, we developed the first mouse model of severe MR. Valvular damage was achieved by severing the mitral valve leaflets and chords with iridectomy scissors, and MR was confirmed by echocardiography. Serial echocardiography was performed to follow up LV morphology and systolic function. Analysis of cardiac tissues was subsequently performed to evaluate valve deformation, cardiomyocyte morphology, LV fibrosis, and cell death. Finally, dysregulated pathways were assessed by RNA-sequencing analysis and immunofluorescence.. In the ensuing 15 weeks after the induction of MR, gradual LV dilatation and dysfunction occurred, resulting in severe systolic dysfunction. Further analysis revealed that severe MR resulted in a marked increase in cardiac mass and increased cardiomyocyte length but not width, with electron microscopic evidence of sarcomere disarray and the development of sarcomere disruption. From a mechanistic standpoint, severe MR resulted in activation of multiple components of both the mammalian target of rapamycin and calcineurin pathways. Inhibition of mammalian target of rapamycin signaling preserved sarcomeric structure and prevented LV remodeling and systolic dysfunction. Immunohistochemical analysis uncovered a differential pattern of expression of the cell polarity regulator Crb2 (crumbs homolog 2) along the longitudinal axis of cardiomyocytes and close to the intercalated disks in the MR hearts. Electron microscopy images demonstrated a significant increase in polysome localization in close proximity to the intercalated disks and some areas along the longitudinal axis in the MR hearts.. These results indicate that LV dysfunction in response to severe MR is a form of maladaptive eccentric cardiomyocyte hypertrophy and outline the link between cell polarity regulation and spatial localization protein synthesis as a pathway for directional cardiomyocyte growth.

Topics: Animals; Cell Adhesion Molecules; Cell Shape; Cell Size; Disease Models, Animal; Echocardiography; Fibrosis; Gene Expression Profiling; Hypertrophy; Infusion Pumps, Implantable; Magnetic Resonance Imaging; Male; Mice; Mitral Valve; Mitral Valve Insufficiency; Myocytes, Cardiac; Polyribosomes; RNA, Messenger; Sirolimus; Systole; TOR Serine-Threonine Kinases; Ventricular Dysfunction, Left

Bronchopulmonary dysplasia (BPD) is a severe respiratory complication in preterm infants. This study reveals the molecular mechanism of autophagic agonists regulating the Nrf2-ARE pathway via p62 to improve alveolar development in BPD rats.. Newborn Sprague-Dawley rats were randomly exposed to a hyperoxic environment (FiO. At the levels of lung tissue and primary type II alveolar epithelial cells, the enhanced binding between phosphorylated p62 and Keap1 disrupted the nuclear transport of Nrf2. The activated Nrf2 was insufficient to reverse alveolar simplification. The autophagy agonist was able to inhibit p62 phosphorylation, promote Keap1 degradation, increase Nrf2 nuclear transport, augment downstream antioxidant enzyme expression, and enhance antioxidant capacity, thereby improving the simplification of alveolar structure in BPD rats.. The use of autophagy agonists to enhance the Nrf2-ARE pathway activity and promote alveolar development could be a novel target in antioxidant therapy for BPD.

Topics: Animals; Animals, Newborn; Antioxidant Response Elements; Antioxidants; Autophagy; Bronchopulmonary Dysplasia; Disease Models, Animal; Humans; Hyperoxia; Infant, Newborn; Kelch-Like ECH-Associated Protein 1; Mice; NF-E2-Related Factor 2; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Sirolimus

Maternal aging-associated reduction of oocyte viability is a common feature in mammals, but more research is needed to counteract this process. In women, the first aging phenotype appears with a decline in reproductive function, and the follicle number gradually decreases from menarche to menopause. Cows can be used as a model of early human embryonic development and reproductive aging because both species share a very high degree of similarity during follicle selection, cleavage, and blastocyst formation. Recently, it has been proposed that the main driver of aging is the mammalian target of rapamycin (mTOR) signaling rather than reactive oxygen species. Based on these observations, the study aimed to investigate for the first time the possible role of rapamycin on oocyte maturation, embryonic development, and telomere length in the bovine species, as a target for future strategies for female infertility caused by advanced maternal age. The 1nm rapamycin

Topics: Animals; Cattle; Disease Models, Animal; Embryonic Development; Female; In Vitro Oocyte Maturation Techniques; Infertility, Female; Metaphase; Oocytes; Ovarian Follicle; Pregnancy; Pregnancy, Animal; Sirolimus; Telomere Homeostasis

Myocardial ischemia/reperfusion (I/R) is an important complication of reperfusion therapy for myocardial infarction, and trimetazidine is used successfully for treatment of ischemic cardiomyopathy by regulating mitochondrial function. Moreover, electroacupuncture (EA) preconditioning was demonstrated to be cardioprotective in both in vivo rodent models and in patients undergoing heart valve replacement surgery. However, the mechanisms have not been well elucidated. Mitophagy, mediated by the mTORC1-ULK1-FUNDC1 (mTOR complex 1-unc-51-like autophagy-activating kinase 1-FUN14 domain-containing 1) pathway, can regulate mitochondrial mass and cell survival effectively to restrain the development of myocardial ischemia/reperfusion injury (MIRI). In this study, we hypothesized that EA preconditioning ameliorated MIRI via mitophagy. To test this, rapamycin, an mTOR inhibitor, was used. The results showed that EA preconditioning could reduce the infarct size and risk size, and decrease the ventricular arrhythmia score and serum creatine kinase-myocardial band isoenzyme (CK-MB), lactate dehydrogenase (LDH), and cardiac troponin T (cTnT) in MIRI rats. Moreover, it also attenuated MIRI-induced apoptosis and mitophagy accompanied by elevated mTORC1 level and decreased ULK1 and FUNDC1 levels. However, these effects of EA preconditioning were blocked by rapamycin, which aggravated MIRI, reduced adenosine triphosphate (ATP) production, and antagonized infarct size reduction. In conclusion, our results indicated that EA preconditioning protected the myocardium against I/R injury by inhibiting mitophagy mediated by the mTORC1-ULK1-FUNDC1 pathway.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Autophagy-Related Protein-1 Homolog; Disease Models, Animal; Electroacupuncture; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Sirolimus

Muscle myostatin was reduced in all the therapeutic groups but the MVC group (p <0.001 for RAPA and MVC-RAPA) and in serum samples (p <0.01 for all the groups). Serum CK levels were also significantly lower in MVC and RAPA groups (p <0.01 in both cases). Lower AST levels were observed in all the therapeutic groups (p <0.05 for all of them). The apoptotic effector caspase-3 was significantly lower in MVC and RAPA groups (p<0.05 in both cases). Combined treatment with MVC-RAPA showed a synergistic increase in p-AKT, p-mTOR and SIRT1 levels.. MVC and RAPA show a protective role in some factors involved in frailty. More studies are needed to prove their clinical applications.. Eighty male homozygous IL10KOs were randomly assigned to one of 4 groups (n= 20): i) IL10KO group (IL10KO); ii) IL10KO receiving MVC in drinking water (MVC group), iii) IL10KO receiving RAPA in drinking water (RAPA group), and finally, iv) MVC-RAPA group that received MVC and RAPA in drinking water. Blood and muscle samples were analysed. Survival analysis, frailty index calculation, and functional assessment were also performed.

Topics: Aging; Animals; Cytokines; Disease Models, Animal; Frailty; Interleukin-10; Male; Maraviroc; Mice; Mice, Knockout; Muscle, Skeletal; Random Allocation; Receptors, Chemokine; Sirolimus; Survival Rate

Mouse model of multiple sclerosis (MS) is used for the inflammatory demyelinating disease. Rapamycin (RAPA) may contribute to the reduction of inflammatory responses to experimental autoimmune encephalomyelitis (EAE). Due to its adverse side effects, identifying new therapeutic agents is important. We investigated the transcriptional effects of evening primrose/hemp seed oil (EP/HS oil) compared to RAPA on the expression of immunological factors genes in spleen cells of EAE mouse models. We firstly induced EAE mice by injection of myelin oligodendrocyte glycoprotein (MOG). Then, the EAE mice treated and untreated with EP/HS oil were evaluated and compared with naïve mice. The spinal cords were examined histologically. The immunological factors including genes expression of the regulatory-associated protein of mammalian target of rapamycin (RAPTOR), regulatory-associated companion of mammalian target of rapamycin (RICTOR), interferon (IFN)-γ, interleukin (IL)-10, signal transducer and activator of transcription factors (STAT3), forkhead box P3 (FOXP3), and IL-17 of splenocytes were evaluated by real time-polymerase chain reaction (RT-PCR). The data showed that EP/HS oil was able to reduce the severity of EAE and inhibited the development of the disease. EP/HS oil treatment significantly inhibited the expression of RAPTOR, IFN-γ, IL-17, and STAT3 genes and promoted the expression of RICTOR, IL-10, and FOXP3 genes. In conclusion, the EP/HS oil is likely to be involved in transcription of factors in favor of EAE improvement as well as participating in remyelination in the EAE spinal cord and that it suggests to be effective in therapeutic approaches for MS.

Topics: Animals; Cannabis; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; gamma-Linolenic Acid; Humans; Linoleic Acids; Linseed Oil; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Oenothera biennis; Plant Oils; Regulatory-Associated Protein of mTOR; Seeds; Sirolimus; Spleen

Multiple sclerosis (MS) whose pathogenesis is still unclear is a chronic progressive disease in the central nervous system. Gut microbiota can directly or indirectly affect the immune system through the brain gut axis to engage in the occurrence and development of the disease.. C57BL/6 mice which were immunized by MOG. The results showed that rapamycin and MCC950 could alleviate the progression of the disease by inducing autophagy and inhibiting the immune response. The Alpha diversity of EAE model group was no significant difference compering to control group while the number of OTUs was decreased. After the treatment by rapamycin and MCC950, the abundance and composition of gut microbiota was relatively recovered, which was close to that of normal mice.. Inhibiting immune cell-mediated inflammation and restoring the composition of gut microbiota may help to alleviate the clinical symptoms of multiple sclerosis. Furthermore, to research the regulatory effect between immune response and gut microbiota may be a new strategy for the prevention and treatment of multiple sclerosis.

Topics: Animals; Brain; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Furans; Gastrointestinal Microbiome; Heterocyclic Compounds, 4 or More Rings; Indenes; Inflammation; Intestine, Large; Mice; Mice, Inbred C57BL; Multiple Sclerosis; RNA, Ribosomal, 16S; Sirolimus; Spleen; Sulfonamides; Sulfones

Aortic dissection (AD) is a serious clinical condition that is unpredictable and frequently results in fatal outcome. Although rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has been reported to be effective in preventing aortopathies in mouse models, its mode of action has yet to be clarified. A mouse AD model that was created by the simultaneous administration of β-aminopropionitrile (BAPN) and angiotensin II (AngII) for 14 days. Rapamycin treatment was started either at day 1 or at day 7 of BAPN+AngII challenge, and continued throughout the observational period. Rapamycin was effective both in preventing AD development and in suppressing AD progression. On the other hand, gefitinib, an inhibitor of growth factor signaling, did not show such a beneficial effect, even though both rapamycin and gefitinib suppressed cell cycle activation in AD. Rapamycin suppressed cell cycle-related genes and induced muscle development-related genes in an AD-related gene expression network without a major impact on inflammation-related genes. Rapamycin augmented the activation of Akt1, Akt2, and Stat3, and maintained the contractile phenotype of aortic smooth muscle cells. These findings indicate that rapamycin was effective both in preventing the development and in suppressing the progression of AD, indicating the importance of the mTOR pathway in AD pathogenesis.

Topics: Aminopropionitrile; Angiotensin II; Animals; Aortic Dissection; Cell Cycle Checkpoints; Cell Line; Disease Models, Animal; Gefitinib; Gene Expression Regulation; Gene Ontology; Gene Regulatory Networks; Male; Mice; Muscle, Smooth, Vascular; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

KOP-r agonist U50,488H produces strong aversion and anxiety/depression-like behaviors that enhance alcohol intake and promote alcohol seeking and relapse-like drinking in rodents. Mammalian target of rapamycin complex 1 (mTORC1) pathway in mouse striatum is highly involved in excessive alcohol intake and seeking, and in the U50,488H-induced conditioned place aversion. Therefore, we hypothesized that KOP-r activation increases alcohol consumption through the mTORC1 activation. This study focuses on: (1) how chronic excessive alcohol drinking (4-day drinking-in-the-dark paradigm followed by 3-week chronic intermittent access drinking paradigm [two-bottle choice, 24-h access every other day]) affected nuclear transcript levels of the mTORC1 pathway genes in mouse nucleus accumbens shell (NAcs), using transcriptome-wide RNA sequencing analysis; and (2) whether selective mTORC1 inhibitor rapamycin could alter excessive alcohol drinking and prevent U50,488H-promoted alcohol intake. Thirteen nuclear transcripts of mTORC1 pathway genes showed significant up-regulation in the NAcs, with two genes down-regulated, after excessive alcohol drinking, suggesting the mTORC1 pathway was profoundly disrupted. Single administration of rapamycin decreased alcohol drinking in a dose-dependent manner. U50,488H increased alcohol drinking, and pretreatment with rapamycin, at a dose lower than effective doses, blocked the U50,488H-promoted alcohol intake in a dose-dependent manner, indicating a mTORC1-mediated mechanism. Our results provide supportive and direct evidence relevant to the transcriptional profiling of the critical mTORC1 genes in mouse NAc shell: with functional and pharmacological effects of rapamycin, altered nuclear transcripts in the mTORC1 signaling pathway after excessive alcohol drinking may contribute to increased alcohol intake triggered by KOP-r activation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Binge Drinking; Central Nervous System Depressants; Disease Models, Animal; Ethanol; Gene Expression; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Nucleus Accumbens; Receptors, Opioid, kappa; Signal Transduction; Sirolimus; Transcriptome

Vascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells. However, we found high dose RPM monotherapy prolonged VCA survival by only a few days, leading us to explore the mechanisms responsible.. A mouse orthotopic forelimb transplantation model (BALB/c- > C57BL/6) was established using WT mice, as well as C57BL/6 recipients with conditional deletion of T-bet within their Treg cells. Events in untreated VCA recipients or those receiving RPM or FK506 therapy were analyzed by flow-cytometry, histopathology and real-time qPCR.. Therapy with RPM (2 mg/kg/d, p < .005) or FK506 (2 mg/kg/d, p < .005) each prolonged VCA survival. In contrast to FK506, RPM increased the ratio of splenic Treg to Teff cells (p < .05) by suppressing Teff and expanding Treg cells. While the proportion of activated splenic CD4 + Foxp3- T cells expressing IFN-γ were similar in control and RPM-treated groups, RPM decreased the proportions ICOS+ and CD8+ IFN-γ + splenic T cells. However, RPM also downregulated CXCR3+ expression by Tregs, and forelimb allografts had reduced infiltration by CXCR3+ Treg cells. In addition, allograft recipients whose Tregs lacked T-bet underwent accelerated rejection compared to WT mice despite RPM therapy.. We demonstrate that while RPM increased the ratio of Treg to Teff cells and suppressed CD8+ T cell allo-activation, it failed to prevent CD4 Teff cell activation and impaired CXCR3-dependent Treg graft homing, thereby limiting the efficacy of RPM in VCA recipients.

Topics: Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Disease Models, Animal; Forelimb; Gene Knockdown Techniques; Humans; Immune Tolerance; Immunosuppressive Agents; Interferon-gamma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Receptors, CXCR3; Sirolimus; T-Box Domain Proteins; Tacrolimus; Vascularized Composite Allotransplantation

Surgical decompression after spinal cord injury (SCI) is a conventional treatment. Although it has been proven to have clinical effects, there are certain limitations, such as the surgical conditions that must be met and the invasive nature of the treatment. Therefore, there is an urgent need to develop a simple and maneuverable therapy for the emergency treatment of patients with SCI before surgery. Rapamycin (RAPA) has been reported to have potential as a therapeutic agent for SCI. In this study, we observed the therapeutic effects of rapamycin and surgical decompression, in combination or separately, on the histopathology in rabbits with SCI. After combination therapy, intramedullary pressure (IMP) decreased significantly, autophagic flux increased, and apoptosis and demyelination were significantly reduced. Compared with RAPA/surgical decompression alone, the combination therapy had a significantly better effect. In addition, we evaluated the effects of mechanical pressure on autophagy after SCI by assessing changes in autophagic initiation, degradation, and flux. Increased IMP after SCI inhibited autophagic degradation and impaired autophagic flux. Decompression improved autophagic flux after SCI. Our findings provide novel evidence of a promising strategy for the treatment of SCI in the future. The combination therapy may effectively improve emergency treatment after SCI and promote the therapeutic effect of decompression. This study also contributes to a better understanding of the effects of mechanical pressure on autophagy after neurotrauma.

Topics: Animals; Apoptosis; Autophagy; Cell Count; Decompression, Surgical; Demyelinating Diseases; Disease Models, Animal; Female; Neurons; Rabbits; Sirolimus; Spinal Cord; Spinal Cord Injuries

Sirtuin 1 (SIRT1) is involved in the pathogenesis of allergic asthma. This study aimed to investigate whether EX‑527, a specific SIRT1 inhibitor, exerted suppressive effects on allergic airway inflammation in mice submitted to ovalbumin (OVA) inhalation. In addition, this study assessed whether such a protective role was mediated by autophagy suppression though mammalian target of rapamycin (mTOR) activation. Female C57BL/6 mice were sensitized to OVA and EX‑527 (10 mg/kg) was administered prior to OVA challenge. The study found that EX‑527 reversed OVA‑induced airway inflammation, and reduced OVA‑induced increases in inflammatory cytokine expression, and total cell and eosinophil counts in bronchoalveolar lavage fluid. In addition, EX‑527 enhanced mTOR activation, thereby suppressing autophagy in allergic mice. To assess whether EX‑527 inhibited airway inflammation in asthma through the mTOR‑mediated autophagy pathway, rapamycin was administered to mice treated with EX‑527 after OVA sensitization. All effects induced by EX‑527, including increased phosphorylated‑mTOR and decreased autophagy, were abrogated by rapamycin treatment. Taken together, the present findings indicated that EX‑527 may inhibit allergic airway inflammation by suppressing autophagy, an effect mediated by mTOR activation in allergic mice.

Topics: Administration, Inhalation; Animals; Asthma; Autophagy; Carbazoles; Cytokines; Disease Models, Animal; Down-Regulation; Female; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphorylation; Sirolimus; Sirtuin 1; TOR Serine-Threonine Kinases

The activation of autophagy was shown to shrink infarct size and mitigate cardiac dysfunction caused by myocardial infarction (MI). However, the underlying mechanisms remain largely unknown. As excessive generation of reactive oxygen species (ROS) deteriorates MI process and Nrf2 signaling exerts an antioxidant role, we explored whether autophagy assuaged MI through Nrf2 signaling activation-mediated ROS clear.. MI models were induced by ligation of the left descending coronary artery (LAD) in C57BL/6J mice or Nrf2 knockout mice (Nrf2-KO). Rapamycin and 3-methyladenine (3-MA) were used to activate and repress autophagy in MI mice, respectively. Aspirin, a cardioprotective drug was given to MI mice to evaluate its effects on autophagy.. Compared with the MI group, rapamycin treatment remarkably decreased the infarct size, cell apoptosis and blood troponin I level, accompanied by reduced redox potential (Eh), ROS, malondialdehyde (MDA) and cytochrome C levels, and increased reduced glutathione (GSH) level. Also, rapamycin treatment increased the expressions of bcl-2, bcl-xL, HSP70, and HSP90. In addition, rapamycin treatment promoted the nuclear accumulation of Nrf2 protein. However, Nrf2 downregulation significantly impaired the effects of rapamycin on the reductions of infarct size, cell apoptosis, troponin I and ROS levels. Similarly, to rapamycin roles, aspirin treatment also remarkably reduced infarct size, cell apoptosis and troponin I in mice with MI surgery, as well as increased the expression level of LC3II/LC3I.. This study demonstrated that autophagy enhancement contributed to the improvement of MI through Nrf2 signaling activation-mediated ROS clear.

Topics: Animals; Apoptosis; Aspirin; Autophagy; Autophagy-Related Proteins; Disease Models, Animal; Male; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocytes, Cardiac; NF-E2-Related Factor 2; Reactive Oxygen Species; Signal Transduction; Sirolimus

Glaucoma is a progressive optic neuropathy that has become the most common cause of irreversible blindness worldwide. Studies have shown that the protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in regulating numerous functions, such as growth, proliferation, cytoskeletal organization, metabolism, and autophagy. Clinical trials have shown that Rho-associated protein kinase (ROCK) inhibitors reduced intraocular pressure (IOP) in patients with glaucoma and ocular hypertension (OHT). In this study, we explored whether rapamycin (RAPA) eye drops can reduce IOP and protect retinal ganglion cells (RGCs). Our results indicated that in rats treated with RAPA, the drug was detected in the aqueous humor (AH), and the IOP was reduced. This may be related to the inhibition of RhoA protein activation by RAPA and regulation of the actin cytoskeleton in trabecular meshwork (TM) cells. In addition, the retinal thickness and the survival rate of RGCs were significantly reduced in the OHT group compared with the control group. These changes in the OHT group were significantly improved after treatment with RAPA. This may be because RAPA inhibited the activation of glial cells and the release of proinflammatory factors, thereby attenuating further damage to the retina and RGCs. Taken together, the results of this study demonstrated that RAPA not only reduced IOP but also protected RGCs, suggesting that RAPA is likely to be an effective strategy for the treatment of glaucoma.

Topics: Administration, Ophthalmic; Animals; Cell Survival; Cells, Cultured; Disease Models, Animal; Glaucoma; Humans; Inflammation Mediators; Intraocular Pressure; Male; Neuroglia; Ophthalmic Solutions; Rats, Sprague-Dawley; Retinal Ganglion Cells; rho GTP-Binding Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trabecular Meshwork

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a group of severe, tissue-specific diseases of childhood with unknown pathogenesis. Brain-specific MDS manifests as devastating spongiotic encephalopathy with no curative therapy. Here, we report cell type-specific stress responses and effects of rapamycin treatment and ketogenic diet (KD) in mice with spongiotic encephalopathy mimicking human MDS, as these interventions were reported to improve some mitochondrial disease signs or symptoms. These mice with astrocyte-specific knockout of

Topics: Animals; Astrocytes; Brain Diseases; Diet, Ketogenic; Disease Models, Animal; DNA Helicases; DNA Replication; DNA, Mitochondrial; Female; Ketosis; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Mutation; Neurons; Sirolimus; Stress, Physiological

PI3K/Akt/mTOR signaling pathway activity is highly elevated in glioblastoma (GBM). Although rapamycin is known to inhibit this pathway, GBM patients are resistant to rapamycin monotherapy. This may be related to mutations of tumor suppressor phosphatase and tensin homolog (PTEN). Here, we show that higher expression of E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) in GBM is correlated with poor prognosis. Smurf1 promotes cell growth and colony formation by accelerating cell cycle and aberrant signaling pathways. In addition, we show that Smurf1 ubiquitylates and degrades PTEN. We further demonstrate that the oncogenic role of Smurf1 is dependent on PTEN. Upregulated Smurf1 impairs PTEN activity, leading to consistent activation of PI3K/Akt/mTOR signaling pathway; and depletion of Smurf1 dramatically inhibits cell proliferation and tumor growth. Moreover, loss of Smurf1 abolishes the aberrant regulation of PTEN, causing negative feedback on PI3K/Akt/mTOR signaling pathway, and thus leading to rescue of tumor sensitivity to rapamycin in an orthotopic GBM model. Taken together, we show that Smurf1 promotes tumor progression via PTEN, and combined treatment of Smurf1 knockdown with mammalian target of rapamycin (mTOR) inhibition reduces tumor progression. These results identify a unique role of Smurf1 in mTOR inhibitor resistance and provide a strong rationale for combined therapy targeting GBM.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioblastoma; Heterografts; Humans; Immunohistochemistry; Mice; Mutation; Oncogenes; Prognosis; PTEN Phosphohydrolase; Sirolimus; Synthetic Lethal Mutations; Ubiquitin-Protein Ligases; Ubiquitination

A drug-eluting coronary stent is being developed at the National Institute of Cardiology of Mexico for the treatment of ischemic heart disease.. To establish the best animal model for the tests, to show the advances in the drug-eluting stent prototype, to assess two drugs' antiproliferative activity and histological results.. Smooth muscle cell culture tests were performed in order to assess sirolimus and paclitaxel antiproliferative properties. The drugs were encapsulated inside the polymeric matrix of the stents. Rabbits and pigs were used as animal models.. Sirolimus and paclitaxel showed an inhibitory effect, which was higher for the latter. Infrared spectroscopy and light and optical microscopy showed that the drug/polymer layer properly adhered to the stent. At a four-week follow-up, both animal models showed satisfactory clinical evolution and adequate histological response, although the porcine model was shown to be more suitable for future protocols.. Preliminary tests of the drug-eluting stent provided bases for the development of a study protocol with an adequate number of pigs and with clinical angiographic and histopathological three-month follow-up.. En el Instituto Nacional de Cardiología de México se desarrolla una endoprótesis (stent) coronaria liberadora de fármacos para el tratamiento de la cardiopatía isquémica.. Establecer el mejor modelo animal para las pruebas, mostrar los avances en el prototipo del stent liberador de fármacos, evaluar la actividad antiproliferativa de dos fármacos y los resultados histológicos.. Se realizaron cultivos de células de músculo liso para evaluar las propiedades antiproliferativas de sirolimus y paclitaxel. Los fármacos fueron encapsulados en el interior de la matriz polimérica de los stents. Se emplearon conejos y cerdos como modelos animales.. Sirolimus y paclitaxel mostraron efecto inhibitorio, mayor en el segundo. La espectroscopia infrarroja y la microscopia óptica y electrónica mostraron que la capa del polímero con el fármaco se adhería adecuadamente al stent. A las cuatro semanas de seguimiento, ambos modelos animales mostraron evolución clínica satisfactoria y adecuada respuesta histológica, si bien el modelo porcino resultó más conveniente para protocolos futuros.. Las pruebas preliminares del stent liberador de fármaco brindó bases para desarrollar el protocolo con un número adecuado en cerdos y con seguimiento clínico angiográfico e histopatológico a tres meses.

Topics: Animals; Disease Models, Animal; Drug-Eluting Stents; Female; Follow-Up Studies; Male; Microscopy; Paclitaxel; Prosthesis Design; Rabbits; Sirolimus; Spectrophotometry, Infrared; Swine

Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1

Topics: Animals; Disease Models, Animal; Epilepsies, Partial; Everolimus; Immunosuppressive Agents; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Knockout; Sirolimus; Treatment Outcome; Tuberous Sclerosis

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.

Topics: Aging; Animals; Cell Line; Disease Models, Animal; Electromyography; Gene Expression Regulation; Humans; Laser Capture Microdissection; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Muscle Fibers, Skeletal; Myoblasts; Neuromuscular Junction; Patch-Clamp Techniques; RNA-Seq; Sarcopenia; Signal Transduction; Sirolimus

Despite recent progress in the treatment of rheumatoid arthritis (RA), many patients still fail to achieve remission or low disease activity. An imbalance between auto-reactive effector T cells (Teff) and regulatory T cells (Treg) may contribute to joint inflammation and damage in RA. Therefore, restoring this balance is a promising approach for the treatment of inflammatory arthritis. Accordingly, our group has previously shown that the combination of TGF-β-releasing microparticles (MP), rapamycin-releasing MP, and IL-2-releasing MP (TRI MP) can effectively increase the ratio of Tregs to Teff in vivo and provide disease protection in several preclinical models. In this study TRI MP was evaluated in the collagen-induced arthritis (CIA) model. Although this formulation has been tested previously in models of destructive inflammation and transplantation, this is the first model of autoimmunity for which this therapy has been applied. In this context, TRI MP effectively reduced arthritis incidence, the severity of arthritis scores, and bone erosion. The proposed mechanism of action includes not only reducing CD4+ T cell proliferation, but also expanding a regulatory population in the periphery soon after TRI MP administration. These changes were reflected in the CD4+ T cell population that infiltrated the paws at the onset of arthritis and were associated with a reduction of immune infiltrate and inflammatory myeloid cells in the paws. TRI MP administration also reduced the titer of collagen antibodies, however the contribution of this reduced titer to disease protection remains uncertain since there was no correlation between collagen antibody titer and arthritis score.

Topics: Animals; Arthritis, Experimental; Autoantibodies; CD4-Positive T-Lymphocytes; Cell Count; Cytokines; Disease Models, Animal; Drug Liberation; Interleukin-2; Male; Mice; Microspheres; Sirolimus; Transforming Growth Factor beta

Mutations in

Topics: Animals; Apoptosis; Bacterial Proteins; Bacterial Toxins; Cells, Cultured; Disease Models, Animal; Enterotoxins; Familial Mediterranean Fever; Humans; Inflammasomes; Macrophages; Mice; Mice, Knockout; Mutation; Necroptosis; Peritonitis; Phosphorylation; Primary Cell Culture; Pyrin; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptional Activation; Up-Regulation

Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4

Topics: Animals; Anti-Bacterial Agents; Disease Models, Animal; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Multiple Sclerosis; Sirolimus

Rabies is an important zoonotic disease in Iran. Autophagy is a process that maintains homeostasis and can be used as an innate defense mechanism against viruses. Apoptosis is the process of programmed cell death induced by physiological and pathological conditions. The crosstalk of autophagy and apoptosis plays a key role in rabies virus infection. In the current study, NMRI mice intra-cranially received 3-Methyl Adenine (3-MA), rapamycin, street rabies virus (SRABV) and drugs plus SRABV. SRABV and Map1lc3, Beclin-1, Atg5 gene expression were assayed by real-time PCR. Immunohistochemistry was carried out via LC3 protein staining as an autophagy marker, and apoptotic cell death was measured using a TUNEL assay. Map1lc3, Beclin-1 and Atg5 genes expression was significantly increased in drug-plus-SRBV-treated tissues compared to control at 24 hpi. Map1lc3 and Atg5 gene expression showed a slight change in the drugs-plus-virus group compared with the control at 72 hpi. The presence of LC3 in the tissues of the group treated with rapamycin plus SRBV confirmed induction of autophagy, but it was not present in the tissues treated with 3-MA plus SRBV. Our data revealed that apoptosis was induced only in the groups receiving the SRBV or rapamycin or both at 24 hpi. Apoptosis was observed after 72 hours, when the drugs' effect had disappeared in all but the autophagy inhibitor group. Understanding the interaction of SRABV with autophagy pathway genes and its effect on host cell apoptosis may open a new horizon for human intervention and allow a deeper understanding of rabies infections.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain; Disease Models, Animal; Fluorescent Antibody Technique, Direct; Mice; Neurons; Rabies; Rabies virus; Sirolimus; Viral Proteins; Virus Replication

Immune homeostasis in peripheral tissues is, to a large degree, maintained by the differentiation and action of regulatory T cells (Treg) specific for tissue Ags. Using a novel mouse model, we have studied the differentiation of naive CD4

Topics: Animals; Autoimmune Diseases; Cell Differentiation; Disease Models, Animal; Forkhead Transcription Factors; Humans; Lymphocyte Activation; Mice; Mice, Transgenic; Ovalbumin; Receptors, Antigen, T-Cell; Signal Transduction; Sirolimus; Skin; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Retinopathy of prematurity (ROP) is a vision-threatening disorder characterized with retinal vaso-obliteration in phase 1 and pathological neovascularization (NV) in phase 2. However, there has been no effective and safe treatment for ROP. Current management is mainly focused on the reduction of abnormal NV in phase 2, and anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment, yet, with great risks of late recurrence and systemic side effects. It has been reported that the severity of vaso-obliteration in phase 1 largely influences subsequent NV, suggesting that it may be a promising target to develop novel treatments for ROP. Here, we investigated the therapeutic potential and safety of early rapamycin intervention in treating phase 1 ROP and possible underlying mechanisms using the mouse model of oxygen-induced retinopathy (OIR). We found that intravitreal injection of rapamycin at postnatal day 7 (P7) significantly reduced retinal avascular area, increased vascular density, and reversed the suppression of deep capillaries development in phase 1 of OIR mice. Rapamycin treatment not only reduced vascular apoptosis, but also promoted proliferation and tip cell functions. Additionally, rapamycin did not interfere with normal retinal vascular development. Further investigation showed that Ang1/Tie2 pathway might be involved in rapamycin's vascular protection in phase 1 OIR retinas. Moreover, early rapamycin treatment at P7 had long-term protective effects of reducing retinal NV and avascular area, as well as enhancing vascular maturity in phase 2 of OIR mice. Together, our data suggest that rapamycin may be a safe and promising strategy for early intervention of ROP.

Topics: Animals; Disease Models, Animal; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Oxygen; Protective Agents; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Signal Transduction; Sirolimus; Vascular Endothelial Growth Factor A

Dry eye disease (DED), one of the most prevalent conditions among the elderly, is a chronic inflammatory disorder that disrupts tear film stability and causes ocular surface damage. Aged C57BL/6J mice spontaneously develop DED. Rapamycin is a potent immunosuppressant that prolongs the lifespan of several species. Here, we compared the effects of daily instillation of eyedrops containing rapamycin or empty micelles for three months on the aged mice. Tear cytokine/chemokine profile showed a pronounced increase in vascular endothelial cell growth factor-A (VEGF-A) and a trend towards decreased concentration of Interferon gamma (IFN)-γ in rapamycin-treated groups. A significant decrease in inflammatory markers in the lacrimal gland was also evident (

Topics: Aging; Animals; Autophagy-Related Protein-1 Homolog; CD4 Antigens; Cell Lineage; Conjunctiva; Cornea; Disease Models, Animal; Dry Eye Syndromes; Forkhead Transcription Factors; Goblet Cells; Humans; Inflammation; Interferon-gamma; Leukocyte Common Antigens; Mice; Ophthalmic Solutions; Sirolimus; Tears; Vascular Endothelial Growth Factor A

The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied.. Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3-9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug.. Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD.. The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.

Topics: 3-Hydroxybutyric Acid; Animals; Antineoplastic Agents; Blood Glucose; Breast Neoplasms; Diet, Ketogenic; Disease Models, Animal; Female; Insulin; Ketosis; Mice; Sirolimus

Osteogenesis imperfecta (OI) type V is an autosomal dominant disorder caused by the c.-14C > T mutation in the interferon-induced transmembrane protein 5 gene (IFITM5), however, its onset mechanism remains unclear. In this study, heterozygous c.-14C > T mutant mice were developed to investigate the effect of immunosuppressants (FK506 and rapamycin) on OI type V. Among the mosaic mice generated by Crispr/Cas9-based technology, mice with less than 40% mosaic ratio of c.-14C > T mutation survived, whereas those with more than 48% mosaic ratio exhibited lethal skeletal abnormalities with one exception. All heterozygous mutants obtained by mating mosaic mice with wild-type mice exhibited a perinatal lethal phenotype due to severe skeletal abnormalities. Administration of FK506, a calcineurin inhibitor, in the heterozygous fetuses improved bone mineral content (BMC) of the neonates, although it did not save the neonates from the lethal effects of the mutation, whereas rapamycin, an mTOR inhibitor, reduced BMC, suggesting that mTOR signaling is involved in the bone mineralization of heterozygous mutants. These findings could clarify certain aspects of the onset mechanism of OI type V and enable development of therapeutics for this condition.

Topics: Animals; Disease Models, Animal; Genes, Lethal; Heterozygote; Immunosuppressive Agents; Male; Membrane Proteins; Mice; Mice, Knockout; Mosaicism; Mutation; Osteogenesis Imperfecta; Sirolimus; Tacrolimus

Intraocular pressure-sensitive retinal ganglion cell degeneration is a hallmark of glaucoma, the leading cause of irreversible blindness. Here, we used RNA-sequencing and metabolomics to examine early glaucoma in DBA/2J mice. We demonstrate gene expression changes that significantly impact pathways mediating the metabolism and transport of glucose and pyruvate. Subsequent metabolic studies characterized an intraocular pressure (IOP)-dependent decline in retinal pyruvate levels coupled to dysregulated glucose metabolism prior to detectable optic nerve degeneration. Remarkably, retinal glucose levels were elevated 50-fold, consistent with decreased glycolysis but possibly including glycogen mobilization and other metabolic changes. Oral supplementation of the glycolytic product pyruvate strongly protected from neurodegeneration in both rat and mouse models of glaucoma. Investigating further, we detected mTOR activation at the mechanistic nexus of neurodegeneration and metabolism. Rapamycin-induced inhibition of mTOR robustly prevented glaucomatous neurodegeneration, supporting a damaging role for IOP-induced mTOR activation in perturbing metabolism and promoting glaucoma. Together, these findings support the use of treatments that limit metabolic disturbances and provide bioenergetic support. Such treatments provide a readily translatable strategy that warrants investigation in clinical trials.

Topics: Animals; Disease Models, Animal; Glaucoma; Glucose; Intraocular Pressure; Mice, Inbred C57BL; Mice, Inbred DBA; Nerve Degeneration; Neuroprotection; Neuroprotective Agents; Pyruvic Acid; Rats, Sprague-Dawley; Retina; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Differentiation; Disease Models, Animal; Female; Humans; Interferon-gamma; Interleukin-17; Lung; Lymphocyte Activation; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Neutrophils; Ovalbumin; Receptors, Antigen, T-Cell; Signal Transduction; Sirolimus; Th17 Cells

Human colon inflammation is associated with changes in the diverse and abundant microorganisms in the gut. As important beneficial microbes, Lactobacillus contributes to the immune responses and intestinal integrity that may alleviate experimental colitis. However, the mechanisms underlying probiotic benefits have not been fully elucidated.. Dextran sodium sulfate or rapamycin-challenged mice were used as model for colon inflammation evaluation. Histological scores of the colon, levels of colonic myeloperoxidase, serum tumor necrosis factor-α and interleukin-6 were assessed as inflammatory markers and the gut microbiota profiles of each mouse were studied.. We found that Lactobacillus casei Zhang (LCZ) can prevent experimental colitis and rapamycin-induced inflammation in intestinal mucosa by improving histological scores, decreasing host inflammatory cytokines, modulating gut-dominated bacteria, enhancing cystic fibrosis transmembrane conductance regulator (CFTR) expression and downregulating the expression of p-STAT3 (phosphorylated signal transducer and activator of transcription 3) or Akt/NF-κB (AKT serine/threonine kinase and nuclear factor kappa B).. Our results suggest that LCZ may provide effective prevention against colitis.

Topics: Animals; Anti-Bacterial Agents; Colitis; Dextran Sulfate; Disease Models, Animal; Lacticaseibacillus casei; Male; Mice; Mice, Inbred C57BL; Sirolimus

Topics: Animals; Antibiotics, Antineoplastic; Carcinogenesis; Disease Models, Animal; Gene Deletion; Mice; Neuroendocrine Tumors; Pancreatic Neoplasms; Proto-Oncogene Proteins; PTEN Phosphohydrolase; Sirolimus

Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution.. Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction.. Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir.. We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

Topics: Animals; Antifungal Agents; Bone Marrow Transplantation; Cytomegalovirus; Cytomegalovirus Infections; Disease Models, Animal; Graft Rejection; Immune Reconstitution; Immune Tolerance; Macaca fascicularis; Sirolimus; Transplant Recipients; Virus Activation

Traumatic optic neuropathy is an injury to the optic nerve that leads to vision loss. Autophagy is vital for cell survival and cell death in central nervous system injury, but the role of autophagy in traumatic optic nerve injury remains uncertain. Optic nerve crush is a robust model of traumatic optic nerve injury. p62 siRNA and rapamycin are autophagy inducers and have different neuroprotective effects in the central nervous system. In this study, p62 and rapamycin induced autophagy, but only p62 siRNA treatment provided a favorable protective effect in visual function and retinal ganglion cell (RGC) survival. Moreover, the number of macrophages at the optic nerve lesion site was lower in the p62-siRNA-treated group than in the other groups. p62 siRNA induced more M2 macrophage polarization than rapamycin did. Rapamycin inhibited both mTORC1 and mTORC2 activation, whereas p62 siRNA inhibited only mTORC1 activation and maintained mTORC2 and Akt activation. Inhibition of mTORC2-induced Akt activation resulted in blood-optic nerve barrier disruption. Combined treatment with rapamycin and the mTORC2 activator SC79 improved RGC survival. Overall, our findings suggest that mTORC2 activation after autophagy induction is necessary for the neuroprotection of RGCs in traumatic optic nerve injury and may lead to new clinical applications.

Topics: Animals; Autophagy; Disease Models, Animal; Male; Mechanistic Target of Rapamycin Complex 2; Neuroprotection; Neuroprotective Agents; Optic Nerve Injuries; Proto-Oncogene Proteins c-akt; Rats; Rats, Transgenic; Rats, Wistar; Retinal Ganglion Cells; RNA, Small Interfering; Sequestosome-1 Protein; Signal Transduction; Sirolimus

The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM).. Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated.. The IC. MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers; Cell Cycle; Cell Line, Tumor; Disease Models, Animal; Drug Interactions; Drug Resistance, Neoplasm; Gene Amplification; Humans; Mice; Molecular Structure; N-Myc Proto-Oncogene Protein; Neuroblastoma; Pyrroles; Sirolimus; Spectrum Analysis; Xenograft Model Antitumor Assays

To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel.. Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement.. Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.

Topics: Administration, Intravesical; Animals; Antineoplastic Agents; Autophagy; Cell Line, Tumor; Cell Proliferation; Colloids; Disease Models, Animal; Drug Liberation; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Hydrogels; Liposomes; Mice; Particle Size; Sirolimus; Temperature; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid-induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3-methyladenine (3-MA) and decreased by the autophagy activator rapamycin. In addition, pre-treatment with 3-MA increased acid-induced chondrocyte apoptosis, while pre-treatment with rapamycin reduced acid-induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.

Topics: Adenine; Animals; Apoptosis; Arthritis, Experimental; Autophagy; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; Male; Rats, Sprague-Dawley; Sirolimus

Topics: Animals; Autophagy; Brain; Cognitive Dysfunction; Disease Models, Animal; Male; Mitochondria; Mitochondrial Dynamics; Rats, Wistar; Rilmenidine; Rosiglitazone; Sepsis; Sirolimus; Survival Analysis; Up-Regulation

We recently observed reduced autophagy in Crohn's disease patients and an anti-inflammatory effect of autophagy stimulation in murine colitis, but both anti- and pro-fibrotic effects are associated with autophagy stimulation in different tissues, and fibrosis is a frequent complication of Crohn's disease. Thus, we analyzed the effects of pharmacological modulation of autophagy in a murine model of intestinal fibrosis and detected that autophagy inhibition aggravates, while autophagy stimulation prevents, fibrosis. These effects are associated with changes in inflammation and in collagen degradation in primary fibroblasts. Thus, pharmacological stimulation of autophagy may be useful against intestinal fibrosis.

Topics: Animals; Autophagy; Collagen; Crohn Disease; Disease Models, Animal; Fibroblasts; Fibrosis; Immunosuppressive Agents; Inflammation; Intestines; Mice; Mice, Inbred C57BL; Sirolimus

A brief episode of transient ischemia (TI) can confer cerebral ischemic tolerance against a subsequent severer TI under standard condition. The brain under obesity's conditions is more sensitive to ischemic injury. However, the impact of a brief episode of TI under obesity's conditions has not been fully addressed yet. Thus, the objective of this study was to determine the effect of a brief TI in the hippocampus of high-fat diet (HFD)-induced obese gerbils and related mechanisms. Gerbils were maintained on HFD or normal diet (ND) for 12 weeks and subjected to 2 min TI. HFD gerbils were heavier, with higher blood glucose, serum total cholesterol, triglycerides, and leptin levels. Massive loss of pyramidal neurons occurred in the hippocampal cornu ammonis 1 (CA1) field of HFD animals at 5 days after 2 min of TI, but 2 min of TI did not elicit death of pyramidal neurons in ND gerbils. The HFD group showed significantly increased levels of oxidative stress indicators (dihydroethidium and 4-hydroxynonenal) and proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) and microglial activation in pre- and/or post-ischemic phases compared to the ND group. Levels of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR in the CA1 field of the HFD group were also significantly higher than the ND group. On the other hand, inhibition of mTOR activation by rapamycin (an allosteric mTOR inhibitor) significantly attenuated neuronal death induced by HFD, showing reduction of HFD-induced increases of oxidative stress indicators and proinflammatory cytokines, and microglia activation. Taken together, a brief episode of TI can evoke neuronal death under obesity's conditions. It might be closely associated with an abnormal increase of mTOR activation-mediated, severe oxidative stress and neuroinflammation in pre- and/or post-ischemic phases.

Topics: Animals; Case-Control Studies; Cell Death; Diet, High-Fat; Disease Models, Animal; Gerbillinae; Hippocampus; Interleukin-1beta; Ischemic Attack, Transient; Male; Neurons; Obesity; Oxidative Stress; Phosphorylation; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Up-Regulation

Pre-ischemic hyperglycemia increases the occurrence of post-ischemic seizures both in experimental and clinical settings. The underlying mechanisms are not fully delineated; however, activation of mammalian target of rapamycin (mTOR) has been shown to be engaged in the pathogenesis of epilepsy, in which seizures are a regular occurrence. Therefore, we wanted to explore specifically the capacity of an mTOR inhibitor, rapamycin, in preventing post-ischemic seizures in hyperglycemic rats and to explore the underlying molecular mechanisms. The results showed that none of the rats in the sham control, EG ischemic, or within 3 h of I/R in hyperglycemic ischemic groups experienced seizures. Generalized tonic-clonic seizures were observed in all 8/8 of hyperglycemic ischemic rats at 16 h of I/R. Treatment with rapamycin successfully blocked post-ischemic seizures in 7/8 hyperglycemic ischemic animals. Rapamycin also lessened the neuronal death extraordinarily in hyperglycemic ischemic animals as revealed by histopathological studies. Protein analysis revealed that transient ischemia resulted in increases in p-mTOR and p-S6, especially in the hippocampi of the hyperglycemic ischemic rats. Rapamycin treatment completely blocked mTOR activation. Furthermore, hyperglycemic ischemia induced a much prominent rise of p-ERK1/2 both in the cortex and the hippocampi compared with EG counterparts; whereas rapamycin suppressed it. We conclude that the development of post-ischemic seizures in the hyperglycemic animals may be associated with activations of mTOR and ERK1/2 pathways and that rapamycin treatment inhibited the post-ischemic seizures effectively by suppressing the mTOR and ERK1/2 signaling.

Topics: Animals; Anticonvulsants; Brain Ischemia; Cell Death; Cerebral Cortex; Cytosol; Disease Models, Animal; Hippocampus; Hyperglycemia; Male; MAP Kinase Signaling System; Neurons; Phosphorylation; Rats; Rats, Wistar; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn's-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn's-associated intestinal fibrosis.

Topics: Animals; Colitis; Crohn Disease; Disease Models, Animal; Intestinal Mucosa; Intestines; Mice; Sirolimus; Trinitrobenzenesulfonic Acid

Topics: Animals; Cell Death; Disease Models, Animal; Female; Hippocampus; N-Methylaspartate; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Spasm; TOR Serine-Threonine Kinases; Wortmannin

Inhibition of mammalian target of rapamycin complex I (mTORC1) by rapamycin improves cardiac function in both aging and heart failure. While the protective mechanisms are not fully understood in mammals, they are presumably mediated through metabolic regulation and suppression of protein translation by reduced phosphorylation of 4EBP1, a target of mTORC1. Using transverse aortic constriction (TAC) and Gαq overexpression-induced heart failure models, we examined the effect of cardiac-specific heterozygous deletion (het) of Raptor, a component of mTORC1, and cardiac-specific transgenic overexpression of wild type or phosphorylation site mutant 4EBP1. In wild-type mice with TAC-induced heart failure, quantitative shotgun proteomics revealed decreased abundance of proteins of mitochondrial metabolism and increased abundance of proteins in oxidative stress response, ubiquitin, and other pathways. The Raptor het ameliorated both TAC- and Gαq overexpression-induced heart failure and the associated proteomic remodeling, especially those pathways involved in mitochondrial function, citric acid cycle, and ubiquitination. In contrast, transgenic overexpression of either wild type or mutant 4EBP1 aggravated TAC and Gαq, consistent with reduced adaptive hypertrophy by suppression of protein translation, in parallel with adverse remodeling of left ventricular proteomes. Partial mTORC1 inhibition by Raptor heterozygous deletion ameliorates heart failure and is associated with better preservation of the mitochondrial proteome; however, this effect does not appear to be mediated through suppression of protein translation by increased 4EBP1. Increased activity of 4EBP1 reduced adaptive hypertrophy and aggravated heart failure, suggesting that protein translation is essential for adaptive hypertrophy in pressure overload.

Topics: Animals; Blotting, Western; Disease Models, Animal; DNA; Gene Expression Regulation; Heart Failure; Immunosuppressive Agents; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Proteome; Signal Transduction; Sirolimus

MAGEL2 is a maternally imprinted, paternally expressed gene, located in the Prader-Willi region of human chromosome 15. Pathogenic variants in the paternal copy of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG), a neurodevelopmental disorder related to Prader-Willi syndrome (PWS). Patients with SHFYNG, like PWS, manifest neonatal hypotonia, feeding difficulties, hypogonadism, intellectual disability and sleep apnea. However, individuals with SHFYNG have joint contractures, greater cognitive impairment, and higher prevalence of autism than seen in PWS. Additionally, SHFYNG is associated with a lower prevalence of hyperphagia and obesity than PWS. Previous studies have shown that truncating variants in MAGEL2 lead to SHFYNG. However, the molecular pathways involved in manifestation of the SHFYNG disease phenotype are still unknown. Here we show that a Magel2 null mouse model and fibroblast cell lines from individuals with SHFYNG exhibit increased expression of mammalian target of rapamycin (mTOR) and decreased autophagy. Additionally, we show that SHFYNG induced pluripotent stem cell (iPSC)-derived neurons exhibit impaired dendrite formation. Alterations in SHFYNG patient fibroblast lines and iPSC-derived neurons are rescued by treatment with the mTOR inhibitor rapamycin. Collectively, our findings identify mTOR as a potential target for the development of pharmacological treatments for SHFYNG.

Topics: Animals; Autophagy; Dendrites; Disease Models, Animal; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Phenotype; Prader-Willi Syndrome; Proteins; RNA, Long Noncoding; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

The environmentally ubiquitous fungus

Topics: Animals; Antifungal Agents; Disease Models, Animal; Drug Resistance, Fungal; Epigenesis, Genetic; Humans; Male; Mice; Mice, Inbred BALB C; Mucor; Mucormycosis; RNA Interference; Sirolimus; Tacrolimus

Tumor microenvironments expose cancer cells to heterogeneous, dynamic environments by shifting availability of nutrients, growth factors, and metabolites. Cells integrate various inputs to generate cellular memory that determines trajectories of subsequent phenotypes. Here we report that short-term exposure of triple-negative breast cancer cells to growth factors or targeted inhibitors regulates subsequent tumor initiation. Using breast cancer cells with different driver mutations, we conditioned cells lines with various stimuli for 4 hours before implanting these cells as tumor xenografts and quantifying tumor progression by means of bioluminescence imaging. In the orthotopic model, conditioning a low number of cancer cells with fetal bovine serum led to enhancement of tumor-initiating potential, tumor volume, and liver metastases. Epidermal growth factor and the mTORC1 inhibitor ridaforolimus produced similar but relatively reduced effects on tumorigenic potential. These data show that a short-term stimulus increases tumorigenic phenotypes based on cellular memory. Conditioning regimens failed to alter proliferation or adhesion of cancer cells in vitro or kinase signaling through Akt and ERK measured by multiphoton microscopy in vivo, suggesting that other mechanisms enhanced tumorigenesis. Given the dynamic nature of the tumor environment and time-varying concentrations of small-molecule drugs, this work highlights how variable conditions in tumor environments shape tumor formation, metastasis, and response to therapy.

Topics: Animals; Carcinogenesis; Cell Adhesion; Cell Count; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Luminescent Measurements; Mechanistic Target of Rapamycin Complex 1; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Serum Albumin, Bovine; Sirolimus; Triple Negative Breast Neoplasms; Tumor Microenvironment

Type 2 diabetes mellitus (T2DM) is strongly correlated with Alzheimer's disease (AD). Rapamycin has important uses in oncology, cardiology and transplantation medicine. This study aims to investigate effects of rapamycin on AD in hippocampus of T2DM rat by AMPK/mTOR signaling pathway.. Morris water maze test was applied to evaluate the learning and memory abilities. The fasting plasma glucose (FBG), glycosylated haemoglobin, total cholesterol, triglyceride and serum insulin level were measured. RT-qPCR and Western blot analysis were performed to test expression of AMPK and mTOR. Immunohistochemistry was used to detect the Aβ deposition and immunoblotting to test the total tau, p-tau and Aβ precursor APP expressions.. After treated with rapamycin, T2DM rats and rats with T2DM and AD showed increased learning-memory ability, and decreased levels of FBG, glycosylated hemoglobin, total cholesterol, triglyceride and serum insulin, decreased expression of APP and p-tau, increased AMPK mRNA expression and p-AMPK and decreased Aβ deposition, mTOR mRNA expression and p-mTOR.. The study demonstrated that rapamycin reduces the risk of AD in T2DM rats and inhibits activation of AMPK-mTOR signaling pathway, thereby improving AD lesion in hippocampus of T2DM rats.

Topics: Alzheimer Disease; AMP-Activated Protein Kinases; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Hippocampus; Male; Maze Learning; Phosphorylation; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; tau Proteins; TOR Serine-Threonine Kinases

Topics: Administration, Ophthalmic; Animals; Burns, Chemical; Cell Proliferation; Cells, Cultured; Cornea; Corneal Injuries; Corneal Neovascularization; Corneal Opacity; Disease Models, Animal; Drug Carriers; Drug Compounding; Eye Burns; Fibroblasts; Fibrosis; Humans; Lipids; Male; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Sirolimus; Sodium Hydroxide; Wound Healing

The purpose of this study was to investigate whether rapamycin inhibits the development of thoracic aortic aneurysm and dissection (TAAD) in mice.. Three-week-old C57BL/6J male mice were fed a normal diet and randomized into a control group (n = 6), β-aminopropionitrile fumarate (BAPN) group (Gp A; n = 15), BAPN plus rapamycin (5 mg) group (Gp B; n = 8), and BAPN plus rapamycin (10 mg) group (Gp C; n = 8). Gp A, Gp B, and Gp C were administered BAPN (1 g/kg/d) for 4 weeks. One week after BAPN administration, Gp B and Gp C were treated with rapamycin (5 mg/kg/d or 10 mg/kg/d) through gavage for 21 days. Thoracic aortas were harvested for Western blot and immunofluorescence staining at day 14 and for morphologic and histologic analyses at day 28.. BAPN treatment induced TAAD formation in mice. The incidence of TAAD in control, Gp A, Gp B, and Gp C mice was 0%, 80%, 25%, and 37.5%, respectively. Smaller thoracic aortic diameters (ascending aorta and arch) were observed in Gp B and Gp C mice than in Gp A mice (Gp B vs Gp A: ascending aorta, ex vivo, 1.07 ± 0.21 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.51 ± 0.40 mm vs 2.70 ± 1.06 mm [P < .05]; Gp C vs Gp A: ascending aortas, ex vivo, 1.10 ± 0.33 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.55 ± 0.56 mm vs 2.70 ± 1.06 mm [P < .05]). TAAD mice exhibited elastin fragmentation, abundant inflammatory cell infiltration, and significantly increased matrix metalloproteinase production in the aorta, and rapamycin treatment alleviated these changes. The protein levels of p-S6K and p-S6 in TAAD aortic tissues increased significantly, whereas they were suppressed by rapamycin.. Rapamycin suppressed TAAD formation, probably by inhibition of mechanistic target of rapamycin signaling and reduction of inflammatory cell infiltration and matrix metalloproteinase 9 production. Targeting of the mechanistic target of rapamycin signaling pathway using rapamycin may be a favorable modulation for the clinical treatment of TAAD.

Topics: Aminopropionitrile; Animals; Anti-Inflammatory Agents; Aorta, Thoracic; Aortic Aneurysm, Thoracic; Aortic Dissection; Dilatation, Pathologic; Disease Models, Animal; Male; Matrix Metalloproteinase 9; Mice, Inbred C57BL; Phosphorylation; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Remodeling

Topics: Animals; Conjunctiva; Delayed-Action Preparations; Disease Models, Animal; Dry Eye Syndromes; Female; Immunosuppressive Agents; Mice; Microspheres; Sirolimus; Sjogren's Syndrome

Multiple sclerosis (MS) is a highly disabling demyelinating disease, which mainly affects young adults and is difficult to cure. Activated microglia may be involved in the process of neuronal cell damage and release inflammatory cytokines to injure neurons. Rapamycin (RAPA), an immunosuppressant, can induce autophagy in microglia to delay the process of the disease. As an inhibitor of NLRP3, MCC950 (CP-456773) can regulate the activation of inflammasome. An experimental autoimmune encephalomyelitis model, a disease model of MS, was established to detect the role of activated microglia in the dynamic evolution of MS. Our research showed that RAPA and MCC950 could reduce both the clinical symptom and the release of cytokines in immune cells. MCC950 reduced interleukin-1β (IL-1β) production in vivo and enhanced the effect of RAPA. We hypothesized that inflammation and demyelination in the central nervous system can be reduced by inhibiting the immune response mediated by microglia. This study provides theoretical support to the therapeutic evaluation of RAPA and MCC950 to make the mammalian targets of RAPA and NLRP3 the therapeutic targets of MS.

Topics: Animals; Brain; CD4 Antigens; Disease Models, Animal; Drug Therapy, Combination; Encephalomyelitis, Autoimmune, Experimental; Female; Furans; Glial Fibrillary Acidic Protein; Heterocyclic Compounds, 4 or More Rings; Indenes; Interleukin-1beta; Mice, Inbred C57BL; Microtubule-Associated Proteins; Multiple Sclerosis; Sirolimus; Sulfonamides; Sulfones

Heart failure (HF) progression can be prevented by an inhibitor of the mammalian target of rapamycin and an autophagy enhancer rapamycin. This current study aimed to investigate the effect of rapamycin on HF progression and myocardial cells apoptosis.. HF rats were injected with low-, middle-, and high-dose rapamycin. Echocardiography, hematoxylin-eosin staining, plasma brain natriuretic peptide, myocardial cells apoptosis, and Akt activation in rapamycin-treated rats were detected.. HF rats showed reduced cardiac functions, destructive pathological changes in the myocardium, enhanced Akt activation and myocardial cells apoptosis. However, rapamycin reversed all the changes in a dose-dependent manner. Cardiac functions were enhanced by rapamycin. Myocardial cells apoptotic percentage, Akt expression, and pathological changes of the myocardium in HF rats were inhibited by rapamycin administration.. Rapamycin protected against myocardial hypertrophy and myocardial cells apoptosis in HF rats in a dose-dependent manner.

Topics: Animals; Apoptosis; Autophagy; Disease Models, Animal; Dose-Response Relationship, Drug; Echocardiography; Gene Expression Regulation; Heart; Heart Failure; Humans; Myocardium; Natriuretic Peptide, Brain; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus

Paraquat (PQ) intoxication is frequently associated with a high mortality rate. No specific treatment has been shown to reduce mortality in victims within the first 72 hours. We investigated the protective effects of rapamycin (Rapa) against PQ-induced toxicity in a zebrafish model.. To determine the maximum nonlethal concentration (MNLC) and lethal concentration 50 (LC50) of Rapa, zebrafish were treated at 2-5 days post fertilisation (dpf) and their mortality was recorded every 24 hours. At 5 dpf, the zebrafish were treated with PQ 100 µg/mL or PQ+Rapa (MNLC, 1/3 MNLC or 1/9 MNLC) for 72 hours, and the rate of survival was recorded every 24 hours. Reverse transcription-polymerase chain reaction was used to test the signalling pathway of mTOR (mammalian target of rapamycin).. MNLC and LC50 of Rapa were determined to be 6.7 µg/mL and 28.9 µg/mL, respectively. At 48 hours, the PQ+Rapa groups had much lower mortality than the PQ group. The rates of survival of the PQ+Rapa groups were 43.33% (MNLC), 53.89% (1/3 MNLC) and 44.45% (1/9 MLNC), as compared to 19.45% in the PQ group, with the 1/3 MNLC group showing the highest rate of survival (p < 0.001). atg1 was slightly activated in the PQ group. In the PQ+Rapa groups, the expression of atg1 was markedly increased, suggesting strengthening of the autophagy process.. Rapa can increase the rate of survival of PQ-intoxicated zebrafish by inhibiting mTOR complex 1 and activating autophagy. Rapa could be an alternative first-line drug in the treatment of PQ poisoning.

Topics: Acute Disease; Animals; Disease Models, Animal; Paraquat; Sirolimus; Survival Rate; Zebrafish

The mammalian target of rapamycin (mTOR) signaling pathway has newly been recommended to be a nutrient sensor in the placenta. It is speculated that mTORC1 may be activated in diabetes, associated with increased placental nutrient availability. Thus, we aimed to investigate the mTOR signaling pathway both in diabetic and non-diabetic placenta and searched for the alterations of angiogenic factors VEGF, VEGFR1 and VEGFR2.. Streptozotocin (STZ) was administered by intravenous injection in doses of 60 mg/kg body weight and STZ injected rats were exposed to Everolimus (Rapamycin analog) and sacrificed at gestational days 14 and 20. mTORC1 and mTORC2 target proteins and angiogenic factors were analyzed at protein and mRNA levels in the placenta. Soluble VEGF A and İnsulin protein levels were determined in blood serum.. Placenta and embryo weights were altered after STZ and/or Rapamycin administration. mTOR pathway inhibition was confirmed by decreased p70S6K (Thr389) phosphorylation levels. We found that maternal diabetic environment led to an increase in Akt phosphorylation at 14th and decrease at 20th gestational days. Serum levels of Insulin in 14 th and 20 th days of gestation were decreased in Rapamycin and diabetic groups. On the other side serum levels of Soluble VEGF were increased in 14 th and decreased in 20 th days of pregnancy.. According to our results, it might be suggested that angiogenesis related proteins will be related with placental growth regulation and mTOR may be a candidate pathway mediating the process in normal and diabetic pregnancy.

Topics: Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Female; Immunosuppressive Agents; Neovascularization, Physiologic; Placenta; Pregnancy; Pregnancy Complications; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Topics: Achondroplasia; Animals; Biomarkers; Cartilage Oligomeric Matrix Protein; Cell Line, Tumor; Chondrocytes; Disease Models, Animal; Drug Delivery Systems; Endoplasmic Reticulum Stress; Endoplasmic Reticulum, Rough; Eosinophil Peroxidase; Humans; Inflammation Mediators; Interleukin-16; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Mutation; Protein Phosphatase 2; Proteins; Rats; Signal Transduction; Sirolimus; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases

Vein graft restenosis has an adverse impact on bridge vessel circulation and patient prognosis after coronary artery bypass grafting.. We used the extravascular supporter α-cyanoacrylate (α-CA), the local application rapamycin/sirolimus (RPM), and a combination of the two (α-CA-RPM) in rat models of autogenous vein graft to stimulate vein graft change. The aim of our study was to observe the effect of α-CA, RPM, and α-CA-RPM on vein hyperplasia.. Fifty healthy Sprague Dawley (SD) rats were randomized into the following 5 groups: sham, control, α-CA, RPM, and α-CA-RPM. Operating procedure as subsequently described was used to build models of grafted rat jugular vein on carotid artery on one side. The level of endothelin-1 (ET-1) was determined by enzyme-linked immunosorbent assay (ELISA). Grafted veins were observed via naked eye 4 weeks later; fresh veins were observed via microscope and image-processing software in hematoxylin-eosin (HE) staining and immunohistochemistry after having been fixed and stored" (i.e. First they were fixed and stored, and second they were observed); α-Smooth Muscle Actin (αSMA) and von Willebrand factor (vWF) were measured with reverse transcription-polymerase chain reaction (RT-PCR). Comparisons were made with single-factor analysis of variance and Fisher's least significant difference test, with p < 0.05 considered significant.. We found that intimal thickness of the α-CA, RPM, and α-CA-RPM groups was lower than that of the control group (p < 0.01), and the thickness of the α-CA-RPM group was notably lower than that of the α-CA and RPM groups (p < 0.05).. RPM combined with α-CA contributes to inhibiting intimal hyperplasia in rat models and is more effective for vascular patency than individual use of either α-CA or RPM.

Topics: Actins; Animals; Carotid Arteries; Cell Proliferation; Coronary Artery Bypass; Cyanoacrylates; Disease Models, Animal; Drug Combinations; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Graft Occlusion, Vascular; Hyperplasia; Jugular Veins; Male; Random Allocation; Rats, Sprague-Dawley; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Time Factors; Treatment Outcome; Tunica Intima

The neuroprotective effect of autophagy activation by rapamycin and trehalose was studied in a mouse model of Parkinson's disease (PD) induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both rapamycin (10 mg/kg/day, 7 days) and trehalose (2% in drinking water, 7 days) increased the expression of LC3-II (a marker of autophagy activation) in the frontal cortex and striatum of normal C57Bl/6J mice, with signs of an additive effect. Autophagy stimulation in the striatum was confirmed by a lysosomal osmotic test. In the model of MPTP-induced PD, the two drugs were applied starting from the 2nd day after subchronic daily MPTP administration (20 mg/kg/day, 4 days). A marked increase in LC3-II expression in the striatum was detected under the action of trehalose and in the S. nigra after combined treatment with rapamycin and trehalose. The drugs had a positive effect for recovery of dopaminergic neurons and neuroprotection after MPTP-induced PD-like injury. The therapeutic effect was proven by active restoration of tyrosine hydroxylase (TH) content in the striatum and S. nigra and by improved cognition measured by the passive avoidance learning task. The results revealed the additive effect of the combined treatment with rapamycin and trehalose on dopaminergic deficits (according to the levels of TH expression in the nigrostriatal system) but not on the behavioral performance in the mouse PD model. Thus, the autophagy activation through different pathways by the combination of rapamycin and trehalose reverses both neuronal dopaminergic and behavioral deficits in vivo and seems to be a promising therapy for PD-like pathology.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Autophagy; Behavior, Animal; Cognition; Corpus Striatum; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Drug Therapy, Combination; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; MPTP Poisoning; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Sirolimus; Substantia Nigra; Trehalose; Tyrosine 3-Monooxygenase

mTOR (mammalian target of rapamycin) signaling has emerged as a key regulator in a wide range of cellular processes ranging from cell proliferation, immune responses, and electrolyte homeostasis. mTOR consists of 2 distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) with distinct downstream signaling events. mTORC1 has been implicated in pathological conditions, such as cancer and type 2 diabetes mellitus in humans, and inhibition of this pathway with rapamycin has been shown to attenuate salt-induced hypertension in Dahl salt-sensitive rats. Several studies have found that the mTORC2 pathway is involved in the regulation of renal tubular sodium and potassium transport, but its role in hypertension has remained largely unexplored. In the present study, we, therefore, determined the effect of mTORC2 inhibition with compound PP242 on salt-induced hypertension and renal injury in salt-sensitive rats. We found that PP242 not only completely prevented but also reversed salt-induced hypertension and kidney injury in salt-sensitive rats. PP242 exhibited potent natriuretic actions, and chronic administration tended to produce a negative Na

Topics: Acute Kidney Injury; Animals; Blood Pressure; Cell Proliferation; Disease Models, Animal; Hypertension; Immunosuppressive Agents; Male; Rats; Rats, Inbred Dahl; Signal Transduction; Sirolimus; Sodium Chloride, Dietary; TOR Serine-Threonine Kinases

Osteogenesis imperfecta (OI) is commonly caused by heterozygous type I collagen structural mutations that disturb triple helix folding and integrity. This mutant-containing misfolded collagen accumulates in the endoplasmic reticulum (ER) and induces a form of ER stress associated with negative effects on osteoblast differentiation and maturation. Therapeutic induction of autophagy to degrade the mutant collagens could therefore be useful in ameliorating the ER stress and deleterious downstream consequences. To test this, we treated a mouse model of mild to moderate OI (α2(I) G610C) with dietary rapamycin from 3 to 8 weeks of age and effects on bone mass and mechanical properties were determined. OI bone mass and mechanics were, as previously reported, compromised compared to WT. While rapamycin treatment improved the trabecular parameters of WT and OI bones, the biomechanical deficits of OI bones were not rescued. Importantly, we show that rapamycin treatment suppressed the longitudinal and transverse growth of OI, but not WT, long bones. Our work demonstrates that dietary rapamycin offers no clinical benefit in this OI model and furthermore, the impact of rapamycin on OI bone growth could exacerbate the clinical consequences during periods of active bone growth in patients with OI caused by collagen misfolding mutations.

Topics: Animals; Apoptosis; Bone Density; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Female; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Osteoblasts; Osteogenesis; Osteogenesis Imperfecta; Sirolimus

Demyelination occurs following many neurological insults, most notably in multiple sclerosis (MS). Therapeutics that promote remyelination could slow the neurological decline associated with chronic demyelination; however, in vivo testing of candidate small molecule drugs and signaling cascades known to impact myelination is expensive and labor intensive. Here, we describe the development of a novel zebrafish line which uses the putative promoter of Myelin Protein Zero (mpz), a major structural protein in myelin, to drive expression of Enhanced Green Fluorescent Protein (mEGFP) specifically in the processes and nascent internodes of myelinating glia. We observe that changes in fluorescence intensity in Tg(mpz:mEGFP) larvae are a reliable surrogate for changes in myelin membrane production per se in live larvae following bath application of drugs. These changes in fluorescence are strongly predictive of changes in myelin-specific mRNAs [mpz, 36K and myelin basic protein (mbp)] and protein production (Mbp). Finally, we observe that certain drugs alter nascent internode number and length, impacting the overall amount of myelin membrane synthesized and a number of axons myelinated without significantly changing the number of myelinating oligodendrocytes. These studies demonstrate that the Tg(mpz:mEGFP) reporter line responds effectively to positive and negative small molecule regulators of myelination, and could be useful for identifying candidate drugs that specifically target myelin membrane production in vivo. Combined with high throughput cell-based screening of large chemical libraries and automated imaging systems, this transgenic line is useful for rapid large scale whole animal screening to identify novel myelinating small molecule compounds in vivo.

Topics: Animals; Animals, Genetically Modified; Culture Media, Conditioned; Demyelinating Diseases; Disease Models, Animal; Embryo, Nonmammalian; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Immunosuppressive Agents; Larva; Luminescent Proteins; Myelin Basic Protein; Myelin P0 Protein; Myelin Sheath; Neuroglia; Oligodendroglia; Red Fluorescent Protein; Sirolimus; SOXE Transcription Factors; Spinal Cord; Zebrafish; Zebrafish Proteins

Stroke is one of the leading causes of death in the world, but the underlying molecular mechanism of this disease remains elusive, thus it will be great challenges to finding appropriate protection. MicroRNAs are short, single-stranded, non-coding RNAs and recent studies have shown that they are aberrantly expressed in ischemic condition. Due to the fact that miR-1 has harmful effects on neural damages during brain ischemia, limited miR-1 has been proven to be protective in middle cerebral artery occlusion (MCAO). Here, the possible positive effect of intravenous injection of antagomiR-1 as a post-ischemic treatment on neurological deficits, infarct volume, brain edema and blood-brain barrier (BBB) permeability was evaluated. The rats were divided randomly into three experimental groups, each with 21 animals. MCAO surgery was performed on all groups and one hour later, 0.1 ml normal saline, 0.1 ml rapamycin and 300 pmol/g miR-1 antagomir (soluble in 0.1 ml normal saline), were injected intravenously into control, positive control and treatment group, respectively. After 24 h, neurologic deficits score, infarct volume, brain edema and BBB permeability were measured. The results indicated that post-treatment with miR-1 antagomir significantly improved neurological deficits and reduced infarction volume, brain edema, and BBB permeability. These data proved that there is a positive effects of antagomiR-1 on ischemic neuronal injury and neurological impairment. Due to the fact that microRNAs are able to protect the brain, it would be a promising therapeutic approach to stroke treatment.

Topics: Administration, Intravenous; Animals; Antagomirs; Blood-Brain Barrier; Brain; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Male; MicroRNAs; Neurons; Rats; Rats, Wistar; Sirolimus; Stroke

Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-β-catenin signaling. Activating β-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between β-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were β-catenin-mutated liver tumors. Genetic disruption of β-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in β-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-β-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are β-catenin mutated and GS positive.

Topics: Acetates; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Child; Child, Preschool; Disease Models, Animal; Female; Glutamate-Ammonia Ligase; Glutamine; Hepatocytes; Humans; Infant; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Phenols; Retrospective Studies; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Wnt Signaling Pathway

Glial cell line-derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid β-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid β-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid-induced hepatic lipotoxicity.

Topics: Animals; Autophagy; Cell Death; Diet, High-Fat; Disease Models, Animal; Female; Glial Cell Line-Derived Neurotrophic Factor; Hep G2 Cells; Hepatocytes; Humans; Lipogenesis; Lipolysis; Male; Mice; Mice, Transgenic; Non-alcoholic Fatty Liver Disease; Oxygen Consumption; Palmitates; Random Allocation; Rats; Sensitivity and Specificity; Signal Transduction; Sirolimus

Identifying regulatory mechanisms that influence inflammation in metabolic tissues is critical for developing novel metabolic disease treatments. Here, we investigated the role of microRNA-146a (miR-146a) during diet-induced obesity in mice. miR-146a is reduced in obese and type 2 diabetic patients and our results reveal that miR-146a-/- mice fed a high-fat diet (HFD) have exaggerated weight gain, increased adiposity, hepatosteatosis, and dysregulated blood glucose levels compared to wild-type controls. Pro-inflammatory genes and NF-κB activation increase in miR-146a-/- mice, indicating a role for this miRNA in regulating inflammatory pathways. RNA-sequencing of adipose tissue macrophages demonstrated a role for miR-146a in regulating both inflammation and cellular metabolism, including the mTOR pathway, during obesity. Further, we demonstrate that miR-146a regulates inflammation, cellular respiration and glycolysis in macrophages through a mechanism involving its direct target Traf6. Finally, we found that administration of rapamycin, an inhibitor of mTOR, was able to rescue the obesity phenotype in miR-146a-/- mice. Altogether, our study provides evidence that miR-146a represses inflammation and diet-induced obesity and regulates metabolic processes at the cellular and organismal levels, demonstrating how the combination of diet and miRNA genetics influences obesity and diabetic phenotypes.

Topics: Animals; Blood Glucose; Diet, High-Fat; Disease Models, Animal; Female; Gene Expression; Humans; Hyperglycemia; Inflammation; Insulin; Intra-Abdominal Fat; Macrophages; Male; Metabolic Diseases; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; NF-kappa B; Obesity; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Weight Gain

There are few treatments for patients with recurrent pregnancy loss (RPL) or recurrent implantation failure (RIF). Women with RPL and unexplained infertility have lower T regulatory cell (T

Topics: Animals; Birth Rate; Disease Models, Animal; Embryo Implantation; Female; Immunosuppressive Agents; Infertility, Female; Live Birth; Lymphocyte Depletion; Mice; Sirolimus; T-Lymphocytes, Regulatory

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a highly active mTOR pathway, which makes mTOR a potential therapeutic target. MLN0128 is an ATP-competitive inhibitor of mTOR that has entered clinical trials for solid tumors. Our results demonstrated that MLN0128 has a greater effect on inhibiting proliferation than the allosteric mTOR inhibitor rapamycin. MLN0128 has ∼30 nM 50% inhibitory concentration (IC

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Benzoxazoles; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Heterografts; Humans; Inhibitory Concentration 50; Lymphoma, Primary Effusion; Mice; Neoplasm Transplantation; Pyrimidines; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Huangkui capsule (HKC), an anti-inflammatory Chinese modern patent medicine, has been now widely applied to the clinical therapy of diabetic nephropathy (DN). However, the overall therapeutic mechanisms in vivo are still unclear. Renal tubular epithelial-to-mesenchymal transition (EMT) is one of the major pathogenesis of renal interstitial fibrosis in DN. Recently, the physiological roles of NLRP3 inflammasome activation and toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling are closely linked to EMT. But, it remains elusive whether HKC regulates renal tubular EMT in vivo through targeting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys.. This study thereby aimed to clarify the therapeutic effects of HKC on renal tubular EMT in DN and its underlying mechanisms in vivo, compared to rapamycin (RAP).. Thirty-two rats were randomly divided into 4 groups: the Sham group, the Vehicle group, the HKC group and the RAP group. The early DN rat models were induced by unilateral nephrectomy combined with intraperitoneal injection of streptozotocin, and administered with HKC suspension or RAP suspension or vehicle after modeling for 4 weeks. Changes in the incipient renal lesions-related parameters in urine and blood were analyzed, respectively. Renal interstitial tissues were isolated for histomorphometry, immunohistochemistry and Western blotting at sacrifice.. For the early DN rat models, HKC at the suitable dose of 2 g/kg/day ameliorated the general condition and biochemical parameters partially including kidney weight (KW), urinary albumin (UAlb), serum creatinine (Scr) and serum albumin (Alb), attenuated renal tubular EMT significantly and inhibited the activation of NLRP3 inflammasome in the kidneys obviously, which was superior to RAP generally. In addition to these, HKC also suppressed TLR4/NF-κB signaling in the kidneys of the DN model rats accurately, which was different from RAP specifically.. The results of this study further indicated that HKC, different from RAP, can alleviate renal tubular EMT in the DN model rats, likely by inhibiting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys. Our findings thus provide the more accurate information in vivo about a clinical value of HKC, a traditional anti-inflammatory phytomedicine, in the treatment of the early DN patients.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Fibrosis; Inflammasomes; Kidney; Male; Nephrectomy; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Toll-Like Receptor 4

As an inhibitor of the immune system and a longevity drug, rapamycin has been suggested as a treatment for Alzheimer's disease, although the underlying mechanisms remain to be clarified. To elucidate the mechanisms, we performed a high-throughput quantitative proteomics analysis and bioinformatics analysis of the changes in the proteome profiles of hippocampus and temporal lobe of wild-type mice, APP/PS1 mice and rapamycin-treated APP/PS1 mice (ProteomeXchange: PXD009540). Morris Water Maze tests were used to evaluate the effectiveness of rapamycin in APP/PS1 treatment and Western blot analysis was used to verify the proteomics data. The results of Morris Water Maze tests indicated that rapamycin improved the spatial learning and memory abilities of APP/PS1 mice. Proteome analysis identified 100 significantly changed (SC) proteins in hippocampus and 260 in temporal lobe in APP/PS1 mice. Among these, 57 proteins in hippocampus and 167 proteins in temporal lobe were rescued by rapamycin. STRING analysis indicated relatively more complicated protein interactions of AD-related rapamycin rescued proteins in temporal lobe. Pathway analysis showed that SC proteins in APP/PS1 mice were mainly enriched in cholesterol biosynthesis pathway and cytoplasmic ribosomal proteins. After rapamycin treatment, the expression of most proteins in these signaling pathways were reversed. Overall, our findings demonstrate that rapamycin may be an potential strategy which can effectively delays the progression of AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Cholesterol; Disease Models, Animal; Hippocampus; Immunosuppressive Agents; Maze Learning; Mice; Mice, Transgenic; Presenilin-1; Ribosomal Proteins; Signal Transduction; Sirolimus; Temporal Lobe

Autism spectrum disorders (ASD) form a heterogeneous, neurodevelopmental syndrome characterized by deficits in social interactions and repetitive behavior/restricted interests. Dysregulation of mTOR signaling has been implicated in the pathogenesis of certain types of ASD, and inhibition of mTOR by rapamycin has been demonstrated to be an effective therapeutics for impaired social interaction in Tsc1+/-, Tsc2+/-, Pten-/- mice and valproic acid-induced ASD animal models. However, it is still unknown if dysregulation of mTOR signaling is responsible for the ASD-related deficit caused by other genes mutations. Contactin associated protein-like 2 (CNTNAP2) is the first widely replicated autism-predisposition gene. Mice deficient in Cntnap2 (Cntnap2-/- mice) show core ASD-like phenotypes, and have been demonstrated as a validated model for ASD-relevant drug discovery. In this study, we found hyperactive Akt-mTOR signaling in the hippocampus of Cntnap2-/- mice with RNA sequencing followed with biochemical analysis. Treatment with Akt inhibitor LY294002 or mTOR inhibitor rapamycin rescued the social deficit, but had no effect on hyperactivity and repetitive behavior/restricted behavior in Cntnap2-/- mice. We further showed that the effect of LY294002 and rapamycin on social behaviors is reversible. Our results thus identified hyperactive Akt-mTOR signaling pathway as a therapeutic target for abnormal social behavior in patients with dysfunction of CNTNAP2.

Topics: Animals; Autism Spectrum Disorder; Chromones; Disease Models, Animal; Hippocampus; Humans; Hyperkinesis; Male; Membrane Proteins; Mice; Mice, Knockout; Morpholines; Nerve Tissue Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Social Behavior; Stereotyped Behavior; TOR Serine-Threonine Kinases

Animal experiment: a rat model of lumbar disc herniation (LDH) induced painful radiculopathies.. To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in dorsal root ganglia (DRG) neurons in LDH-induced painful radiculopathies.. Overactivation of multiple pain signals in DRG neurons triggered by LDH is crucial to the development of radicular pain. AMPK is recognized as a cellular energy sensor, as well as a pain sensation modulator, but its function in LDH-induced pain hypersensitivity remains largely unknown.. The LDH rat model was established by autologous nucleus pulposus transplantation into the right lumbar 5 (L5) nerve root. At different time points after AMPK agonist metformin (250 mg/kg/d) or mammalian target of rapamycin (mTOR) inhibitor rapamycin (5 mg/kg) intraperitoneal administration, thermal and mechanical sensitivity were evaluated by measuring paw withdrawal latency (PWL) and 50% paw withdrawal thresholds (PWT). The levels of AMPK, mTOR, and p70S6K phosphorylation were determined by Western blot. We also investigated the proportion of p-AMPK positive neurons in the right L5 DRG neurons using immunofluorescence.. LDH evoked persistent thermal hyperalgesia and mechanical allodynia on the ipsilateral paw, as indicated by the decreased PWL and 50% PWT. These pain hypersensitive behaviors were accompanied with significant inhibition of AMPK and activation of mTOR in the associated DRG neurons. Pharmacological activation of AMPK in the DRG neurons not only suppressed mTOR/p70S6K signaling, but also alleviated LDH-induced pain hypersensitive behaviors.. We provide a molecular mechanism for the activation of pain signals based on AMPK-mTOR axis, as well as an intervention strategy by targeting AMPK-mTOR axis in LDH-induced painful radiculopathies.. N/A.

Topics: AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Male; Metformin; Neurons; Nucleus Pulposus; Pain; Phosphorylation; Radiculopathy; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Spinal Nerve Roots; TOR Serine-Threonine Kinases

Cartilage endplate (CEP) calcification inhibits the transport of metabolites and nutrients in the intervertebral disk and is an important initiating factor of intervertebral disk degeneration. However, the mechanisms governing CEP degeneration have not been thoroughly elucidated. In this study, we established a mouse CEP degeneration model and showed that autophagy insufficiency caused the degeneration of CEP. We found that the inflammatory cytokine tumor necrosis factor-α (TNF-α) increased the level of intracellular reactive oxygen species (ROS) and caused cell senescence and osteogenic differentiation of cartilage endplate stem cells (CESCs), whereas rapamycin-induced autophagy protected CESCs from TNF-α-induced oxidative stress and cell senescence. Furthermore, rapamycin-induced autophagy helped CESCs maintain the chondrogenic properties and inhibited extracellular matrix protease expression and osteogenic differentiation. Further study revealed that autophagy activated by rapamycin or inhibited by chloroquine influenced the expression and nuclear translocation of Nrf2, thereby controlling the expression of antioxidant proteins and the scavenging of ROS. Taken together, the results indicate that rapamycin-induced autophagy enhances Nrf2/Keap1 signaling and promotes the expression of antioxidant proteins, thereby eliminating ROS, alleviating cell senescence, reducing the osteogenic differentiation of CESCs, and ultimately protecting CEPs from chronic inflammation-induced degeneration. Stem Cells 2019;37:828-840.

Topics: Animals; Autophagy; Cartilage; Cell Differentiation; Chloroquine; Chondrogenesis; Disease Models, Animal; Female; Gene Expression Regulation; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Kelch-Like ECH-Associated Protein 1; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Osteogenesis; Reactive Oxygen Species; Signal Transduction; Sirolimus; Stem Cells; Tumor Necrosis Factor-alpha

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.

Topics: Administration, Intranasal; Animals; Autophagy; Biomarkers; Disease Models, Animal; Down Syndrome; Female; Male; Mice; Oxidative Stress; Proteasome Endopeptidase Complex; Proteomics; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Ischemia-reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Numerous therapeutic approaches for I/R injury have been studied, including autophagy, particularly in animal models of renal I/R injury derived from young or adult animals. However, the precise role of autophagy in renal ischemia-reperfusion in the aged animal model remains unclear. The purpose of this study was to demonstrate whether autophagy has similar effects on renal I/R injury in young and aged rats.. All rats were divided into two age groups (3 months and 24 months) with each group being further divided into four subgroups (sham, I/R, I/R+Rap (rapamycin, an activator of autophagy), I/R+3-MA (3-methyladenine, an inhibitor of autophagy)). The I/R+Rap and I/R+3-MA groups were intraperitoneally injected with rapamycin and 3-MA prior to ischemia. We then measured serum levels of urea nitrogen, creatinine and assessed damage in the renal tissue. Immunohistochemistry was used to assess LC3-II and caspase-3, and Western blotting was used to evaluate the autophagy-related proteins LC3-II, Beclin-1 and P62. Apoptosis and autophagosomes were evaluated by TUNEL and transmission electron microscopy, respectively.. Autophagy was activated in both young and aged rats by I/R and enhanced by rapamycin, although the level of autophagy was lower in the aged groups. In young rats, the activation of autophagy markedly improved renal function, reduced apoptosis in the renal tubular epithelial cells and the injury score in the renal tissue, thereby exerting protective effects on renal I/R injury. However, this level of protection was not present in aged rats.. Our data indicated that the activation of autophagy was ineffective in aged rat kidneys. These discoveries may have major implications in that severe apoptosis in aged kidneys might be refractory to antiapoptotic effect induced by the activation of autophagy.

Topics: Acute Kidney Injury; Adenine; Age Factors; Animals; Apoptosis; Autophagosomes; Autophagy; Beclin-1; Blood Urea Nitrogen; Caspase 3; Creatinine; Disease Models, Animal; Kidney; Male; Microtubule-Associated Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis Inducing Factor; Autophagy; Beclin-1; Blotting, Western; Caspase Inhibitors; Cytoprotection; Disease Models, Animal; Drug Combinations; Immunosuppressive Agents; Injections, Intraocular; Male; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Necroptosis; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Retina; Retinal Detachment; Sirolimus

The vast majority of mitochondrial disorders have limited the clinical management to palliative care. Rapamycin has emerged as a potential therapeutic drug for mitochondrial diseases since it has shown therapeutic benefits in a few mouse models of mitochondrial disorders. However, the underlying therapeutic mechanism is unclear, the minimal effective dose needs to be defined and whether this therapy can be generally used is unknown.. We have evaluated whether low and high doses of rapamycin administration may result in therapeutic effects in a mouse model (Coq9. Low dose of rapamycin induces metabolic changes in liver and transcriptomics modifications in midbrain. The high dose of rapamycin induces further changes in the transcriptomics profile in midbrain due to the general inhibition of mTORC1. However, neither low nor high dose of rapamycin were able to improve the mitochondrial bioenergetics, the brain injuries and the phenotypic characteristics of Coq9. These results may be due to the lack of microgliosis-derived neuroinflammation, the limitation to induce autophagy, or the need of a functional CoQ-junction. Therefore, the translation of rapamycin therapy into the clinic for patients with mitochondrial disorders requires, at least, the consideration of the particularities of each mitochondrial disease. FUND: Supported by the grants from "Fundación Isabel Gemio - Federación Española de Enfermedades Neuromusculares - Federación FEDER" (TSR-1), the NIH (P01HD080642) and the ERC (Stg-337327).

Topics: Animals; Autophagy; Cell Respiration; Disease Models, Animal; Gene Expression Profiling; Humans; Metabolomics; Mice; Mitochondria; Mitochondrial Diseases; Mitochondrial Encephalomyopathies; Phenotype; Sirolimus; Treatment Outcome; Ubiquinone

Topics: Animals; Anti-Bacterial Agents; Aortic Aneurysm, Thoracic; Disease Models, Animal; Down-Regulation; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; MicroRNAs; Sirolimus

New generation bioresorbable scaffolds (BRS) promise to improve the outcomes of current generation BRS technologies by decreasing wall thickness while maintaining structural strength. This study aimed to compare the biomechanical behavior and vascular healing profile of a novel thin-walled (98 μm) sirolimus-eluting ultrahigh molecular weight BRS (Magnitude, Amaranth Medical) to the Absorb everolimus-eluting bioresorbable vascular scaffold (Abbott Vascular).. In vitro biomechanical testing showed lower number of fractures on accelerated cycle testing over time (at 21K cycles = 20.0 [19.0-21.0] in Absorb versus 0.0 [0.0-1.0] in Magnitude-BRS). Either Magnitude (n = 43) or Absorb (n = 22) was implanted in 65 coronary segments of 22 swine. Scaffold strut's coverage was evaluated using serial optical coherence tomography (OCT) analysis. At 14 days, Magnitude-BRS demonstrated a higher percentage of embedded struts (97.7% [95.3, 100.0] compared to Absorb (57.2% [48.0, 76.0], p = 0.003) and lower percentage of uncovered struts (0.0% [0.0, 0.0] versus Absorb 5.5% [2.6, 7.7], p = 0.02). Also, it showed a lower percent late recoil (-1.02% [-4.11, 3.21] versus 4.42% [-1.10, 8.74], p = 0.04) at 28 days. Histopathology revealed comparable neointimal proliferation and vascular healing responses between two devices up to 180 days.. A new generation thin walled (98-μm) Magnitude-BRS displayed a promising biomechanical behavior and strut healing profile compared to Absorb at the experimental level. This new generation BRS platform has the potential to improve the clinical outcomes shown by the current generation BRS.

Topics: Absorbable Implants; Animals; Coronary Angiography; Coronary Artery Disease; Disease Models, Animal; Drug-Eluting Stents; Immunosuppressive Agents; Percutaneous Coronary Intervention; Polyesters; Prosthesis Design; Reference Values; Sirolimus; Swine; Tissue Scaffolds; Tomography, Optical Coherence

Asymmetric partitioning of fate determinants is a mechanism that contributes to T cell differentiation. However, it remains unclear whether the ability of T cells to divide asymmetrically is influenced by their differentiation state, as well as whether enforcing asymmetric cell division (ACD) rates would have an impact on T cell differentiation and memory formation. Using the murine LCMV infection model, we established a correlation between cell stemness and the ability of CD8

Topics: Animals; Arenaviridae Infections; Asymmetric Cell Division; CD8-Positive T-Lymphocytes; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Hematopoietic Stem Cells; Humans; Immunologic Memory; Immunotherapy, Adoptive; Lymphocytic choriomeningitis virus; Mice; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To compare the anti-peritoneal fibrotic effects between a mammalian target of rapamycin complex 1-specific blocker and a phosphatidyl-inositol 3-kinase/mammalian target of rapamycin dual-blocker.. A total of 40 male Sprague-Dawley rats were randomly divided into five groups with eight animals per group. The normal group (N group) did not receive any intervention. The normal saline group (NS group) received an intraperitoneal injection of normal saline at 1 ml/100 g daily. The model group (3 W group), rapamycin (RAPA) group and BEZ235 (PI3K/mTOR dual-blocker) group all received an intraperitoneal injection of 0.1% chlorhexidine gluconate at 1 ml/100g daily. And the RAPA and BEZ235 groups also received a 0.5 mg/d RAPA or 2.5 mg/d BEZ235 gavage every day, respectively. Rats in each group were sacrificed after 3 weeks.. Immunohistochemistry, real-time PCR and western blotting analysis of fibrosis-related indicators (FN, Col 1, and α-SMA) confirmed that RAPA and BEZ235 significantly inhibited peritoneal fibrosis and that these two drugs had similar effects. The p-Akt, p-mTOR, p-p70S6K expression levels were significantly up-regulated in the 3 W group compared to the NS group, confirming that the mTOR pathway was significantly activated during peritoneal fibrosis. RAPA significantly inhibited the phosphorylation of mTOR and p70S6K but did not have significant effects on p-Akt upstream of mTOR. BEZ235 had significant inhibitory effects on all signaling molecules (p-Akt, p-mTOR, and p-p70S6K) in the mTOR pathway.. RAPA did not up-regulate p-Akt in a negative feedback fashion. Both drugs effectively inhibited peritoneal fibrosis.

Topics: Animals; Chlorhexidine; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Imidazoles; Injections, Intraperitoneal; Kidney Failure, Chronic; Male; Mechanistic Target of Rapamycin Complex 1; Peritoneal Dialysis; Peritoneal Fibrosis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinolines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Although cilostazol was proved to have antitumor biological effects, its function in myocardial ischemia and reperfusion (I/R) injury and the underlying mechanisms were not fully illustrated yet. In this study, a rat model of I/R injury was constructed and quantitative real-time PCR, Western blot, and immunofluorescence (IF) assay were performed. Our results showed that cilostazol increased LC3 II/LC3 I ratio, reduced p62 abundance, and promoted the expressions of LAMP1, LAMP2, cathepsin B, and cathepsin D, indicating that cilostazol could activate autophagy and elevated lysosome activation. Following analysis showed that cilostazol enhanced nuclear protein expression of transcription factor EB (TFEB), an important regulator of autophagy-lysosome pathway. Furthermore, CCI-779, an inhibitor of TFEB, could reverse the effects of cilostazol on autophagic activity and lysosome activation. Importantly, cilostazol suppressed I/R injury-induced apoptosis by decreasing the cleavage of caspase 3 and PARP. Enzyme-linked immunosorbent assay showed that cilostazol reduced the serum levels of CTn1 and CK-MB and decreased infract size caused by I/R injuries. Altogether this study suggested that cilostazol protects against I/R injury by regulating autophagy, lysosome, and apoptosis in a rat model of I/R injury. The protective mechanism of cilostazol was partially through increasing the transcriptional activity of TFEB.

Topics: Animals; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cilostazol; Disease Models, Animal; Lysosomes; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Sirolimus

AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers.

Topics: Administration, Oral; AMP-Activated Protein Kinases; Animals; Cell Proliferation; Disease Models, Animal; Disease-Free Survival; Female; Glycolysis; Hypoglycemic Agents; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenformin; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; PTEN Phosphohydrolase; Signal Transduction; Sirolimus

This study was to observed the different doses of rapamycin on the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. 63 female C57BL/6 mice (6-8 weeks) was chosen and randomly divided into three groups: control, low-dose rapamycin-treated EAE mice (0.3 mg/kg), and high-dose rapamycin-treated EAE mice (1 mg/kg). The EAE mice recovery of neurological function in different concentrations of rapamycin were assessed by neurological function score; The assessment of neurological function was divided into three periods: initial stage (10-13d), peak phase (17-21d), remission phase (25-28d), and calculated the score for each period. The inflammatory cell infiltration of mice was assessed by IL-17 A immunohistochemical staining which produced by Th17 cell and positive cell count. The autoimmune recovery of EAE mice was evaluated by flow cytometry on the expression of CD4+ CD25+ Foxp3+ T cells. The transcription factors of Foxp3+ and RORC (RAR-related orphan receptor C) mRNA expression were evaluated by qRT-PCR in Treg cells and Th17 cells. In the neurological function score, the high-dose group was significantly lower than the other two groups in the peak drug phase and the remission phase (P < 0.05), while there was no significant difference in the initial stage (P > 0.05). The percentage of CD4+CD25+Foxp3+T cells, the number of Th17 cells, and the expression of Foxp3 and RORC mRNA level in the high-dose rapamycin group were greater than those in the vehicle-treated group and the low-dose rapamycin group. High doses of rapamycin (1 mg/kg) have a better relieves inflammation of EAE by altering the balance of Treg/Th17 in a mouse model.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Inflammation; Interleukin-17; Mice; Nuclear Receptor Subfamily 1, Group F, Member 3; Sirolimus; T-Lymphocytes, Regulatory; Th17 Cells

Autosomal dominant polycystic kidney disease (PKD) is characterized by cyst formation and growth, which are partially driven by abnormal proliferation of tubular cells. Proproliferative mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2) are activated in the kidneys of mice with PKD. Sirolimus indirectly inhibits mTORC1. Novel mTOR kinase inhibitors directly inhibit mTOR kinase, resulting in the inhibition of mTORC1 and mTORC2. The aim of the present study was to determine the effects of sirolimus versus the mTOR kinase inhibitor torin2 on cyst growth and kidney function in the

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Cell Cycle Proteins; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; Kidney Tubules; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Mutation; Naphthyridines; Phosphorylation; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

The pathogenesis of the development of IgA nephropathy has not been clear up to now. At present, some studies revealed that the mTOR pathway may participate in IgA nephropathy; however, the mechanism has not been systematically studied. In this study, we established an IgAN rat model to investigate the protective effects of rapamycin as a new type of immunosuppressant, as well as its therapeutic mechanisms.. After the establishment of IgA nephropathy model, rats were treated with different concentrations of rapamycin, and the protective effect of different concentrations of rapamycin on renal function of the rats was observed. The deposition of IgA was observed by immunofluorescence. The kidney expression of Akt and p70S6k proteins in mTOR pathway was examined using the western blot assay after rapamycin treatment.. Morphology and immunofluorescence confirmed that the rat model of IgA nephropathy was successfully established. In particular, the level of proteinuria decreased with the increase of the dose of rapamycin, as well as the deposition of IgA in glomeruli. Moreover, the western blot analysis indicated that the expression of p70S6K in the downstream of mTOR pathway decreased and the upstream protein AKT of the mTOR pathway was overexpressed in the rats model.. We found that rapamycin has protective effects in the IgA nephropathy rat model in a dose-dependent manner. In addition, the result of western blot assay suggested that rapamycin may display its therapeutic effects through interfering the AKT-mTOR-p70S6K signaling pathway.

Topics: Animals; Disease Models, Animal; Disease Progression; Glomerulonephritis, IGA; Humans; Immunoglobulin A; Immunosuppressive Agents; Kidney Glomerulus; Male; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

The sequential reactivation of mechanistic target of rapamycin (mTOR) inhibited autophagic flux in neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R), which was characterized by reduction of autophagosome formation and restriction of autolysosome degradation. However, its detailed molecular mechanism was still unknown. In this study, we further explore the existing form of mTOR and its suppression on the transcriptional levels of related mRNA from neurons exposed to ischemia-reperfusion injury.. The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR.. The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons.. The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.

Topics: Animals; Autophagy; Autophagy-Related Proteins; Cells, Cultured; Disease Models, Animal; Feedback, Physiological; Gene Expression Regulation; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Neurons; Phosphorylation; Reperfusion Injury; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.

Topics: Animals; Cathepsins; Disease Models, Animal; Drug Carriers; Drug Compounding; Drug Liberation; Drug Stability; Elastin; Immunosuppressive Agents; Injections, Subcutaneous; Male; Mice; Mice, Inbred NOD; Peptides; Sirolimus; Sjogren's Syndrome; Tacrolimus Binding Protein 1A

The abnormal activation of glycogen synthase kinase 3β (GSK3β) is one of the mechanisms involved in the pathogenesis of Alzheimer's disease (AD), which results in amyloid β‑peptide (Aβ) plaque overproduction, Tau hyperphosphorylation and neuronal loss. A number of studies have reported that the activation of the mammalian target of rapamycin (mTOR) contributes to the generation and deposition of Aβ, as well as to the formation of neurofibrillary tangles (NFTs) by inhibiting autophagy. GSK3β is also involved in the mTOR signalling pathway. However, whether the inhibition of the activation of mTOR via the regulation of the function of GSK3β affects the pathology of AD remains unclear. In this study, we intraperitoneally injected amyloid precursor protein (APP)/presenilin‑1 (PS1) transgenic mice with rapamycin, a known activator of autophagy that inhibits mTOR. Our results revealed that rapamycin treatment decreased senile plaque deposition by reducing APP generation, and downregulating β‑ and γ‑secretase activity. Rapamycin also increased Aβ clearance by promoting autophagy and reduced Tau hyperphosphorylation by upregulating the levels of insulin‑degrading enzyme. Additionally, rapamycin markedly promoted the proliferation of differentiated SH‑SY5Y cells stably transfected with the APPswe gene and prevented neuronal loss in the brains of mice in a model of AD. Moreover, rapamycin induced autophagy and promoted autolysosome degradation. In this study, we provide evidence that rapamycin inhibits GSK3β activation and elevates β‑catenin expression by improving the Wnt3a expression levels, which facilitates the amelioration of AD pathology. On the whole, our findings indicate that rapamycin inhibits the activation of mTOR and alters the Wnt/GSK3β/β‑catenin signalling pathway; thus, it may serve as a therapeutic target in the treatment of AD.

Topics: Alzheimer Disease; Animals; beta Catenin; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; Mice; Mice, Transgenic; Sirolimus; TOR Serine-Threonine Kinases; Wnt Signaling Pathway; Wnt3A Protein

Lupus nephritis (LN) is one of the principal causes of mortality and disability in patients with systemic lupus erythematosus. Sirolimus has been used to treat patients with LN; however, the effects and mechanism of sirolimus in these patients remains unclear. The present study aimed to elucidate the therapeutic effects and mechanisms of sirolimus in LN mice using low, medium and high doses of sirolimus (0.1, 0.3 and 1 mg/kg, respectively). The survival probability and kidney index were calculated, and renal fibrosis was determined using Masson's Trichrome staining. The expression levels of E‑cadherin, α‑smooth muscle actin (α‑SMA) and vimentin were assessed via immunofluorescence analysis. Transcriptome analysis of control and sirolimus‑treated LN mice was performed using RNA‑sequencing, differentially expressed gene (DEG) identification and annotation, and Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The results suggested that a medium dose of sirolimus alleviated renal fibrosis and increased the survival rates of mice with LN (P<0.05). Furthermore, transcriptome analysis revealed 334 DEGs associated with LN, 176 of which were upregulated and 158 were downregulated. Following GO functional enrichment, 'biological process', 'molecular function' and 'cellular component' terms were identified. A total of 10 KEGG pathways were enriched, with 'cytokine‑cytokine receptor interaction' and 'interleukin‑17 signaling pathway' being significantly enriched (P<0.05). To the best of our knowledge, the present study is the first to conduct transcriptome analysis of LN mice treated with sirolimus, and demonstrated that a dose of 0.3 mg/kg exerted the greatest therapeutic effects.

Topics: Animals; Computational Biology; Disease Models, Animal; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Gene Regulatory Networks; Humans; Lupus Nephritis; Mice; Protein Interaction Mapping; Protein Interaction Maps; Sirolimus; Transcriptome

DEPDC5 is now recognized as one of the genes most often implicated in familial/inherited focal epilepsy and brain malformations. Individuals with pathogenic variants in DEPDC5 are at risk for epilepsy, associated neuropsychiatric comorbidities and sudden unexplained death in epilepsy. Depdc5flox/flox-Syn1Cre (Depdc5cc+) neuronal-specific Depdc5 knockout mice exhibit seizures and neuronal mTORC1 hyperactivation. It is not known if Depdc5cc+ mice have a hyperactivity/anxiety phenotype, die early from terminal seizures or whether mTOR inhibitors rescue DEPDC5-related seizures and associated comorbidities. Herein, we report that Depdc5cc+ mice were hyperactive in open-field testing but did not display anxiety-like behaviors on the elevated-plus maze. Unlike many other mTOR-related models, Depdc5cc+ mice had minimal epileptiform activity and rare seizures prior to seizure-induced death, as confirmed by video-EEG monitoring. Treatment with the mTORC1 inhibitor rapamycin starting after 3 weeks of age significantly prolonged the survival of Depdc5cc+ mice and partially rescued the behavioral hyperactivity. Rapamycin decreased the enlarged brain size of Depdc5cc+ mice with corresponding decrease in neuronal soma size. Loss of Depdc5 led to a decrease in the other GATOR1 protein levels (NPRL2 and NPRL3). Rapamycin failed to rescue GATOR1 protein levels but rather rescued downstream mTORC1 hyperactivity as measured by phosphorylation of S6. Collectively, our data provide the first evidence of behavioral alterations in mice with Depdc5 loss and support mTOR inhibition as a rational therapeutic strategy for DEPDC5-related epilepsy in humans.

Topics: Animals; Disease Models, Animal; Electroencephalography; Epilepsy; Fluorescent Antibody Technique; Genes, Lethal; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; GTPase-Activating Proteins; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Neurons; Phenotype; Signal Transduction; Sirolimus

Sepsis is a life-threatening condition that may develop to multiple organ failure and septic shock. Autophagy is considered to play an important role in the regulation of inflammation. The present study aims to investigate the protective role of mTORC1 inhibitor, rapamycin, on septic death using cecal ligation and puncture (CLP) mice model. Here, results showed that pretreatment with rapamycin reduced the pyroptosis of peritoneal macrophages stimulated by cecal contents and the release of inflammatory factors such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α); In septic mice, rapamycin treatment decreased the activation of inflammasome in lung, and alleviated the pathological injuries in lung, liver and spleen tissues during acute stage of sepsis. Treatment of rapamycin rescued animals from septic death significantly. Our results indicated that activation of autophagy is a potential strategy to regulate the excessive inflammation in acute stage of sepsis.

Topics: Animals; Autophagy; Cecum; Cytokines; Disease Models, Animal; Inflammasomes; Inflammation; Ligation; Lung; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Sepsis; Sirolimus

Splenic plasmacytoid dendritic cells (pDC) possess the capability to harbor live replicative Plasmodium parasite. Isolated splenic pDC from infected mice causes malaria when transferred to naïve mice. Incomplete autophagic degradation might cause poor antigen processing and poor immune response. Induction of autophagic flux by rapamycin treatment led to better prognosis by boosting pDC centered immune response against the pathogen. Splenic pDC from rapamycin-treated infected mice, caused less parasitemia in naïve mice. The downregulation of adhesion with unaltered phagocytic potential of the cells post autophagic induction restricted excessive parasite burden within them. Rapamycin-treated pDC played a better role in antigen presentation. They showed higher expression of co-stimulatory molecules CD80, CD86, DEC205, MHCI. Rapamycin-treated pDC induced CD28 expression on CD8

Topics: Animals; Autophagy; CD8-Positive T-Lymphocytes; Cell Adhesion; Cytokines; Dendritic Cells; Disease Models, Animal; Malaria, Cerebral; Male; Mice; Sirolimus; Spleen; T-Lymphocytes, Regulatory; Th1-Th2 Balance; Th17 Cells

Old livers are more damaged by hepatic ischemia and reperfusion (IR) injury than young livers. The aim of this study was to investigate the effects of ischemic and rapamycin preconditioning on IR injury in old livers. Young (8-week-old) and aged (60-week-old) mice were subjected to IR or a sham control procedure. The aged mice were randomly divided into six groups: IR (CON), IR with ischemic preconditioning (IPC), IR with rapamycin preconditioning (RAPA), IR with combined ischemic and rapamycin preconditioning (IPC + RAPA), IR with 3-methyladenine (3-MA), IR with combined ischemic and rapamycin preconditioning with 3-MA pretreatment (IPC + RAPA+3-MA). Liver injury was evaluated 6 h after reperfusion. Hepatocellular autophagy induction was also analyzed by western blotting. The results revealed that aged mice had aggravated liver IR injury as compared to young mice. In aged mice following IR, IPC + RAPA but not IPC or RAPA alleviated liver injury, as evidenced by lower levels of serum ALT, improved preservation of liver architecture with lower Suzuki scores, and decreased caspase-3 activity compared with CON. In addition, western blot analysis revealed increased LC3B II but decreased p62 protein expression levels in the IPC + RAPA group, indicating that autophagic flux was restored by combined ischemic and rapamycin preconditioning. Furthermore, autophagy inhibition by the inhibitor 3-MA abrogated the protective role in the IPC + RAPA group, while no significant effects were observed in the CON group. In conclusions, our results demonstrated that combined ischemic and rapamycin preconditioning protected old livers against IR injury, which was likely attributed to restored autophagy activation.

Topics: Aging; Animals; Autophagy; Caspase 3; Cells, Cultured; Combined Modality Therapy; Disease Models, Animal; Humans; Immunosuppressive Agents; Ischemic Preconditioning; Liver; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Reperfusion Injury; Sirolimus

Topics: Abatacept; Animals; Cell Transplantation; Cells, Cultured; Disease Models, Animal; Feasibility Studies; Graft Rejection; Histocompatibility Antigens; Humans; Immune Tolerance; Immunosuppressive Agents; Kidney Transplantation; Macaca mulatta; Myeloid-Derived Suppressor Cells; Sirolimus; T-Lymphocytes; Transplantation, Autologous; Transplantation, Homologous

In an adult healthy liver, hepatocytes are in a quiescent stage unless a physical injury, such as ablation, or a toxic attack occur. Indeed, to maintain their crucial organismal homeostatic role, the damaged or remaining hepatocytes will start proliferating to restore their functional mass. One of the limiting conditions for cell proliferation is amino-acid availability, necessary both for the synthesis of proteins important for cell growth and division, and for the activation of the mTOR pathway, known for its considerable role in the regulation of cell proliferation. The overarching aim of our present work was to investigate the role of amino acids in the regulation of the switch between quiescence and growth of adult hepatocytes. To do so we used non-confluent primary adult rat hepatocytes as a model of partially ablated liver. We discovered that the absence of amino acids induces in primary rat hepatocytes the entrance in a quiescence state together with an increase in Drosha protein, which does not involve the mTOR pathway. Conversely, Drosha knockdown allows the hepatocytes, quiescent after amino-acid deprivation, to proliferate again. Further, hepatocyte proliferation appears to be independent of miRNAs, the canonical downstream partners of Drosha. Taken together, our observations reveal an intriguing non-canonical action of Drosha in the control of growth regulation of adult hepatocytes responding to a nutritional strain, and they may help to design novel preventive and/or therapeutic approaches for hepatic failure.

Topics: Amino Acids; Animals; Autophagy; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Gene Knockdown Techniques; Hepatocytes; Liver Failure; Male; MicroRNAs; Mitochondria; Rats; Rats, Wistar; Ribonuclease III; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Cardiac pressure overload (for example due to aortic stenosis) induces irreversible myocardial dysfunction, cardiomyocyte hypertrophy and interstitial fibrosis in patients. In contrast to adult, neonatal mice can efficiently regenerate the heart after injury in the first week after birth. To decipher whether insufficient cardiac regeneration contributes to the progression of pressure overload dependent disease, we established a transverse aortic constriction protocol in neonatal mice (nTAC). nTAC in the non-regenerative stage (at postnatal day P7) induced cardiac dysfunction, myocardial fibrosis and cardiomyocyte hypertrophy. In contrast, nTAC in the regenerative stage (at P1) largely prevented these maladaptive responses and was in particular associated with enhanced myocardial angiogenesis and increased cardiomyocyte proliferation, which both supported adaptation during nTAC. A comparative transcriptomic analysis between hearts after regenerative versus non-regenerative nTAC suggested the transcription factor GATA4 as master regulator of the regenerative gene-program. Indeed, cardiomyocyte specific deletion of GATA4 converted the regenerative nTAC into a non-regenerative, maladaptive response. Our new nTAC model can be used to identify mediators of adaptation during pressure overload and to discover novel potential therapeutic strategies.

Topics: Angiogenesis Inducing Agents; Animals; Cell Proliferation; Cytokinesis; Disease Models, Animal; Female; Fibrosis; GATA4 Transcription Factor; Gene Expression; Heart; Heart Failure; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Myocytes, Cardiac; Pressure; Rats; Sirolimus; Transcriptome

Arteriovenous fistulae (AVF) are the most common access created for hemodialysis, but up to 60% do not sustain dialysis within a year, suggesting a need to improve AVF maturation and patency. In a mouse AVF model, Akt1 regulates fistula wall thickness and diameter. We hypothesized that inhibition of the Akt1-mTORC1 axis alters venous remodeling to improve AVF patency. Daily intraperitoneal injections of rapamycin reduced AVF wall thickness with no change in diameter. Rapamycin decreased smooth muscle cell (SMC) and macrophage proliferation; rapamycin also reduced both M1 and M2 type macrophages. AVF in mice treated with rapamycin had reduced Akt1 and mTORC1 but not mTORC2 phosphorylation. Depletion of macrophages with clodronate-containing liposomes was also associated with reduced AVF wall thickness and both M1- and M2-type macrophages; however, AVF patency was reduced. Rapamycin was associated with improved long-term patency, enhanced early AVF remodeling and sustained reduction of SMC proliferation. These results suggest that rapamycin improves AVF patency by reducing early inflammation and wall thickening while attenuating the Akt1-mTORC1 signaling pathway in SMC and macrophages. Macrophages are associated with AVF wall thickening and M2-type macrophages may play a mechanistic role in AVF maturation. Rapamycin is a potential translational strategy to improve AVF patency.

Topics: Animals; Arteriovenous Shunt, Surgical; Disease Models, Animal; Kidney Diseases; Mechanistic Target of Rapamycin Complex 1; Mice; Proto-Oncogene Proteins c-akt; Renal Dialysis; Signal Transduction; Sirolimus; Vascular Remodeling

The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers.. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined.. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS.. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine.

Topics: Aminoacetonitrile; Animals; Antidepressive Agents; Brain; Chronic Disease; Depressive Disorder; Disease Models, Animal; Dominance-Subordination; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Ketamine; Male; Mice, Inbred C57BL; Morpholines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stress, Psychological; TOR Serine-Threonine Kinases

Drug-eluting stents (DES) have evolved to using bioresorbable polymers as a method of drug delivery. The impact of bioresorbable polymer on long-term neointimal formation, inflammation, and healing has not been fully characterised. This study aimed to evaluate the biological effect of polymer resorption on vascular healing and inflammation.. A comparative DES study was performed in the familial hypercholesterolaemic swine model of coronary stenosis. Permanent polymer DES (zotarolimus-eluting [ZES] or everolimus-eluting [EES]) were compared to bioresorbable polymer everolimus-eluting stents (BP-EES) and BMS. Post implantation in 29 swine, stents were explanted and analysed up to 180 days. Area stenosis was reduced in all DES compared to BMS at 30 days. At 180 days, BP-EES had significantly lower area stenosis than EES or ZES. Severe inflammatory activity persisted in permanent polymer DES at 180 days compared to BP-EES or BMS. Qualitative para-strut inflammation areas (graded as none to severe) were elevated but similar in all groups at 30 days, peaked at 90 days in DES compared to BMS (p<0.05) and, at 180 days, were similar between BMS and BP-EES but were significantly greater in DES.. BP-EES resulted in a lower net long-term reduction in neointimal formation and inflammation compared to permanent polymer DES in an animal model. Further study of the long-term neointima formation deserves study in human clinical trials.

Topics: Absorbable Implants; Animals; Coronary Stenosis; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Inflammation; Neointima; Percutaneous Coronary Intervention; Polymers; Sirolimus; Swine; Wound Healing

To investigate the role of glucagon-like peptide-1 analog (GLP-1) in high-fat diet-induced obesity-related glomerulopathy (ORG). Male C57BL/6 mice fed a high-fat diet for 12 wk were treated with GLP-1 (200 μg/kg) or 0.9% saline for 4 wk. Fasting blood glucose and insulin and the expression of podocin, nephrin, phosphoinositide 3-kinase (PI3K), glucose transporter type (Glut4), and microtubule-associated protein 1A/1B-light chain 3 (LC3) were assayed. Glomerular morphology and podocyte foot structure were evaluated by periodic acid-Schiff staining and electron microscopy. Podocytes were treated with 150 nM GLP-1 and incubated with 400 μM palmitic acid (PA) for 12 h. The effect on autophagy was assessed by podocyte-specific Glut4 siRNA. Insulin resistance and autophagy were assayed by immunofluorescence and Western blotting. The high-fat diet resulted in weight gain, ectopic glomerular lipid accumulation, increased insulin resistance, and fusion of podophyte foot processes. The decreased translocation of Glut4 to the plasma membrane and excess autophagy seen in mice fed a high-fat diet and in PA-treated cultured podocytes were attenuated by GLP-1. Podocyte-specific Glut4 siRNA promoted autophagy, and rapamycin-enhanced autophagy worsened the podocyte injury caused by PA. Excess autophagy in podocytes was induced by inhibition of Glut4 translocation to the plasma membrane and was involved in the pathology of ORG. GLP-1 restored insulin sensitivity and ameliorated renal injury by decreasing the level of autophagy.

Topics: Animals; Autophagy; Blood Glucose; Cell Line; Cytoprotection; Diet, High-Fat; Disease Models, Animal; Glucagon-Like Peptide 1; Glucose Transporter Type 4; Insulin; Insulin Resistance; Kidney Diseases; Male; Mice, Inbred C57BL; Obesity; Palmitic Acid; Podocytes; Protein Transport; Signal Transduction; Sirolimus

Resolvins are bioactive lipid mediators that are generated from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). We recently demonstrated that the DHA-derived resolvins D1 and D2 exert antidepressant effects. However, whether the EPA-derived resolvins E1 (RvE1) and E2 (RvE2) produce antidepressant effects is not clear.. We examined the antidepressant effects of RvE1/RvE2 in a murine lipopolysaccharide (LPS)-induced depression model using the tail suspension and forced swim tests. RvE1/RvE2 reportedly possesses both chemerin receptor ChemR23 agonistic activity and leukotriene B. Intracerebroventricular infusions of RvE1 (1 ng)/RvE2 (10 ng) produced significant antidepressant effects. An intracerebroventricular infusion of chemerin (500 ng), but not U75302 (a BLT1 antagonist; 10 or 50 ng), produced antidepressant effects. Intraperitoneal rapamycin (an mTORC1 inhibitor; 10 mg/kg) blocked the antidepressant effect of intracerebroventricular RvE1. Bilateral intra-mPFC and intra-DG infusions of RvE1 (50 pg/side) exerted antidepressant effects.. The results of this study demonstrate that (1) RvE1/RvE2 produce antidepressant effects likely via ChemR23, (2) mTORC1 signaling mediates the antidepressant effect of RvE1, and (3) mPFC and DG are the key brain regions involved in these actions. RvE1/RvE2 and their receptors may be promising targets for the development of novel antidepressants.

Topics: Analysis of Variance; Animals; Antidepressive Agents; Brain; Depressive Disorder; Disease Models, Animal; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Alcohols; Glycols; Hindlimb Suspension; Lipopolysaccharides; Locomotion; Male; Mice; Prefrontal Cortex; Signal Transduction; Sirolimus; Swimming; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR), a serine/threonine kinase regulate variety of cellular functions including cell growth, differentiation, cell survival, metabolism, and stress response, is now appreciated to be a central regulator of immune responses. Because mTOR inhibitors enhanced the anti-inflammatory activities of regulatory T cells and decreased the production of proinflammatory cytokines by macrophages, mTOR has been a pharmacological target for inflammatory diseases. In this study, we examined the role of mTOR in the production of proinflammatory and vasodilator mediators in zymosan-induced non-septic shock model in rats. To elucidate the mechanism by which mTOR contributes to non-septic shock, we have examined the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system caused by mTOR/mitogen-activated protein kinase kinase (MEK1)/extracellular signal-regulated kinase (ERK1/2)/inhibitor κB kinase (IKKβ)/inhibitor of κB (IκB-α)/nuclear factor-κB (NF-κB) signalling pathway activation. After 1 h of zymosan (500 mg/kg, i.p.) administration to rats, mean arterial blood pressure (MAP) was decreased and heart rate (HR) was increased. These changes were associated with increased expression and/or activities of ribosomal protein S6, MEK1, ERK1/2, IKKβ, IκB-α and NF-κB p65, and NADPH oxidase system activity in cardiovascular and renal tissues. Rapamycin (1 mg/kg, i.p.), a selective mTOR inhibitor, reversed these zymosan-induced changes in these tissues. These observations suggest that activation of mTOR/MEK1/ERK1/2/IKKβ/IκB-α/NF-κB signalling pathway with proinflammatory and vasodilator mediator formation and NADPH oxidase system activity contributes to systemic inflammation in zymosan-induced non-septic shock. Thus, mTOR may be an optimal target for the treatment of the diseases characterized by the severe systemic inflammatory response.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Arterial Pressure; Cyclooxygenase 2; Cytokines; Disease Models, Animal; I-kappa B Proteins; Inflammation; Inflammation Mediators; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Nitrosative Stress; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Rats, Wistar; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Vasodilation; Zymosan

CHARGE syndrome-which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies-is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in

Topics: Alternative Splicing; Animals; Antibiotics, Antineoplastic; Argonaute Proteins; CHARGE Syndrome; Chlorocebus aethiops; COS Cells; Disease Models, Animal; DNA-Binding Proteins; Drug Evaluation, Preclinical; Female; Humans; Male; Mice; Mice, Transgenic; Neural Crest; Pregnancy; Proteins; Rabbits; Rats; Sirolimus

The effects of bleomycin and rapamycin on cellular senescence and differentiation of rabbit annulus fibrosus stem cells (AFSCs) were investigated using a cell culture model. The results showed that bleomycin induced cellular senescence in AFSCs as evidenced by senescence-associated secretory phenotype. The morphology of AFSCs was changed from cobblestone-like cells to pancake-like cells. The senescence-associated β-galactosidase activity, the protein expression of P16 and P21, and inflammatory-related marker gene levels IL-1β, IL-6, and TNF-α were increased in bleomycin-treated AFSCs in a dose-dependent manner. Rapamycin treatment decreased the gene expression of MMP-3, MMP-13, IL-1β, IL-6, TNF-α, and protein levels of P16 and P21 in bleomycin-treated AFSCs. Furthermore, neither bleomycin nor rapamycin changed the ribosomal S6 protein level in AFSCs. However, the phosphorylation of the ribosomal S6 protein was increased in bleomycin-treated AFSCs and decreased in rapamycin-treated AFSCs. AFSCs differentiated into adipocytes, osteocytes, and chondrocytes when they were cultured with respective differentiation media. Rapamycin inhibited multi-differentiation potential of AFSCs in a concentration-dependent manner. Our findings demonstrated that mammalian target of rapamycin (mTOR) signaling affects cellular senescence, catabolic and inflammatory responses, and multi-differentiation potential, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.

Topics: Analysis of Variance; Animals; Annulus Fibrosus; Bleomycin; Blotting, Western; Cellular Senescence; Disease Models, Animal; Gene Expression; Humans; Intervertebral Disc Degeneration; Lumbar Vertebrae; Rabbits; Real-Time Polymerase Chain Reaction; Sirolimus; Stem Cells

Topics: Alzheimer Disease; Animals; Behavior, Animal; Blood-Brain Barrier; Cell Line; Cognition; Dementia, Vascular; Disease Models, Animal; Female; Male; Matrix Metalloproteinase 9; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C57BL; Mice, Knockout; Protein Kinase Inhibitors; Receptors, LDL; Sirolimus; Tight Junction Proteins; Tight Junctions; TOR Serine-Threonine Kinases

Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.

Topics: Activating Transcription Factor 4; Animals; Aorta; Aortic Diseases; Cell Death; Cell Proliferation; Cell Transdifferentiation; Disease Models, Animal; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mice, Mutant Strains; Muscle, Smooth, Vascular; Osteogenesis; Phosphorylation; Receptor for Advanced Glycation End Products; S100 Proteins; Signal Transduction; Sirolimus; Taurochenodeoxycholic Acid; Unfolded Protein Response; Uremia; Vascular Calcification

Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5-0.75 μM) and stimulated apoptosis at higher doses (1.0-2.0 μM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse.

Topics: Adolescent; Adult; Animals; Apoptosis; Arsenic Trioxide; Autophagy; Cell Line, Tumor; Disease Models, Animal; Drug Synergism; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Mice, SCID; Middle Aged; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Reactive Oxygen Species; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Young Adult

Topics: Animals; Blood Glucose; Body Composition; Body Weight; Cardiomyopathies; Cardiotonic Agents; Diabetes Mellitus, Type 2; Diet; Disease Models, Animal; Echocardiography; Female; Insulin; Insulin Resistance; Longevity; Male; Mice; Mice, Inbred C57BL; Sirolimus; Weight Gain

Lupus nephritis (LN) is one of the most common and severe complications in Systemic lupus erythematosus patients, and the mechanism underlining the pathogenesis of LN is still unknown. Autophagy plays vital roles in maintaining cell homeostasis and is involved in the pathogenesis of many diseases. In this study, we investigated the role of autophagy in the progression of LN.. Autophagic activities in podocytes of both LN patients (Class IV and V) and mice were evaluated. Podocytes were observed by electron microscopy, and autophagic activity was evaluated by immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by immunohistochemistry, TUNEL assays and flow cytometric analysis.. Significantly greater podocyte injury and discrepant autophagic levels were observed in LN patients. Differentiated mouse podocytes in the LN group showed reduced nephrin expression and increased apoptosis, as well as significantly higher levels of apoptosis-related proteins (cleaved caspase-3 and Bax). In the mice LN group, the increased number of autophagosomes was accompanied by increased LC3-II/LC3-I ratios and decreased p62, suggesting increased autophagic and apoptotic activity in podocytes. Blockade of autophagic activity by 3-MA or siRNA-mediated silencing of Atg5 resulted in decreases in LC3-II/LC3-I ratios, podocyte apoptosis and damage in the mice LN group. Futhermore, Rapamycin treatment increased LC3-II/LC3-I ratios, and enhanced LN-induced apoptosis in podocyte from modal animal.. This study demonstrates that autophagic activity of podocytes is a crucial factor in renal injury by directly affecting the function of podocyte; thus, inhibiting this activity during the early stages of LN is implicated as a potential therapeutic strategy for delaying the progression of LN. Also, clinical application in LN needs to consider patients' pathological type and drugs' comprehensive effectiveness.

Topics: Adenine; Adolescent; Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Disease Progression; Female; Humans; Lupus Nephritis; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Podocytes; Sirolimus; Young Adult

Cortical dysplasia (CD) is a common cause for intractable epilepsy. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in CD; however, the mechanisms by which mTOR hyperactivation contribute to the epilepsy phenotype remain elusive. Here, we investigated whether constitutive mTOR hyperactivation in the hippocampus is associated with altered voltage-gated ion channel expression in the neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with epilepsy. We found that the protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kvβ2, potassium channel subunits were increased, along with altered Kv1.1 distribution, within the hippocampus of NS-Pten KO mice. The aberrant Kv1.1 protein levels were present in young adult (≥postnatal week 6) but not juvenile (≤postnatal week 4) NS-Pten KO mice. No changes in hippocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inhibition with rapamycin treatment at early and late stages of the pathology normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Together, these studies demonstrate altered Kv1.1 protein expression in association with mTOR hyperactivation in NS-Pten KO mice and suggest a role for mTOR signaling in the modulation of voltage-gated ion channel expression in this model.

Topics: Animals; Disease Models, Animal; Epilepsy; Gene Expression Regulation; Hippocampus; Humans; Kv1.1 Potassium Channel; Kv1.2 Potassium Channel; Kv1.4 Potassium Channel; Malformations of Cortical Development; Mice; Mice, Knockout; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Stroke is considered an underlying etiology of the development of seizures. Stroke leads to glucose and oxygen deficiency in neurons, resulting in brain dysfunction and injury. Mild hypothermia is a therapeutic strategy to inhibit stroke‑induced seizures, which may be associated with the regulation of energy metabolism of the brain. Mammalian target of rapamycin (mTOR) signaling and solute carrier family 2, facilitated glucose transporter member (GLUT)‑1 are critical for energy metabolism. Furthermore, mTOR overactivation and GLUT‑1 deficiency are associated with genetically acquired seizures. It has been hypothesized that mTOR and GLUT‑1 may additionally be involved in seizures elicited by stroke. The present study established global cerebral ischemia (GCI) models of rats. Convulsive seizure behaviors frequently occurred during the first and the second days following GCI, which were accompanied with seizure discharge reflected in the EEG monitor. Expression of phosphor (p)‑mTOR and GLUT‑1 were upregulated in the cerebral cortex and hippocampus, as evidenced by immunohistochemistry and western blot analyses. Mild hypothermia and/or rapamycin (mTOR inhibitor) treatments reduced the number of epileptic attacks, seizure severity scores and seizure discharges, thereby alleviating seizures induced by GCI. Mild hypothermia and/or rapamycin treatments reduced phosphorylation levels of mTOR and the downstream effecter p70S6 in neurons, and the amount of GLUT‑1 in the cytomembrane of neurons. The present study revealed that mTOR is involved in stroke‑induced seizures and the anti‑seizure effect of mild hypothermia. The role of GLUT‑1 in stroke‑elicited seizures appears to be different from the role in seizures induced by other reasons. Further studies are necessary in order to elucidate the exact function of GLUT-1 in stroke‑elicited seizures.

Topics: Animals; Biomarkers; Brain Ischemia; Disease Models, Animal; Electroencephalography; Glucose; Glucose Transporter Type 1; Hypothermia, Induced; Immunohistochemistry; Male; Neurons; Rats; Seizures; Severity of Illness Index; Signal Transduction; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Glucocorticoids have excellent therapeutic properties; however, they cause significant adverse atrophogenic effects. The mTORC1 inhibitor REDD1 has been recently identified as a key mediator of glucocorticoid-induced atrophy. We performed computational screening of a connectivity map database to identify putative REDD1 inhibitors. The top selected candidates included rapamycin, which was unexpected because it inhibits pro-proliferative mTOR signaling. Indeed, rapamycin inhibited REDD1 induction by glucocorticoids dexamethasone, clobetasol propionate, and fluocinolone acetonide in keratinocytes, lymphoid cells, and mouse skin. We also showed blunting of glucocorticoid-induced REDD1 induction by either catalytic inhibitor of mTORC1/2 (OSI-027) or genetic inhibition of mTORC1, highlighting role of mTOR in glucocorticoid receptor signaling. Moreover, rapamycin inhibited glucocorticoid receptor phosphorylation, nuclear translocation, and loading on glucocorticoid-responsive elements in REDD1 promoter. Using microarrays, we quantified a global effect of rapamycin on gene expression regulation by fluocinolone acetonide in human keratinocytes. Rapamycin inhibited activation of glucocorticoid receptor target genes yet enhanced the repression of pro-proliferative and proinflammatory genes. Remarkably, rapamycin protected skin against glucocorticoid-induced atrophy but had no effect on the glucocorticoid anti-inflammatory activity in different in vivo models, suggesting the clinical potential of combining rapamycin with glucocorticoids for the treatment of inflammatory diseases.

Topics: Animals; Atrophy; Disease Models, Animal; Female; Immunosuppressive Agents; Keratinocytes; Mice; Mice, Inbred C57BL; Receptors, Glucocorticoid; Sirolimus; Skin; Steroids; Transcription Factors

Chronic pain can be initiated by one or more acute stimulations to sensitize neurons into the primed state. In the primed state, the basal nociceptive thresholds of the animal are normal, but, in response to another hyperalgesic stimulus, the animal develops enhanced and prolonged hyperalgesia. The exact mechanism of how primed state is formed is not completely understood. Here, we showed that spinal protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signal pathway is required for neuronal plasticity change, hyperalgesic priming formation, and the development of chronic hyperalgesia using acid-induced muscle pain model in mice. We discovered that phosphorylated extracellular signal-regulated kinase-positive neurons in the amygdala, spinal cord, and dorsal root ganglion were significantly increased after first acid injection. Inhibition of the phosphorylated extracellular signal-regulated kinase activity intrathecally, but not intracerebroventricularly or intramuscularly before first acid injection, prevented the development of chronic pain induced by second acid injection, which suggests that hyperalgesic priming signal is stored at spinal cord level. Furthermore, intrathecal injection of PKC but not protein kinase A blocker prevented the development of chronic pain, and PKC agonist was sufficient to induce prolonged hyperalgesia response after acid injection. We also found that mammalian target of rapamycin-dependent protein synthesis was required for the priming establishment. To test whether hyperalgesic priming leads to synaptic plasticity change, we recorded field excitatory postsynaptic potentials from spinal cord slices and found enhanced long-term potentiation in mice that received one acid injection. This long-term potentiation enhancement was prevented by inhibition of extracellular signal-regulated kinase. These findings show that the activation of PKC/ERK signal pathway and downstream protein synthesis is required for hyperalgesic priming and the consolidation of pain singling.

Topics: Acids; Animals; Disease Models, Animal; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Extracellular Signal-Regulated MAP Kinases; Female; Ganglia, Spinal; Hyperalgesia; Mice; Mice, Inbred C57BL; Myalgia; Neurons; Pain Measurement; Protein Kinases; Sirolimus; Spinal Cord; Stilbamidines

Akt activation in human cancers exerts chemoresistance, but pan-Akt inhibition elicits adverse consequences. We exploited the consequences of Akt-mediated mitochondrial and glucose metabolism to selectively eradicate and evade chemoresistance of prostate cancer displaying hyperactive Akt. PTEN-deficient prostate cancer cells that display hyperactivated Akt have high intracellular reactive oxygen species (ROS) levels, in part, because of Akt-dependent increase of oxidative phosphorylation. High intracellular ROS levels selectively sensitize cells displaying hyperactive Akt to ROS-induced cell death enabling a therapeutic strategy combining a ROS inducer and rapamycin in PTEN-deficient prostate tumors in mouse models. This strategy elicited tumor regression, and markedly increased survival even after the treatment was stopped. By contrast, exposure to antioxidant increased prostate tumor progression. To increase glucose metabolism, Akt activation phosphorylated HK2 and induced its expression. Indeed, HK2 deficiency in mouse models of Pten-deficient prostate cancer elicited a marked inhibition of tumor development and extended lifespan.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Disease Models, Animal; Humans; Male; Mice; Models, Biological; Neoplasm Transplantation; Oncogene Protein v-akt; Oxidative Phosphorylation; Prostatic Neoplasms; Reactive Oxygen Species; Sirolimus; Treatment Outcome

Epithelial-mesenchymal transition (EMT) plays a pivotal role in idiopathic pulmonary fibrosis (IPF). In bleomycin-induced pulmonary fibrosis mice, we observed that inhibition of mTOR (mammalia target of rapamycin) attenuated IPF. Rapamycin suppressed the down-regulation of E-cadherin and up-regulation of fibronectin in bleomycin-induced pulmonary fibrosis mice. In addition, dual immunofluorescence staining for E-cadherin and fibronectin demonstrated that rapamycin pretreatment decreased the proportions of AECs undergoing EMT in bleomycin-induced pulmonary fibrosis, indicating that mTOR inhibition suppressed EMT in vivo. In the setting of transforming growth factor (TGF)-β1-induced EMT in AECs, we found that mTOR inhibitor attenuated TGF-β1-induced EMT in AECs. This EMT was characterized by morphology and cell skeleton changes and the expression of EMT phenotype markers. Finally, mTOR blockade decreased S6k and TGF-β1-induced Smad2/3 phosphorylation. Bleomycin induced pulmonary fibrosis and EMT in mice, while mTOR repression inhibited bleomycin-induced pulmonary fibrosis and attenuated EMT in vivo. Hence, our study provided evidence of a novel mechanism by which mTOR inhibitor ameliorates pulmonary fibrosis. Suppression of mTOR and EMT may be a target for treatment of pulmonary fibrosis.

Topics: Animals; Bleomycin; Cadherins; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation; Male; Mice; Phosphorylation; Pulmonary Alveoli; Pulmonary Fibrosis; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus; Smad2 Protein; Smad3 Protein; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Stroke, as a kind of acute cerebrovascular diseases, has greatly influenced the patients' quality of life and left a huge public health burden. Vitexin is a flavone C-glycoside (apigenin-8-C-?-D-glucopyranoside) present in several medicinal and other plants. This study aims to explore the role of vitexin in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic stroke. The results showed that the MCAO-induced brain infarction was obviously decreased by vitexin. And the abnormal protein levels of Caspase-3, Bcl-2-associated X protein (Bax), antigen identified by monoclonal antibody (Ki-67) and B cell lymphoma 2 (Bcl-2) in MCAO model rats were reversed by vitexin. Further research indicated that vitexin alleviated MCAO-induced oxidative injury by reducing the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric Oxide (NO). In addition, vitexin attenuated the secretion of pro-inflammatory cytokine (interleukin (IL)-6 and tumor necrosis factor alpha (TNF-?)) and increased anti-inflammatory cytokine (IL-10) production to ameliorate MCAO-induced inflammation. What's more, vitexin repressed the MCAO-induced autophagy through mechanistic target of rapamycin (mTOR)/Ulk1 pathway. Specifically, the MCAO-induced decreased expression of mTOR, peroxisome proliferator-activated receptor ? (PPAR?) and p62 were inhibited by vitexin. At the same time, MCAO-induced increased expression of Ulk1, Beclin1 and rate of LC3?/LC3? also were repressed by vitexin. But the inhibition of vitexin on the MCAO-induced oxidative injury, apoptosis and inflammation were reversed by rapamycin. These results implied that vitexin suppressed the autophagy dysfunction to attenuate MCAO-induced cerebral ischemic stroke via mTOR/Ulk1 pathway.

Topics: Animals; Apigenin; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Brain Ischemia; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Sirolimus; Stroke; TOR Serine-Threonine Kinases

Topics: Animals; Animals, Newborn; Cell Division; Disease Models, Animal; Ependymoglial Cells; Hamartoma Syndrome, Multiple; Mice, Knockout; Mosaicism; Mutation; Neuroglia; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Retina; Retinal Pigment Epithelium; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autosomal-dominant polycystic kidney disease (ADPKD) is a very common genetic disease leading to renal failure. Numerous aberrantly regulated signaling pathways have been identified as promising molecular drug targets for ADPKD therapy. In rodent models, many small-molecule drugs against such targets have proven effective in reducing renal cyst growth. For example, mammalian target of rapamycin (mTOR) inhibition with rapamycin greatly ameliorates renal cystic disease in several rodent models. However, clinical trials with mTOR inhibitors were disappointing largely due to the intolerable extrarenal side effects during long-term treatment with these drugs. Most other potential drug targets in ADPKD are also widely expressed in extrarenal tissues, which makes it likely that untargeted therapies with small-molecule inhibitors against such targets will lead to systemic adverse effects during the necessary long-term treatment of years and decades in ADPKD patients. To overcome this problem, we previously demonstrated that folate-conjugated rapamycin (FC-rapa) targets polycystic kidneys due to the high expression of the folate receptor (FRα) and that treatment of a nonortholgous PKD mouse model leads to inhibition of renal cyst growth. Here we show, in a head-to-head comparison with unconjugated rapamycin, that FCrapa inhibits renal cyst growth, mTOR activation, cell cycling, and fibrosis in an orthologous Pkd1 mouse model. Both unconjugated rapamycin and FC-rapa are similarly effective on polycystic kidneys in this model. However, FC-rapa lacks the extrarenal effects of unconjugated rapamycin, in particular immunosuppressive effects. We conclude that folate-conjugation is a promising avenue for increasing the tissue specificity of small-molecule compounds to facilitate very long-term treatment in ADPKD.

Topics: A549 Cells; Animals; Disease Models, Animal; Drug Compounding; Folate Receptor 1; Folic Acid; Humans; Integrases; Kidney; Mice, Knockout; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Tissue Distribution; TOR Serine-Threonine Kinases; TRPP Cation Channels

Benign biliary stricture (BBS) is highly refractory. Currently, there is no effective strategy for prevention of BBS recurrence. The aim of this study is to establish a novel BBS rabbit model and to investigate the efficacy of biliary infusion with anti-proliferative medications for treating BBS.. A BBS model was established via surgical injury and biliary infection. The biliary infusion tube was inserted into the common bile duct via the stump of cystic duct after cholecystectomy. Biliary infusions with Rapamycin, Pirfenidone and Fasudil were performed daily during the 4 weeks following the surgery. The wall thickness and luminal area of the bile duct were assessed.. All rabbits formed BBS after surgery. The mortality rate was 13% (8/60) and tube withdrawal rate was 4% (2/48). The thickness of the bile duct wall was significantly reduced; whereas the luminal area of the bile duct was dramatically enlarged in the Rapamycin or the Pirfenidone treated group, compared to the saline treated group. Furthermore, the local treatment significantly decreased the levels of proliferation makers, including PCNA, Collagen I and fibrogenic mediators, including ACTA2 and TGF-beta.. We have established a novel animal model for BBS formation. We have further demonstrated that biliary infusion with Rapamycin or Pirfenidone limits the biliary strictures through inhibiting the proliferation of the bile duct wall in this model. This may represent a new avenue for preventing biliary restenosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antineoplastic Agents; Biliary Tract Diseases; Constriction, Pathologic; Disease Models, Animal; Drug Evaluation, Preclinical; Pyridones; Rabbits; Secondary Prevention; Sirolimus; Vasodilator Agents

This study evaluated the manufacturing method and anti-adhesion properties of a new composite mesh in the rat model, which was made from sirolimus (SRL) grafts on a poly(L-lactic acid) (PLLA)-modified polypropylene (PP) hernia mesh.. PLLA was first grafted onto argon-plasma-treated native PP mesh through catalysis of stannous chloride. SRL was grafted onto the surface of PP-PLLA meshes using catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) in a CH. Attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy indicated the existence of a C=O group absorption peak (1724.1 cm. The SRL-coated composite mesh showed minimal formation of intra-abdominal adhesions in a rat model of abdominal wall defect repair.

Topics: Abdominal Wall; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Female; Lactic Acid; Laparotomy; Peritoneum; Polypropylenes; Rats; Rats, Sprague-Dawley; Sirolimus; Surgical Mesh; Surgical Wound; Tissue Adhesions

Mutations in DEPDC5 are causal factors for a broad spectrum of focal epilepsies, but the underlying pathogenic mechanisms are still largely unknown. To address this question, a zebrafish depdc5 knockout model showing spontaneous epileptiform events in the brain, increased drug-induced seizure susceptibility, general hypoactivity, premature death at 2-3 weeks post-fertilization, as well as the expected hyperactivation of mTOR signaling was developed. Using this model, the role of DEPDC5 in brain development was investigated using an unbiased whole-transcriptomic approach. Surprisingly, in addition to mTOR-associated genes, many genes involved in synaptic function, neurogenesis, axonogenesis, and GABA network activity were found to be dysregulated in larval brains. Although no gross defects in brain morphology or neuron loss were observed, immunostaining of depdc5

Topics: Animals; Disease Models, Animal; Epilepsies, Partial; Gene Knockout Techniques; Intracellular Signaling Peptides and Proteins; Loss of Function Mutation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish; Zebrafish Proteins

Calcium-permeable transient receptor potential (TRP) channels exist in eukaryotic cells from yeasts to animals and plants. and they act as sensors for various stresses. Arabidopsis thaliana calcium permeable stress-gated cation channel 1 (AtCSC1) was the first plant calcium-permeable TRP to be described and can be activated by hyperosmotic shock. Candida albicans CaPHM7 is one of the sequence homologs of AtCSC1, but its function remains unknown.. We show here that CaPhm7 is localized to the plasma membrane in both the yeast and hyphal cells of C. albicans. C. albicans cells lacking CaPHM7 are sensitive to SDS and ketoconazole but tolerant to rapamycin and zinc. In addition, deletion of CaPHM7 leads to a filamentation defect, reduced colony growth and attenuated virulence in the mouse model of systemic infection.. CaPhm7 is involved in the regulation of ion homeostasis, drug tolerance, filamentation and virulence in this important human fungal pathogen. CaPhm7 could be a potential target of antifungal drugs.

Topics: Animals; Candida albicans; Candidiasis; Disease Models, Animal; Drug Resistance, Fungal; Fungal Proteins; Homeostasis; Hyphae; Ketoconazole; Mice; Sirolimus; Sodium Dodecyl Sulfate; Transient Receptor Potential Channels; Virulence; Zinc

Insulin resistance (IR) is a hallmark of type 2 diabetes mellitus (T2DM). This study aimed to explore the effects of rapamycin, a specific inhibitor of kinase mammalian target of rapamycin (mTOR), on IR in T2DM rats, and to validate whether the underlying mechanism was associated with autophagy. In this study, the model of T2DM rats was established by feeding the animals with a high-fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ). Diabetic rats were randomly divided into model of T2DM control group (DM-C, n = 15), metformin group (DM-M, n = 15), rapamycin group (DM-Rapa, n = 15), 3-methyladenine (3-MA) group (DM-3-MA, n = 15), and rapamycin + 3-MA group (DM-Rapa-3-MA, n = 15). Rats in different treatment groups were given by corresponding therapy from gastric tube. Meanwhile, normal control group was established (n = 10). As expected, HFD- and STZ- induced T2DM rats exhibited significantly impaired glucose tolerance, reduced insulin sensitivity, dysglycemia and dyslipidemia, aggravated hepatic steatosis, enhanced hepatic inflammation, elevated p-mTOR, and suppressed hepatic autophagy. Importantly, rapamycin and metformin significantly ameliorated IR, relieved disorders of glucose and lipid metabolism, reduced inflammatory level, inhibited mTOR, and promoted autophagy. Importantly, the autophagy inhibitor 3-MA significantly reversed the effects exerted by rapamycin. Collectively, our study suggests that rapamycin improved IR and hepatic steatosis in T2DM rats via activation of autophagy.

Topics: Animals; Autophagy; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Glucose; Hypoglycemic Agents; Insulin; Insulin Resistance; Lipid Metabolism; Liver; Male; Rats; Rats, Sprague-Dawley; Sirolimus

Bortezomib (BTZ) is largely used as a chemotherapeutic agent for the treatment of cancer. However, one of the significant limiting complications of BTZ is painful peripheral neuropathy during BTZ therapy. Drugs preventing and/or treating the painful symptoms induced by BTZ are lacking since the underlying mechanisms leading to neuropathic pain remain largely unclear. The purposes of this study were to examine 1) the effects of blocking mammalian target of rapamycin (mTOR) on mechanical pain and cold hypersensitivity evoked by BTZ and 2) the underlying mechanisms responsible for the role of mTOR in regulating BTZ-induced neuropathic pain.. Behavioral test was performed to determine mechanical pain and cold sensitivity in a rat model. Western blot analysis and ELISA were used to examine expression of mTOR and phosphatidylinositide 3-kinase (p-PI3K) signals, and the levels of substance P and calcitonin gene-related peptide (CGRP).. Systemic injection of BTZ significantly increased mechanical pain and cold sensitivity as compared with control animals (P< 0.05 vs. control rats). The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4E-binding protein 4 (p-4E-BP1) as well as p-PI3K was amplified in the dorsal horn of spinal cord of BTZ rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated mechanical pain and cold hypersensitivity. Blocking PI3K signal also attenuated activities of mTOR, which was accompanied with decreasing neuropathic pain. Inhibition of either mTOR or PI3K blunted enhancement of the spinal substance P and CGRP in BTZ rats.. The data for the first time revealed specific signaling pathways leading to BTZ-induced peripheral neuropathic pain, including the activation of mTOR and PI3K. Inhibition of these signal pathways alleviates pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of peripheral painful neuropathy observed during chemotherapeutic application of BTZ.

Topics: Animals; Behavior, Animal; Bortezomib; Calcitonin Gene-Related Peptide; Carrier Proteins; Chromones; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Male; Morpholines; Neuralgia; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Spinal Cord; Substance P; TOR Serine-Threonine Kinases; Up-Regulation

Previous studies in various rodent epilepsy models have suggested that mammalian target of rapamycin (mTOR) inhibition with rapamycin has anti-epileptogenic potential. Since treatment with rapamycin produces unwanted side effects, there is growing interest to study alternatives to rapamycin as anti-epileptogenic drugs. Therefore, we investigated curcumin, the main component of the natural spice turmeric. Curcumin is known to have anti-inflammatory and anti-oxidant effects and has been reported to inhibit the mTOR pathway. These properties make it a potential anti-epileptogenic compound and an alternative for rapamycin.. To study the anti-epileptogenic potential of curcumin compared to rapamycin, we first studied the effects of both compounds on mTOR activation, inflammation, and oxidative stress in vitro, using cell cultures of human fetal astrocytes and the neuronal cell line SH-SY5Y. Next, we investigated the effects of rapamycin and intracerebrally applied curcumin on status epilepticus (SE)-induced inflammation and oxidative stress in hippocampal tissue, during early stages of epileptogenesis in the post-electrical SE rat model for temporal lobe epilepsy (TLE).. Rapamycin, but not curcumin, suppressed mTOR activation in cultured astrocytes. Instead, curcumin suppressed the mitogen-activated protein kinase (MAPK) pathway. Quantitative real-time PCR analysis revealed that curcumin, but not rapamycin, reduced the levels of inflammatory markers IL-6 and COX-2 in cultured astrocytes that were challenged with IL-1β. In SH-SY5Y cells, curcumin reduced reactive oxygen species (ROS) levels, suggesting anti-oxidant effects. In the post-SE rat model, however, treatment with rapamycin or curcumin did not suppress the expression of inflammatory and oxidative stress markers 1 week after SE.. These results indicate anti-inflammatory and anti-oxidant properties of curcumin, but not rapamycin, in vitro. Intracerebrally applied curcumin modified the MAPK pathway in vivo at 1 week after SE but failed to produce anti-inflammatory or anti-oxidant effects. Future studies should be directed to increasing the bioavailability of curcumin (or related compounds) in the brain to assess its anti-epileptogenic potential in vivo.

Topics: Animals; Anti-Inflammatory Agents; Astrocytes; Brain; Cells, Cultured; Curcumin; Cytokines; Disease Models, Animal; Fetus; Gene Expression Regulation; Humans; Inflammation; Male; Neuroblastoma; Neurons; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Status Epilepticus

Previous studies indicate that the transcription factor upstream stimulating factor 1 (USF1) is involved in the regulation of lipid and glucose metabolism. However, the role of USF1 in lipid-induced autophagy remains unknown. Interestingly, we found that USF1 overexpression suppresses autophagy-related gene expression in HepG2 cells. Further assays confirmed that USF1 could transcriptionally activate mTOR expression, thereby suppressing rapamycin-induced autophagy in HepG2 cells. Moreover, pharmacological activation of autophagy with rapamycin decreases the numbers and sizes of lipid droplets (LDs) in HepG2 cells exposed to an oleate/palmitate mixture. Of note, USF1 upregulation decreases colocalization of LDs and autophagosomes. In conclusion, our data provide evidence that USF1 contributes to abnormal lipid accumulation in the liver by suppressing autophagy via regulation of mTOR transcription.

Topics: Animals; Autophagy; Autophagy-Related Proteins; Diet, High-Fat; Disease Models, Animal; Fatty Acids, Monounsaturated; Gene Expression Regulation; Hep G2 Cells; Humans; Lipid Droplets; Liver; Mice; Non-alcoholic Fatty Liver Disease; Particle Size; Sirolimus; TOR Serine-Threonine Kinases; Transcriptional Activation; Up-Regulation; Upstream Stimulatory Factors

Microglial abnormalities have been reported in pathologic specimens from patients with tuberous sclerosis complex (TSC), a genetic disorder characterized by epilepsy, intellectual disability, and autism. However, the pathogenic role of microglia in epilepsy in TSC is poorly understood, particularly whether microglia defects may be a primary contributor to epileptogenesis or are secondary to seizures or simply epiphenomena. In this study, we tested the hypothesis that Tsc1 gene inactivation in microglia is sufficient to cause epilepsy in mouse models of TSC.. Using a chemokine receptor, Cx3cr1, to target microglia, conventional Tsc1. Tsc1. Microglia abnormalities may contribute to epileptogenesis in the context of neuronal involvement in TSC mouse models, but selective Tsc1 gene inactivation in microglia alone may not be sufficient to cause epilepsy, suggesting that microglia have more supportive roles in the pathogenesis of seizures in TSC.

Topics: Animals; Animals, Newborn; Brain; Calcium-Binding Proteins; CX3C Chemokine Receptor 1; Disease Models, Animal; Electroencephalography; Estrogen Antagonists; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microglia; Phosphopyruvate Hydratase; Sirolimus; Statistics, Nonparametric; Tamoxifen; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Video Recording

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.

Topics: Abatacept; Animals; Antibodies, Monoclonal; CD28 Antigens; Disease Models, Animal; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Lymphocyte Activation; Macaca mulatta; Sirolimus; Systems Biology; T-Lymphocytes

Rapamycin (Rp), the main mammalian target of rapamycin complex inhibitor, is a promising therapeutic agent for breast cancer. However, metabolic disorders and drug resistance reduce its efficacy. Epidemiological, clinical, and experimental studies have demonstrated that omega-3 polyunsaturated fatty acids (ω-3 PUFAs) significantly reduce the incidence and mortality of breast cancer and improve metabolic disorders.. Three breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Rp plus ω-3 PUFA treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics, seahorse experiments, and western blot analysis.. We found that ω-3 PUFAs and Rp synergistically induced cell cycle arrest and apoptosis in vitro and in vivo, accompanied by autophagy blockage. In addition, Rp-induced hypertriglyceridemia and hypercholesterolemia were completely abolished by ω-3 PUFA supplementation. Moreover, the combined treatment of ω-3 PUFA and Rp significantly inhibited glycolysis and glutamine metabolism. The anti-tumor effects of this combination treatment were dependent on ROS production, which was increased by β-oxidation and oxidative phosphorylation.. Our study revealed that ω-3 PUFA enhanced the anti-tumor activity of Rp while minimizing its side effects in vitro and in vivo. These results provide novel insights into the mechanisms underlying the potential beneficial effects of Rp combined with ω-3 PUFAs on the prevention of breast cancer.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Fatty Acids, Omega-3; Female; Humans; Lactic Acid; Malondialdehyde; MCF-7 Cells; Metabolomics; Mice; Microtubule-Associated Proteins; Oxidative Phosphorylation; Reactive Oxygen Species; Sirolimus

Cerebral ischemia/reperfusion (I/R) injury is a common pathological process after cardiac arrest, shock and acute cerebral infarction recanalization, which causes serious injury in brain function. Pinocembrin (Pino), a natural flavonoid at the highest concentration in propolis, exhibited a variety of biological effects, including antitumor, antimicrobial and anti-inflammatory activities. However, the effects of Pino on brain injured after I/R and the mechanisms of its neuroprotective effects remain elusive.. In the present study, we used I/R model rats underwent transient cerebral ischemia inducing by four-vessel occlusion and reperfusion. Pino alone or in combination with autophagy inducer rapamycin (RAPA) was administered to I/R rats. The behavior and cognitive function were evaluated by open field test and Morris water maze test. HE staining was used to determine the survival of hippocampus CA1 pyramidal cells. Three key proteins of autophagy, LC3, Beclin1 and p62, were detected by Western blot.. Our results showed that Pino could significantly reduce the damage of hippocampus CA1 pyramidal neurons and alleviate the impairments of behavior and cognitive function in I/R rats. Pino also decreased the expression of LC3II and Beclin1 and increased the level of p62 in hippocampus CA1 of I/R rats. In addition, Pino also decreased RAPA-induced neuronal damage and excessive activation of autophagy in I/R rats.. Taken together, these results suggested that Pino could protect the brain injury induced by I/R and the potential mechanisms might attribute to inhibition of autophagy activity.

Topics: Animals; Autophagy; Beclin-1; Behavior, Animal; Brain Ischemia; CA1 Region, Hippocampal; Cognition; Disease Models, Animal; Flavanones; Male; Maze Learning; Microtubule-Associated Proteins; Motor Activity; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Sequestosome-1 Protein; Sirolimus; Time Factors

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.

Topics: Animals; Bone Morphogenetic Proteins; Cranial Sutures; Craniosynostoses; Disease Models, Animal; Mice; Mice, Inbred C57BL; Neural Crest; Phenotype; Signal Transduction; Sirolimus; Skull; TOR Serine-Threonine Kinases

Rapamycin is employed as an immunosuppressant following organ transplant and, in patients with hepatocellular carcinoma, to inhibit cancer cell regrowth following liver surgery. Preconditioning the liver with rapamycin to induce the expression of antioxidant enzymes is a potential strategy to reduce ischemia reperfusion (IR) injury. However, pre-treatment with rapamycin inhibits bile flow, especially following ischemia. The aim was to investigate the mechanisms involved in this inhibition. In a rat model of segmental hepatic ischemia and reperfusion, acute administration of rapamycin by intravenous injection did not inhibit the basal rate of bile flow. Pre-treatment of rats with rapamycin for 24 h by intraperitoneal injection inhibited the expression of mRNA encoding the sinusoidal influx transporters Ntcp, Oatp1 and 2 and the canalicular efflux transporter Bsep, and increased expression of canalicular Mrp2. Dose-response curves for the actions of rapamycin on the expression of Bsep and Ntcp in cultured rat hepatocytes were biphasic, and monophasic for effects on Oatp1. In cultured tumorigenic H4IIE liver cells, several bile acid transporters were not expressed, or were expressed at very low levels compared to hepatocytes. In H4IIE cells, rapamycin increased expression of Ntcp, Oatp1 and Mrp2, but decreased expression of Oatp2. It is concluded that the inhibition of bile flow recovery following ischemia observed in rapamycin-treated livers is principally due to inhibition of the expression of sinusoidal bile acid transporters. Moreover, in tumorigenic liver tissue the contribution of tumorigenic hepatocytes to total liver bile flow is likely to be small and is unlikely to be greatly affected by rapamycin.

Topics: Animals; Bile; Bile Ducts; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cholestasis; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatocytes; Humans; Immunosuppressive Agents; Ischemia; Liver; Liver Neoplasms; Liver Transplantation; Male; Membrane Glycoproteins; Rats; Rats, Sprague-Dawley; Rats, Zucker; Reperfusion Injury; Sirolimus; Transplantation Conditioning

Erectile dysfunction (ED), which is common in patients with diabetes mellitus (DM), seriously affects quality of life. Previous studies on the treatment of DM-induced ED (DMED) involve autophagy, but the specific effect and mechanism of treatment are not yet clear.. To investigate the effect and mechanism of rapamycin, an autophagy inducer, in ameliorating DMED.. 45 male Sprague-Dawley rats (7 weeks old) were used in the experiment. 8 rats were randomly selected as the control group; the other rats were treated with streptozotocin to induce type 1 DM. After 10 weeks, an apomorphine test was used to confirm DMED. Rats with DMED were intraperitoneally injected with rapamycin or vehicle for 3 weeks. Rats in the control group were injected with saline. Erectile function in rats was measured by electrically stimulating the cavernous nerve. The penises were then harvested for histologic examinations, ribonucleic acid (RNA), and protein levels of related factors by immunohistochemistry, immunofluorescence, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot.. Erectile function was evaluated by maximum intracavernous pressure and mean arterial pressure. Penile tissues were used to perform histologic examinations and to determine the RNA and protein levels.. Erectile function, which was impaired in rats with DMED, was significantly ameliorated in the DMED + rapamycin group. The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway was inhibited in the DMED group, and rapamycin significantly reduced this inhibition. The DMED group showed increased autophagy and apoptosis level compared with the non-diabetic group, and rapamycin increased the autophagy level and decreased the apoptosis level in the penis. Penile fibrosis was more severe in the DMED group than in the control group and was partially but significantly improved in the DMED + rapamycin group compared with the DMED group. The adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin kinase (mTOR) and PI3K/AKT/mTOR pathways were activated, and the mTOR (regulatory associated protein of mTOR, complex 1 [raptor])/p70 ribosomal protein S6 kinase (p70S6K) pathway was inhibited in the DMED group. Compared with DMED group, rapamycin led to lower AMPK/mTOR and AKT/mTOR pathways expression, a higher degree of mTOR (raptor)/p70S6K pathway inhibition, and no change in the mTORC2-related pathway.. Rapamycin was effective in restoring erectile function in type 1 DMED models.. This study suggested for the first time that rapamycin, an autophagy inducer, is effective in restoring erectile function in rats with diabetes. However, the rat model might not represent the human condition.. Rapamycin improved erectile function in rats with DMED, likely by promoting autophagy, inhibiting apoptosis and fibrotic activity, and ameliorating endothelial function. These findings provide evidence of a potential treatment option for DMED. Lin H, Wang T, Ruan Y, et al. Rapamycin supplementation may ameliorate erectile function in rats with streptozotocin-induced type 1 diabetes by inducing autophagy and inhibiting apoptosis, endothelial dysfunction, and corporal fibrosis. J Sex Med 2018;15:1246-1259.

Topics: Animals; Apoptosis; Autophagy; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Erectile Dysfunction; Injections, Intraperitoneal; Male; Penile Erection; Rats; Rats, Sprague-Dawley; Sirolimus; Streptozocin

Semaphorin 3A (sema 3A) is one of a class of secretory proteins belonging to a family of axon-directed factors found in podocytes, distal tubules, and collecting tubes of the kidney. It is considered to be a potential target molecule involved in the mammalian target of the rapamycin (mTOR) pathway in renal injury or renal diseases, but it has an unknown role in the course of hexavalent chromium-Cr(VI) induced nephrotoxicity. In the present study, an acute kidney injury (AKI) model in rats or cultured tubular epithelial HK-2 cells was employed for Cr(VI) exposure alone or in combination with rapamycin (Rap) or N-acetyl-l-cysteine (NAC) or recombinant sema 3A. The methods of histopathology, biochemics, and western blotting were applied to evaluate tubular injury and the role of sema 3A. The results showed that a significant increase of urinary sema 3A indicates an early occurrence of AKI exposed to Cr(VI), accompanied with a significant increase of tubular injury score and phosphorylated mTOR proteins. Further, Cr(VI) treatment, in combination with pretreatment of the mTOR pathway inhibitor, Rap, showed a considerably stronger protective effect of Rap in protecting against Cr(VI)-induced nephrotoxicity than that seen with the free radical scavenger NAC, highlighting the dominant renal protective role of the mTOR pathway in inhibiting toxicity by downregulating the expressed levels of sema 3A in renal tissue. This study has demonstrated that an increased expression of sema 3A occurs in Cr(VI)-induced AKI resulting from activation of the mTOR pathway, and that inhibition of this pathway has been shown to decrease the severity of the toxicity. In conclusion, this study has shown that increased urinary sema 3A is indicative of an activated mTOR pathway and is a valuable biomarker of the early AKI induced by Cr(VI) exposure.

Topics: Acute Kidney Injury; Animals; Biomarkers; Cell Line; Cell Survival; Chromium; Disease Models, Animal; Humans; Kidney Function Tests; Kidney Tubules, Proximal; Male; Oxidative Stress; Rats, Sprague-Dawley; Semaphorin-3A; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Adding rapamycin or acarbose to diet at 9-10 months of age has been shown to significantly increase life span in both male and female UM-HET3 mice. The current study examined cochleae of male and female UM-HET3 mice at 22 months of age to determine if either treatment also influenced age-related loss of cochlear hair cells. A large loss of cochlear outer hair cells was observed at 22 months of age in untreated mice in both apical and basal halves of the cochlear spiral. Addition of acarbose to diet had no significant effect on the amount of outer hair cell loss at 22 months of age or in its pattern, with large loss in both apical and basal halves. The addition of rapamycin to diet, however, significantly reduced outer hair cell loss in the basal half of the cochlea at 22 months of age when compared to untreated mice. There was no significant difference between male and female mice in any of the conditions. Age-related outer hair cell loss in the apical cochlea precedes outer hair cell loss in the base in many mouse strains. The results of the present study suggest that rapamycin but not acarbose treatment can delay age-related loss of outer hair cells at doses at which each drug increases life span.

Topics: Acarbose; Age Factors; Animals; Cell Death; Cytoprotection; Disease Models, Animal; Female; Genetic Predisposition to Disease; Hair Cells, Auditory, Outer; Hearing; Male; Mice, Transgenic; Phenotype; Presbycusis; Sirolimus

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Chemical and Drug Induced Liver Injury; Chromones; Cobalt; Disease Models, Animal; Erythropoietin; Humans; Liver; Liver Function Tests; Mice; Mice, Inbred C57BL; Morpholines; Peptide Fragments; Proto-Oncogene Proteins c-akt; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Human DNA-methylation data have been used to develop highly accurate biomarkers of aging ("epigenetic clocks"). Recent studies demonstrate that similar epigenetic clocks for mice (

Topics: Age Factors; Animals; Biological Clocks; Caloric Restriction; CpG Islands; Databases, Genetic; Disease Models, Animal; DNA Methylation; Dwarfism; Epigenesis, Genetic; Female; Genome-Wide Association Study; Longevity; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Sirolimus; Species Specificity

Fat embolism syndrome (FES) is a serious complication after trauma, surgery and fat emulsion input and can lead to serious pulmonary injury. Autophagy controls the cell survival and homeostasis by removing the mis-folded proteins and damaged organelles as well as intracellular pathogens through a lysosomal degradation pathway. Increasing research documented that autophagy was wildly involved in variety of human diseases and had huge therapeutic potential. However, the role and mechanism of autophagy in FES remains largely unknown. The rat model of FES was established by tail vein injection with fat and was assessed by Wet-to-Dry (W/D) ratio analysis, hematoxylin-eosin (HE) analysis, staining Oil red staining analysis and qPCR analysis. Western blots were employed to detect the expression of autophagy markers. The changes of pulmonary injury were observed after premedication of rapamycin (an autophagy activator). The alveolar structural damage, red free fat substances in the blood vessels of lung, increased the lung ratio, and the up-regulated MPO expression and activity were showed in the FES models. The expressions of autophagy markers were decreased and meanwhile, apoptosis markers were increased in the FES model. Rapamycin restored the expression of autophagy markers and inhibited the apoptosis and further, resulting in the improvement of the pulmonary injury. Thus, our study demonstrated that autophagy was inhibited and apoptosis was promoted in FES and further Rapamycin alleviated the pulmonary damage in FES via restoring the autophagy and inhibiting the apoptosis.

Topics: Adipose Tissue; Allografts; Animals; Apoptosis; Autophagy; Disease Models, Animal; Embolism, Fat; Lung Injury; Male; Rats, Wistar; Sirolimus

Severe influenza A virus infection causes high mortality and morbidity worldwide due to delayed antiviral treatment and inducing overwhelming immune responses, which contribute to immunopathological lung injury. Sirolimus, an inhibitor of mammalian target of rapamycin (mTOR), was effective in improving clinical outcomes in patients with severe H1N1 infection; however, the mechanisms by which it attenuates acute lung injury have not been elucidated. Here, delayed oseltamivir treatment was used to mimic clinical settings on lethal influenza A (H1N1) pdm09 virus (pH1N1) infection mice model. We revealed that delayed oseltamivir plus sirolimus treatment protects mice against lethal pH1N1 infection by attenuating severe lung damage. Mechanistically, the combined treatment reduced viral titer and pH1N1-induced mTOR activation. Subsequently, it suppressed the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated secretion of interleukin (IL)-1β and IL-18. It was noted that decreased NLRP3 inflammasome activation was associated with inhibited nuclear factor (NF)-κB activation, reduced reactive oxygen species production and increased autophagy. Additionally, the combined treatment reduced the expression of other proinflammatory cytokines and chemokines, and decreased inflammatory cell infiltration in lung tissue and bronchioalveolar lavage fluid. Consistently, it inhibited the mTOR-NF-κB-NLRP3 inflammasome-IL-1β axis in a lung epithelial cell line. These results demonstrated that combined treatment with sirolimus and oseltamivir attenuates pH1N1-induced severe lung injury, which is correlated with suppressed mTOR-NLRP3-IL-1β axis and reduced viral titer. Therefore, treatment with sirolimus as an adjuvant along with oseltamivir may be a promising immunomodulatory strategy for managing severe influenza.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Dogs; Drug Therapy, Combination; Epithelial Cells; Female; Inflammasomes; Influenza A Virus, H1N1 Subtype; Interleukin-18; Interleukin-1beta; Lung; Lung Injury; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred BALB C; NLR Family, Pyrin Domain-Containing 3 Protein; Oseltamivir; Reactive Oxygen Species; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Although liver regeneration has been intensively studied in various ways, the mechanisms underlying liver regeneration remain elusive. Apoptosis-stimulating protein two of p53 (ASPP2) was discovered as a binding partner of p53 and plays an important role in regulating cell apoptosis and growth. However, the role of ASPP2 in hepatocyte proliferation and liver regeneration has not been reported. The expression profile of ASPP2 was measured in a mouse model with 70% partial hepatectomy (PH

Topics: Adenine; Animals; Autophagy; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation; Haploinsufficiency; Hepatocytes; Liver Regeneration; Mechanistic Target of Rapamycin Complex 1; Mice; Sirolimus; Tumor Suppressor Proteins

To investigate the efficacy of brain-targeted rapamycin (T-Rap) in treatment of epilepsy in rats.. Rapamycin nanoparticles targeting brain were prepared. The epilepsy model was induced by injection of pilocarpine in rats. The rats with pilocarpine-induced epilepsy were treated with rapamycin (Rap group) or brain-targeted rapamycin (T-Rap group). Seizure activity was observed by electroencephalography; the effect on mTOR signaling pathway was detected by Western blot; neuronal death and moss fiber sprouting were analyzed by Fluoro-Jade B (FJB) and Timm's staining, respectively.. Electroencephalography showed that both preparation of rapamycin significantly reduced the frequency of spontaneous seizures in rats, and the effect of T-Rap was stronger than that of conventional rapamycin (. T-Rap has a better therapeutic effect on epilepsy than conventional rapamycin with a less adverse effects in rats.

Topics: Animals; Brain; Disease Models, Animal; Epilepsy; Neurons; Pilocarpine; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Treatment Outcome

Attributing to their antiproliferative effect, both rapamycin and peroxisome proliferator-activated receptor-γ (PPARγ) can halt the progression of autosomal dominant polycystic kidney disease (ADPKD). Whether combined use could enhance this effect is unknown. The present study used rapamycin and the PPARγ agonist rosiglitazone concomitantly to observe their combined effects on the proliferation of ADPKD cyst-lining epithelial cells and the progression of ADPKD in Han:SPRD rats. Concomitant use of the two drugs inhibited the proliferation of WT9-12 cells significantly through a superimposition effect. Rosiglitazone inhibited the phosphorylation of mammalian target of rapamycin p70S6K. Concomitant use of rosiglitazone and rapamycin further downregulated the p-p70S6K level. Rosiglitazone also inhibited the phosphorylation of Akt and antagonized the activation of Akt induced by rapamycin. Concomitant use of rosiglitazone and rapamycin significantly retarded the deterioration of renal function, decreased cyst cell proliferation and interstitial fibrosis in Han:SPRD rats. Rapamycin significantly increased cholesterol levels in the blood, whereas rosiglitazone mitigated rapamycin-induced hyperlipidemia. These results indicate that the effects of concomitant use of rosiglitazone and rapamycin in inhibiting the proliferation of WT9-12 cells and delaying the progression of ADPKD in Han:SPRD rats are stronger than those of either drug alone. The present study may provide a new strategy for the long-term treatment of ADPKD.

Topics: Animals; Cell Line; Cell Proliferation; Disease Models, Animal; Disease Progression; Drug Therapy, Combination; Epithelial Cells; Fibrosis; G1 Phase Cell Cycle Checkpoints; Humans; Kidney; Lipids; Male; Phosphorylation; Polycystic Kidney, Autosomal Dominant; PPAR gamma; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Renal Agents; Ribosomal Protein S6 Kinases, 70-kDa; Rosiglitazone; Signal Transduction; Sirolimus

The mammalian target of rapamycin (mTOR) signal controls innate and adaptive immune response in multiple immunoregulatory contexts. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells of potent immunosuppressive capacity. In this study, we aimed to investigate the role of MDSCs in the protection of acute kidney injury (AKI) and the regulation of mTOR signal on MDSC's protective role in this context. In mice AKI model, rapamycin administration was associated with improved renal function, restored histological damage and decreased CD4

Topics: Acute Kidney Injury; Animals; Arginase; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Immunosuppressive Agents; Interferon-gamma; Interleukin-1beta; Interleukin-6; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Myeloid Cells; Myeloid-Derived Suppressor Cells; Nitric Oxide Synthase Type II; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Vascular remodeling resulting from pulmonary arterial hypertension (PAH) leads to endothelial fenestrations. This feature can be exploited by nanoparticles (NP), allowing them to extravasate from circulation and accumulate in remodeled pulmonary vessels. Hyperactivation of the mTOR pathway in PAH drives pulmonary arterial smooth muscle cell proliferation. We hypothesized that rapamycin (RAP)-loaded NPs, an mTOR inhibitor, would accumulate in diseased lungs, selectively targeting vascular mTOR and preventing PAH progression. RAP poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) NPs were fabricated. NP accumulation and efficacy were examined in a rat monocrotaline model of PAH. Following intravenous (IV) administration, NP accumulation in diseased lungs was verified via LC/MS analysis and confocal imaging. Pulmonary arteriole thickness, right ventricular systolic pressures, and ventricular remodeling were determined to assess the therapeutic potential of RAP NPs. Monocrotaline-exposed rats showed increased NP accumulation within lungs compared to healthy controls, with NPs present to a high extent within pulmonary perivascular regions. RAP, in both free and NP form, attenuated PAH development, with histological analysis revealing minimal changes in pulmonary arteriole thickness and no ventricular remodeling. Importantly, NP-treated rats showed reduced systemic side effects compared to free RAP. This study demonstrates the potential for nanoparticles to significantly impact PAH through site-specific delivery of therapeutics.

Topics: Administration, Intravenous; Animals; Disease Models, Animal; Hypertension, Pulmonary; Lung; Nanoparticles; Rats; Rats, Sprague-Dawley; Sirolimus

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6.. Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Biopsy, Needle; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Disease Models, Animal; Genes, myc; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Phosphoproteins; Phosphorylation; Proportional Hazards Models; Proto-Oncogene Mas; Random Allocation; Signal Transduction; Sirolimus; Statistics, Nonparametric; TOR Serine-Threonine Kinases

Podocytes are highly differentiated epithelial cells wrapping glomerular capillaries to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classic model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, whereas inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared with wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Autophagy; Autophagy-Related Protein 7; Cells, Cultured; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Doxorubicin; Male; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Proteinuria; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; Time Factors

Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.

Topics: Animals; Antiviral Agents; Apoptosis; Cell Line; Chlorocebus aethiops; Disease Models, Animal; DNA Replication; Eukaryotic Initiation Factor-4G; Female; Immunohistochemistry; Liver; Mice; Mice, Inbred BALB C; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; Rift Valley Fever; Rift Valley fever virus; Signal Transduction; Sirolimus; Survival Analysis; Vero Cells; Viral Load; Virus Replication

The pathogenesis of some types of preeclampsia is related to fatty acid oxidation disorders. Rapamycin can regulate fatty acid metabolism. This study aimed to investigate the effects of rapamycin on the clinical manifestations and blood lipid parameters in different preeclampsia-like mouse models.. Two preeclampsia-like mouse models and a control group were established: L-NA (injected with Nω-nitro-L-arginine methyl ester), LPS (injected with lipopolysaccharide), and the control group with normal saline (NS). The mouse models were established at preimplantation (PI), early- and late-pregnancy (EP, LP) according to the time of pregnancy. The administration of rapamycin (RA; L-NA+RA, LPS+RA, and NS+RA) or vehicle as controls (C; L-NA+C, LPS+C, NS+C) were followed on the 2nd day after the mouse models' establishment. Each subgroup consisted of eight pregnant mice. The mean arterial pressure (MAP), 24-h urinary protein, blood lipid, fetus, and placental weight were measured. The histopathological changes and lipid deposition of the liver and placenta were observed. Student's t-test was used for comparing two groups. Repeated measures analysis of variance was used for blood pressure analysis. Qualitative data were compared by Chi-square test.. The MAP and 24-h urinary protein in the PI, EP, and LP subgroups of the L-NA+C and LPS+C groups were significantly higher compared with the respective variables in the NS+C group (P < 0.05). The preeclampsia-like mouse models were established successfully. There was no significant difference in the MAP between the PI, EP, and LP subgroups of the L-NA+RA and L-NA+C groups and the LPS+RA and LPS+C groups. The 24-h urine protein levels in the PI and EP subgroups of the L-NA+RA group were significantly lower compared with the respective levels in the L-NA+C groups (1037 ± 63 vs. 2127 ± 593 μg; 976 ± 42 vs. 1238 ± 72 μg; bothP < 0.05), also this effect appeared similar in the PI and EP subgroups of the LPS+RA and LPS+C groups (1022 ± 246 vs. 2141 ± 432 μg; 951 ± 41 vs. 1308 ± 30 μg; bothP < 0.05). The levels of serum-free fatty acid (FFA) in the PI and EP subgroups of the L-NA+RA groups were significantly lower compared with the respective levels in the L-NA+C group (2.49 ± 0.44 vs. 3.30 ± 0.18 mEq/L; 2.23 ± 0.29 vs. 2.84 ± 0.14 mEq/L; bothP < 0.05). The levels of triglycerides (TG) and total cholesterol in the PI subgroup of the L-NA+RA group were significantly lower compared with the respective levels in the L-NA+C (1.51 ± 0.16 vs. 2.41 ± 0.37 mmol/L; 2.11 ± 0.17 vs. 2.47 ± 0.26 mmol/L; bothP < 0.05), whereas high-density lipoprotein serum concentration was significantly higher (1.22 ± 0.19 vs. 0.87 ± 0.15 mmol/L;P < 0.05) and low-density lipoprotein serum concentration did not exhibit a significant difference. There were no significant differences in the FFA of the PI, EP, and LP subgroups between the LPS+RA and the LPS+C groups. The levels of TG in the PI subgroup of the LPS+RA group were significantly lower compared with the respective levels in the LPS+C group (0.97 ± 0.05 vs. 1.22 ± 0.08 mmol/L;P < 0.05).. Rapamycin can improve clinical manifestations and blood lipid profile in part of the preeclampsia-like mouse models.

Topics: Animals; Blood Pressure; Chi-Square Distribution; Cholesterol; Disease Models, Animal; Female; Lipid Metabolism; Lipids; Lipoproteins, HDL; Lipoproteins, LDL; Mice; Mice, Inbred C57BL; Placenta; Pre-Eclampsia; Pregnancy; Pregnancy Outcome; Sirolimus; Triglycerides

Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype.. TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1β and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice.. In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1β and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE. TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.

Topics: ADAM10 Protein; ADAM17 Protein; Adult; Aged; Aging; Animals; Apolipoproteins E; Atherosclerosis; Biomarkers; Disease Models, Animal; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Inflammation; Interleukin-18; Interleukin-1beta; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophages; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Multiprotein Complexes; Neovascularization, Physiologic; NLR Family, Pyrin Domain-Containing 3 Protein; Peptide Fragments; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Sirolimus; Thioredoxins; TOR Serine-Threonine Kinases

Pulmonary arterial hypertension (PAH) is a common complication of congenital heart disease. However, effective treatments for PAH are rare. This study aimed to investigate the inhibitory effects of rapamycin on PAH in the carotid artery-jugular vein (CA-JV) shunt PAH rat model as well as the mechanism underlying these effects.. Twenty-four Sprague-Dawley rats were randomized into the following 3 groups: a control group, a CA-JV shunt group and a treatment group. Rapamycin (2 mg/kg/day) was administered to the treatment group, and placebo was administered to the CA-JV shunt group. Haemodynamic evaluations, pulmonary tissue samplings for morphometry and immunofluorescence and western blot analyses were performed to evaluate the effects of rapamycin on PAH.. Rapamycin attenuated the increase of right ventricular systolic pressure (RVSP) and the right ventricular (RV) hypertrophy (RVSP: CA-JV vs CA-JV + rapamycin, P = 0.017; RV: CA-JV vs CA-JV + rapamycin, P = 0.022), as well as the intrapulmonary vessel thickening (thickness index: CA-JV vs CA-JV + rapamycin, P = 0.028; area index: CA-JV vs CA-JV + rapamycin, P = 0.014), induced by overcirculation of the pulmonary vasculature in the CA-JV shunt-induced PAH rat model. Rapamycin decreased the expression level of the indicated cell proliferation marker (α-smooth muscle actin) in the lung vessel and mechanistic target of rapamycin (mTOR) pathway components (p-mTOR: CA-JV vs CA-JV + rapamycin, P = 0.004; p-Raptor: CA-JV vs CA-JV + rapamycin, P = 0.000; p-S6K1: CA-JV vs CA-JV + rapamycin, P = 0.000; p-Akt: CA-JV vs CA-JV + rapamycin, P = 0.001; p-Rheb: CA-JV vs CA-JV + rapamycin, P = 0.000) in pulmonary tissue.. Rapamycin reduced pulmonary vascular remodelling by inhibiting cell proliferation via Akt/mTOR signalling pathway down-regulation in the CA-JV shunt-induced PAH model in rats. Thus, rapamycin may be a novel candidate drug for the treatment of PAH.

Topics: Animals; Arteriovenous Shunt, Surgical; Blotting, Western; Carotid Artery, Common; Cell Proliferation; Disease Models, Animal; Down-Regulation; Hypertension, Pulmonary; Immunosuppressive Agents; Jugular Veins; Male; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Remodeling

Rapamycin (RAPA), an inhibitor of mammalian target of rapamycin (mTOR), exhibits a high neuroprotective action against neurodegenerative diseases in mouse models. Since neuroinflammation has been shown to be involved in Alzheimer's disease (AD) development and progression, the aim of this study was to examine the anti-inflammatory role of RAPA in AD in vivo and in vitro, and investigate the underlying mechanisms. We found that amyloid-β (Aβ) induced neuronal inflammation and a remarkable increase in mTOR activity in in-vivo and in-vitro models of inflammation, suggesting the critical role of mTOR signaling in neuronal inflammation. In addition, administration of RAPA was found to down-regulate mTOR, p-mTOR, Nuclear factor kappa B (NF-κB) p65, p-p65, TNF-α, IL-1β and Bax protein expression in Aβ

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Brain; Cell Line, Tumor; Cell Proliferation; Cytokines; Disease Models, Animal; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neuroblastoma; Neuroglia; NF-kappa B; Peptide Fragments; Signal Transduction; Sirolimus; Transfection

SNORD44 is a C/D box small nucleolar RNA, and exhibits low expression in breast cancer and head and neck squamous cell carcinoma tissues. Its host gene is growth arrest specific transcript 5 (GAS5), which is a long noncoding RNA. GAS5 is downregulated in colorectal cancer (CRC), and overexpression of GAS5 suppresses cell proliferation. However, the function of SNORD44 in CRC remains largely unknown, and the application of SNORD44 combined with GAS5 in CRC treatment has not been reported. In this study, the expression levels of SNORD44 and GAS5 were measured in CRC tissues by quantitative RT-PCR. The correlation between SNORD44 and GAS5 was evaluated by Pearson correlation analysis. An oncolytic adenovirus expressing SNORD44 and GAS5 (SPDD-UG) was constructed. The biological effects of SPDD-UG were investigated in CRC cell line SW620 and LS174T in vitro and in xenografts. The synergistic effect of rapamycin and SPDD-UG was explored in SW620 and LS174T cells and tumors. We demonstrated that SNORD44 expression level was markedly decreased in CRC tissues and positively correlated with GAS5 expression. SPDD-UG significantly inhibited SW620 and LS174T cell growth and induced cell apoptosis. Intratumoral injection of SPDD-UG significantly suppressed xenografts growth in nude mice. Moreover, the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin, enhanced the antitumor effect through antagonizing the PI3K/Akt pathway activated by SPDD-UG. These results suggest that overexpression of SNORD44 and GAS5 by oncolytic adenovirus provides a promising method for CRC therapy.

Topics: Adenoviridae; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Gene Expression; Gene Order; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Lentivirus; Mice; Oncolytic Viruses; RNA, Long Noncoding; RNA, Small Nucleolar; Signal Transduction; Sirolimus; Tumor Burden; Xenograft Model Antitumor Assays

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation and fibrosis. Our previous research has indicated that Sirtuin1 (Sirt1) plays a role in the regulation of TNF-α-induced inflammation; however, whether Sirt1 may inhibit the progress of SSc by blocking inflammation remains unknown.. We aimed to investigate the function of Sirt1 in SSc.. The function and its mechanism of Sirt1 were evaluated in fibroblasts or scleroderma mice. The expression of Sirt1 and cytokines was analyzed using real-time PCR, western blot, ELISA and immunohistochemistry.. We determined that fibroblasts of SSc patients were activated to exhibit inflammation. Sirt1, activated by resveratrol (Res), ameliorated cutaneous inflammation and fibrosis in bleomycin (BLM)-induced scleroderma mice. An improvement in mammalian target of rapamycin (mTOR) was identified in the fibroblasts of SSc patients and the skin lesions of BLM mice. Rapamycin, an mTOR specific inhibitor, substantially inhibited the induced inflammation and fibrosis. The enhancement of mTOR expression in the skin lesions of the BLM-treated mice was significantly inhibited by Sirt1 activation. However, in both the BLM-treated cells and mice, Res exerted an inhibitory function on the expression of inflammatory factors, and collagen was diminished following mTOR knockdown. These findings suggest that Res may inhibit inflammation and fibrosis via mTOR.. The modulation of Sirt1 activity may represent a potential therapeutic method for SSc. The mechanism may involve the inhibition of mTOR phosphorylation, whereas mTOR activity was shown to be a pathogenic culprit of SSc.

Topics: Animals; Biopsy; Bleomycin; Cells, Cultured; Collagen; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Gene Knockdown Techniques; Humans; Immunohistochemistry; Mice; Mice, Inbred C3H; Phosphorylation; Real-Time Polymerase Chain Reaction; Resveratrol; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Sirolimus; Sirtuin 1; Skin; Stilbenes; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Astrocytes have been implicated in epileptogenesis and seizure-induced brain injury. Pathological studies reveal a variety of structural abnormalities in astrocytes, such as vacuolization and astrogliosis. While in vivo imaging methods have demonstrated rapid changes in astrocytes under a variety of physiological and pathological conditions, the acute effects of seizures on astrocyte morphology in vivo and corresponding mechanisms of seizure-induced astrocytic injury have not been documented. In this study, we utilized in vivo two-photon imaging to directly monitor the acute structural effects of kainate-induced seizures on cortical astrocytes. Kainate seizures cause an immediate, but transient, vacuolization of astrocytes, followed over several days by astrogliosis. These effects are prevented by pre- or post-treatment with rapamycin, indicating the mTOR pathway is involved in mediating seizure-induced astrocyte injury. These finding have clinical implications for mechanisms of seizure-induced astrocyte injury and potential therapeutic applications with mTOR inhibitors.

Topics: Animals; Astrocytes; Disease Models, Animal; Kainic Acid; Mice; Seizures; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Vacuoles

Despite its rarity (1%-2%), acute graft-versus-host disease after liver transplantation (LT-aGVHD) has a high mortality rate (85%). A gradual decrease in regulatory T cells (Tregs) correlates with disease progression in a rat LT-GVHD model, and treatments which increase Tregs exert therapeutic effects on LT-aGVHD. In this study, LT-aGVHD model rats were treated with rapamycin (RAPA), OSI-027, or an equal quantity of vehicle. Rats treated with OSI-027 survived longer (>100 days) than those in the RAPA (70 ± 8 days) or control (24 ± 3 days) groups. Flow cytometric analysis showed that the Treg ratios in peripheral blood mononuclear cells in the OSI-027 group were higher than those in the RAPA or control groups. The proportions of donor-derived lymphocytes in the OSI-027 group were lower than those in the RAPA or control groups. Hematoxylin-eosin staining of skin tissue demonstrated less severe lymphocyte infiltration in the OSI-027 group than that in the RAPA or control groups. In vitro, OSI-027 induced differentiation of CD4

Topics: Acute Disease; Allografts; Animals; Cell Differentiation; Disease Models, Animal; Disease Progression; Female; Forkhead Transcription Factors; Graft vs Host Disease; Humans; Imidazoles; Immunosuppressive Agents; Leukocyte Count; Leukocytes, Mononuclear; Liver Transplantation; Postoperative Complications; Rats; Rats, Inbred Lew; Sirolimus; Specific Pathogen-Free Organisms; T-Lymphocytes, Regulatory; Triazines

The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.

Topics: Administration, Topical; Aminoquinolines; Animals; Caspase 14; Dendritic Cells; Disease Models, Animal; Imiquimod; Immunosuppressive Agents; Keratin-10; Keratin-14; Ki-67 Antigen; Langerhans Cells; Lymph Nodes; Macrophages; Membrane Proteins; Mice, Inbred BALB C; Neovascularization, Physiologic; Protein Precursors; Psoriasis; Sirolimus; Skin; TOR Serine-Threonine Kinases

The present study was designed to examine the effect of autophagy and apoptosis on intestinal injury in mice after severe burns.. Kunming mice were subjected to third degree burns over 30% of the total body surface area. Damage to the intestine was assessed by examining changes in intestinal mucosal morphology, enzyme-linked immunosorbent assay of serum d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α (marker of intestinal damage), hematoxylin and eosin staining, and Western blotting under 4 experimental conditions: control group, burn only (burn group), burn and administration of rapamycin to stimulate intestinal autophagy (rapamycin group), or burn and administration of 3-methyladenine to inhibit intestinal autophagy (3-methyladenine group).. At day 1 postburn, the expression levels of light chain 3 II, beclin-1, and cleaved caspase-3 were significantly greater in all 3 groups of mice subjected to the burn injury than in the control group 1 day postburn; while the levels of light chain 3 II and beclin-1 were significantly greater and those of cleaved caspase-3 were significantly less in the rapamycin group than in the burn group. In contrast, light chain 3 II and beclin-1 levels were significantly less and those of cleaved caspase-3 significantly greater in the 3-methyladenine group. All 3 groups subjected to burn injury showed significantly increased levels of d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α. Of the 3 groups, the rapamycin group exhibited the least observed levels, the 3-methyladenine group the greatest, and the burn group intermediate. Pathologic sections of the intestinal tissue showed that all 3 burn groups exhibited severe intestinal mucosal damage at 1 day postburn. The condition of the 3-methyladenine treatment group was worse than that of the rapamycin treatment group, but better than that of the burn group.. Intestinal autophagy is activated in response to intestinal apoptosis after severe burns and may alleviate burn-induced intestinal injury.

Topics: Adenine; Animals; Autophagy; Burns; Disease Models, Animal; Female; Interleukin-6; Intestinal Mucosa; Intestines; Male; Mice; Sirolimus; Tumor Necrosis Factor-alpha

Autophagy is an intracellular bulk self-degrading process in which cytoplasmic contents of abnormal proteins and excess or damaged organelles are sequestered into autophagosomes, and degraded upon fusion with lysosomes. Although autophagy is generally considered to be pro-survival, it also functions in cell death processes. We recently reported on the hippocampal, higher vulnerability to cerebral ischemia in mice lacking the circadian clock protein PERIOD1 (PER1), a phenomenon we found to be linked to a PER1-dependent modulation of the expression patterns of apoptotic/autophagic markers.. To exclude the contribution of vascular or glial factors to the innate vulnerability of Per1 knockout-mice (Per1-/--mice) to cerebral ischemia in vivo, we compared the autophagic machinery between primary hippocampal cultures from wild-type (WT)- and Per1-/--mice, using the lipophilic macrolide antibiotic, Rapamycin to induce autophagy.. Development of autophagy in WT cells involved an increased LC3-II-to-LC3-I ratio (microtubule-associated protein 1 light chain 3) and an overall increase in the level of LC3-II. In addition, immunostaining of LC3 in WT cells revealed the typical transformation of LC3 localization from a diffused staining to a dot- and ring-like pattern. In contrast, Per1-/--hippocampal cells were resistant to Rapamycin induced alterations of autophagy hallmarks.. Our in vitro data suggests that basal activity of autophagy seems to be modulated by PER1, and confirms the in vivo data by showing that the autophagic machinery is depressed in Per1-/--hippocampal neurons.The implication of both autophagy and circadian dysfunction in the pathogenesis of cerebral ischemia suggests that a functional connection between the two processes may exist.

Topics: Animals; Autophagy; Brain Ischemia; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Immunosuppressive Agents; Lysosomal-Associated Membrane Protein 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Neurons; Period Circadian Proteins; Sirolimus; Time Factors

Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin.. The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures.. In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin.. Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy.

Topics: Animals; Astrocytes; Cells, Cultured; Disease Models, Animal; Epilepsy, Temporal Lobe; Fetus; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Hippocampus; Humans; Interleukin-1beta; Male; Phosphopyruvate Hydratase; Proteasome Endopeptidase Complex; Protein Subunits; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Influenza virus infection often causes severe disease and acute respiratory distress syndrome. It is a common belief that overwhelming immune response contributes to the severe illness. Physicians and researchers have put forth immune modulation as salvage therapy for better recovery. However, empiric corticosteroid failed in both humans and animal models. Reported success with Rapamycin in humans prompted a comprehensive animal study and mechanistic dissection. Here we report the effect of Rapamycin alone or in combination with Oseltamivir for severe influenza in BALB/c mice. We found that Rapamycin had no antiviral effect against H1N1, H3N2 and novel-H1N1 influenza viruses in vitro. Rapamycin alone aggravated the severe disease of PR8 H1N1 influenza virus infection in mice. Timely Oseltamivir anti-viral therapy abolished the disease. Delayed Oseltamivir treatment could not prevent severe illness and Rapamycin adjuvant was associated with exacerbated disease. Rapamycin adjuvant suppressed influenza hemagglutinin antigen-specific T cell immunity and impaired virus clearance from the lungs. It also resulted in intensified lung pathology with increased intra-alveolar edema and hyaline deposition. Rapamycin may work as the salvage therapy for severe influenza but it is very difficult to define the appropriate window for such treatment to take effect.

Topics: Animals; Antiviral Agents; Disease Models, Animal; Disease Progression; Immunity, Cellular; Influenza A virus; Lung; Mice; Mice, Transgenic; Orthomyxoviridae Infections; Oseltamivir; Severity of Illness Index; Sirolimus; T-Lymphocytes

Studies about reconsolidation of conditioned fear memories have shown that pharmacological manipulation at memory reactivation can attenuate or enhance the subsequent expression of the conditioned fear response. Here we examined the effects of a single injection of the mTOR inhibitor rapamycin (Rap) into the infralimbic (IL) and prelimbic (PL) areas [which compose the ventromedial prefrontal cortex (PFC)] on reconsolidation and extinction of a traumatic fear memory. We found opposite effects of Rap infused into the PL and IL on reconsolidation and extinction: intra-PL Rap and systemic Rap impaired reconsolidation and facilitated extinction whereas intra-IL Rap enhanced reconsolidation and impaired extinction. These effects persisted at least 10 days after reactivation. Shock exposure induced anxiety-like behavior and impaired working memory and intra-IL and -PL Rap normalized these effects. Finally, when measured after fear retrieval, shocked rats exhibited reduced and increased phosphorylated p70s6K levels in the IL and basolateral amygdala, respectively. No effect on phosphorylated p70s6K levels was observed in the PL. The study points to the differential roles of the IL and PL in memory reconsolidation and extinction. Moreover, inhibiting mTOR via rapamycin following reactivation of a fear memory may be a novel approach in attenuating enhanced fear memories.

Topics: Animals; Anxiety; Avoidance Learning; Basolateral Nuclear Complex; Conditioning, Psychological; Disease Models, Animal; Electroshock; Extinction, Psychological; Fear; Male; Memory Consolidation; Memory, Short-Term; Nootropic Agents; Phosphorylation; Prefrontal Cortex; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Stress Disorders, Post-Traumatic; TOR Serine-Threonine Kinases

Myasthenia gravis (MG) is an autoimmune disease commonly treated with immunosuppressants. We evaluated the novel immunosuppressant, rapamycin (RAPA), in a rat model of experimental autoimmune MG (EAMG). Mortality rates in the RAPA (12%) were significantly down compared with the EAMG (88%) or cyclophosphamide (CTX) (68%) intervention groups. Muscular weakness decreased after both RAPA and CTX treatment. However, Lennon scores were lower (1.74 ± 0.49, 3.39 ± 0.21, and 3.81 ± 0.22 in RAPA, CTX, and EAMG groups, respectively), and body weights (203.12 ± 4.13 g, 179.23 ± 2.13 g, and 180.13 ± 5.13 g in RAPA, CTX, and EAMG groups, respectively) were significantly higher, only in the RAPA group. The proportion of regulatory T cells (Treg) significantly increased, while that of Th17 cells significantly decreased in the RAPA group compared with the EAMG group. In comparison, CTX intervention resulted in increased Th17 but significantly decreased Tregs. Hence, RAPA can be more effectively used in comparison with CTX to treat MG, with an efficacy higher than that of CTX. In addition, our results suggest RAPA's efficacy in alleviating symptoms of MG stems from its ability to correct the Treg/Th17 imbalance observed in MG.

Topics: Animals; Disease Models, Animal; Female; Inflammation; Male; Muscle Weakness; Myasthenia Gravis; Rats; Sirolimus; T-Lymphocytes, Regulatory; Th17 Cells

Mild traumatic brain injury (mTBI), common in juveniles, has been reported to be caused by sports-related concussion. Many young children may suffer from post-concussion syndrome. mTBI, in early stages of life, could play a part in neuron apoptosis and degeneration, cognitive and motor coordination impairment, as well as dementia. Our study was aimed at further investigating the post-therapeutic efficacy of rapamycin in the recuperation of mTBI while at the same time investigating the metamorphosis in both autophagy and mitophagy in mTBI. We created a weight-drop rat mTBI model with the administration of rapamycin at 4 h after every mTBI. Behavioral tests of beam walking and open field task indicated the expected improvement of cognitive and motor coordination functions. Both Western blot and immunofluorescence examinations revealed increased Beclin-1 and PINK1 in the treated rats as well as reduction of caspase-3 and cytochrome C (Cyt C). More so, the TUNEL staining evidenced curtailment of apoptotic cells following treatment with rapamycin. The upregulation of Beclin-1 and PINK1 and the downregulation of caspase-3 and Cyt C extrapolate that rapamycin plays neuroprotective as well as anti-apoptotic role via interposition of both autophagy and mitophagy.

Topics: Animals; Apoptosis; Autophagy; Brain Concussion; Disease Models, Animal; Male; Mitophagy; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Sirolimus; Up-Regulation

To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3-5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1-2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5-6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.

Topics: Animals; Animals, Newborn; Apoptosis; Autophagosomes; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Protein-1 Homolog; Beclin-1; Brain; Cells, Cultured; Disease Models, Animal; Hypoxia-Ischemia, Brain; Lysosomes; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Neurons; Sequestosome-1 Protein; Sirolimus; Swine

Acquired aplastic anemia, the prototypical bone marrow failure disease, is characterized by pancytopenia and marrow hypoplasia. Most aplastic anemia patients respond to immunosuppressive therapy, usually with anti-thymocyte globulin and cyclosporine, but some relapse on cyclosporine withdrawal or require long-term administration of cyclosporine to maintain blood counts. In this study, we tested efficacy of rapamycin as a new or alternative treatment in mouse models of immune-mediated bone marrow failure. Rapamycin ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of cyclosporine. Rapamycin effectively reduced Th1 inflammatory cytokines interferon-γ and tumor necrosis factor-α, increased the Th2 cytokine interleukin-10, stimulated expansion of functional regulatory T cells, eliminated effector CD8

Topics: Anemia, Aplastic; Animals; Bone Marrow; Bone Marrow Diseases; Bone Marrow Failure Disorders; Disease Models, Animal; Epitopes, T-Lymphocyte; Hemoglobinuria, Paroxysmal; Immunologic Memory; Immunosuppressive Agents; Mice; Pancytopenia; Signal Transduction; Sirolimus; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Treatment Outcome

Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism.. C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis.. This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway.. This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent.

Topics: Animals; Cell Survival; Cells, Cultured; Disease Models, Animal; Epidermal Cells; Epidermis; Immunohistochemistry; Insulin-Like Growth Factor I; Interleukin-15; Male; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred C57BL; Phosphorylation; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Signal Transduction; Sirolimus; Wound Healing

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is frequently activated in cancers and can be counteracted with the clinical mTORC1 inhibitors everolimus and temsirolimus. Although mTORC1 and dual mTORC1/2 inhibitors are currently under development to treat various malignancies, the emergence of drug resistance has proven to be a major complication. Using the cis-Apc/Smad4 mouse model of locally invasive intestinal adenocarcinoma, we show that administration of everolimus or the dual mTORC1/2 inhibitor AZD8055 significantly reduces the growth of intestinal tumors. In contrast, although everolimus treatment at earlier phase of tumor progression delayed invasion of the tumors, both inhibitors exhibited little effect on blocking invasion of the tumors when administered later in their progression. Biochemical and immunohistochemical analyses revealed that treatment of cis-Apc/Smad4 mice with everolimus or AZD8055 induced marked increases in epidermal growth factor receptor (EGFR) and MEK/ERK signaling in tumor epithelial and stromal cells, respectively. Notably, co-administration of AZD8055 and the EGFR inhibitor erlotinib or the MEK inhibitor trametinib was sufficient to suppress tumor invasion in cis-Apc/Smad4 mice. These data indicate that mTOR inhibitor resistance in invasive intestinal tumors involves feedback signaling from both cancer epithelial and stromal cells, highlighting the role of tumor microenvironment in drug resistance, and support that simultaneous inhibition of mTOR and EGFR or MEK may be more effective in treating colon cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Everolimus; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Intestinal Neoplasms; MAP Kinase Signaling System; Mice, 129 Strain; Mice, Inbred C57BL; Morpholines; Neoplasm Invasiveness; Sirolimus; TOR Serine-Threonine Kinases; Tumor Microenvironment

Tuberous sclerosis complex (TSC) is a rare monogenic disorder characterized by benign tumors in multiple organs as well as a high prevalence of epilepsy, intellectual disability and autism. TSC is caused by inactivating mutations in the. Molecular alterations in the frontal cortex and hippocampus of. LC-MS. Molecular changes in the

Topics: Animals; Disease Models, Animal; Frontal Lobe; Hippocampus; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Proteomics; Sirolimus; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

We estimated the effect of various immunosuppressants (ISs) and metformin (M) to provide theoretical background of optimal therapeutic strategy for de novo colon cancer after liver transplantation (LT). Three colon cancer cell lines (HT29, SW620, and HCT116) were used in in vitro studies. HT29 was also used in BALB/c-nude mice animal models. Following groups were used in both in vitro and in vivo studies: sirolimus (S), tacrolimus (T), cyclosporin A (CsA), M, metformin/sirolimus (Met/S), metformin/tacrolimus (Met/T), and metformin/cyclosporin A (Met/CsA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed and western blot analyses were performed for mTOR pathway proteins, apoptosis proteins, and epithelial-mesenchymal-transition (EMT) proteins. Tumor volume was measured for 4 weeks after inoculation. MTT-assay revealed significant cell viability inhibition in all 3 colon cancer cell lines in groups of S, M, and Met/S. Of note, group Met/S showed synergistic effect compare to M or S group. Western blot analysis showed significant low levels of all investigated proteins in groups of S and Met/S in both in vitro and in vivo experiment. Tumor growth was significantly inhibited only in the Met/S group. Combination of Met and S showed the most potent inhibition in all colon cancer cell lines. This finding might have application for de novo colon cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Cyclosporine; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; HCT116 Cells; HT29 Cells; Humans; Metformin; Mice; Mice, Inbred BALB C; Mice, Nude; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Smad3 Protein; Tacrolimus; Transplantation, Heterologous

Cell-to-cell spreading of misfolded α-synuclein (α-syn) is suggested to contribute to the progression of neuropathology in Parkinson's disease (PD). Compelling evidence supports the hypothesis that misfolded α-syn transmits from neuron-to-neuron and seeds aggregation of the protein in the recipient cells. Furthermore, α-syn frequently appears to propagate in the brains of PD patients following a stereotypic pattern consistent with progressive spreading along anatomical pathways. We have generated a C. elegans model that mirrors this progression and allows us to monitor α-syn neuron-to-neuron transmission in a live animal over its lifespan. We found that modulation of autophagy or exo/endocytosis, affects α-syn transfer. Furthermore, we demonstrate that silencing C. elegans orthologs of PD-related genes also increases the accumulation of α-syn. This novel worm model is ideal for screening molecules and genes to identify those that modulate prion-like spreading of α-syn in order to target novel strategies for disease modification in PD and other synucleinopathies.

Topics: Adenosine Triphosphatases; Aldehyde Oxidoreductases; alpha-Synuclein; Animals; Autophagy; Brain; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Communication; Discoidin Domain Receptor 2; Disease Models, Animal; Endocytosis; Exocytosis; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Neurons; Parkinson Disease, Secondary; Protein Aggregates; Protein Serine-Threonine Kinases; Protein Transport; RNA, Small Interfering; Sirolimus; Spectrometry, Fluorescence; Tryptophan Hydroxylase; Ubiquitin-Protein Ligases

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary cause of death is cardiovascular disease at about 14 years. Loss and dysfunction of smooth muscle cells (SMCs) in the vasculature may cause defects associated with HGPS. Due to limitations of 2D cell culture and mouse models, there is a need to develop improved models to discover novel therapeutics. To address this need, we produced a functional three-dimensional model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived SMCs from an HGPS patient. To isolate the effect of the HGPS iSMCs, the endothelial layer consisted of human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor. TEBVs fabricated from HGPS iSMCs and hCB-EPCs show reduced vasoactivity, increased medial wall thickness, increased calcification and apoptosis relative to TEBVs fabricated from normal iSMCs or primary MSCs. Additionally, treatment of HGPS TEBVs with the proposed therapeutic Everolimus, increases HGPS TEBV vasoactivity and increases iSMC differentiation in the TEBVs. These results show the ability of this iPSC-derived TEBV to reproduce key features of HGPS and respond to drugs.

Topics: Animals; Biomarkers; Blood Vessels; Cell Differentiation; Disease Models, Animal; Everolimus; Fibroblasts; Gene Expression; Humans; Induced Pluripotent Stem Cells; Lamin Type A; Mutation; Myocytes, Smooth Muscle; Phenotype; Progeria; Sirolimus; Tissue Engineering

Lung cancer is the leading cause of cancer-related deaths with small-cell lung cancer (SCLC) as the most aggressive subtype. Preferential occurrence of TP53 missense mutations rather than loss implicates a selective advantage for TP53-mutant expression in SCLC pathogenesis. We show that lung epithelial expression of R270H and R172H (R273H and R175H in humans), common TRP53 mutants in lung cancer, combined with RB1 loss selectively results in two subtypes of neuroendocrine carcinoma, SCLC and large cell neuroendocrine carcinoma (LCNEC). Tumor initiation and progression occur in a remarkably consistent time frame with short latency and uniform progression to lethal metastatic disease by 7 months. R270H or R172H expression and TRP53 loss result in similar phenotypes demonstrating that TRP53 mutants promote lung carcinogenesis through loss-of-function and not gain-of-function mechanisms. Tumor responses to targeted and cytotoxic therapeutics were discordant in mice and corresponding tumor cell cultures demonstrating need to assess therapeutic response at the organismal level. Rapamycin did not have therapeutic efficacy in the mouse model despite inhibiting mTOR signaling and markedly suppressing tumor cell growth in culture. In contrast, cisplatin/etoposide treatment using a patient regimen prolonged survival with development of chemoresistance recapitulating human responses. R270H, but not R172H, expression conferred gain-of-function activity in attenuating chemotherapeutic efficacy. These data demonstrate a causative role for TRP53 mutants in development of chemoresistant lung cancer, and provide tractable preclinical models to test novel therapeutics for refractory disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Disease Models, Animal; Disease Progression; Drug Resistance, Neoplasm; Etoposide; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neuroendocrine Tumors; Sirolimus; Small Cell Lung Carcinoma; Tumor Suppressor Protein p53

Rheumatoid arthritis (RA) is a chronic inflammatory disease, which is associated with symptoms, including synovial membrane inflammatory pain, joint synovitis and stiffness. However, there are no effective methods available to cure this disease. In the present study, rapamycin was used to modulate immunity in RA. To limit the cytotoxicity of rapamycin, rapamycin was loaded into well‑characterized biocompatible nanoparticles. In vitro, rapamycin particles downregulated the activation of dendritic cell surface markers, including CD80+ and CD40+, upon interacting with macrophages. The rapamycin particles reduced the secretion of inflammatory cytokines, including interleukin (IL)‑6, tumor necrosis factor (TNF) and IL‑1β, which are characteristic of RA. In vivo, the rapamycin particles decreased the symptoms of RA in mice, and the production of inflammatory cytokines was associated with the occurrence of RA. The present study partially revealed the interactions between rapamycin and two types of immune cell in RA disease, and may potentially offer a solution to improve the treatment of RA.

Topics: Animals; Arthritis, Rheumatoid; Cell Survival; Cytokines; Dendritic Cells; Disease Models, Animal; Immunity; Male; Mice, Inbred DBA; Nanoparticles; Sirolimus; Spleen

C-reactive protein (CRP), was recently reported to be closely associated with poor renal function in patients with acute kidney injury (AKI), but whether CRP is pathogenic or a mere biomarker in AKI remains largely unclear. Impaired autophagy is known to exacerbate renal ischemia-reperfusion injury (IRI). We examined whether the pathogenic role of CRP in AKI is associated with reduction of autophagy. We mated transgenic rabbit CRP over-expressing mice (Tg-CRP) with two autophagy reporter mouse lines, Tg-GFP-LC3 mice (LC3) and Tg-RFP-GFP-LC3 mice (RG-LC3) respectively to generate Tg-CRP-GFP-LC3 mice (PLC3) and Tg-CRP-RFP-GFP-LC3 mice (PRG-LC3). AKI was induced by IRI. Compared with LC3 mice, PLC3 mice developed more severe kidney damage after IRI. Renal tubules were isolated from LC3 mice at baseline for primary culture. OKP cells were transiently transfected with GFP-LC3 plasmid. CRP addition exacerbated lactate dehydrogenase release from both cell types. Immunoblots showed lower LC-3 II/I ratios and higher levels of p62, markers of reduced autophagy flux, in the kidneys of PLC3 mice compared to LC3 mice after IRI, and in primary cultured renal tubules and OKP cells treated with CRP and H2O2 compared to H2O2 alone. Immunohistochemistry showed much fewer LC-3 punctae, and electron microscopy showed fewer autophagosomes in kidneys of PLC3 mice compared to LC3 mice after IRI. Similarly, CRP addition reduced GFP-LC3 punctae induced by H2O2 in primary cultured proximal tubules and in GFP-LC3 plasmid transfected OKP cells. Rapamycin, an autophagy inducer, rescued impaired autophagy and reduced renal injury in vivo. In summary, it was suggested that CRP be more than mere biomarker in AKI, and render the kidney more susceptible to ischemic/oxidative injury, which is associated with down-regulating autophagy flux.

Topics: Acute Kidney Injury; Animals; Autophagy; Beclin-1; C-Reactive Protein; Disease Models, Animal; Epithelial Cells; Gene Expression; Humans; Kidney Tubules; Mice; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Rabbits; Reperfusion Injury; Severity of Illness Index; Sirolimus

GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia.

Topics: Animals; Autophagy; Disease Models, Animal; Down-Regulation; Endoplasmic Reticulum Stress; Glucose-6-Phosphatase; Glucose-6-Phosphate; Glycogen Storage Disease Type I; Immunosuppressive Agents; Kidney; Mice; Sirolimus

Excessive mechanical loading is a major factor affecting heterotopic ossification (HO), which is a major pathological alteration in calcific tendinopathy. However, physical therapies with mechanical loading as the functional element have exhibited promising results in the treatment of calcific tendinopathy. The dual effects that mechanical loading may have on the pathogenesis and rehabilitation of calcified tendinopathy remain unclear. The present study was designed to investigate the effects of mechanical loading on HO in calcific tendinopathy. In the present study, a tendon cell in vitro stretch model and an Achilles tenotomy rat model were used to simulate different elongation mechanical loading scenarios in order to investigate the effects of mechanical loading on HO of the tendon. In addition, rapamycin, a selective mammalian target of rapamycin complex‑1 (mTORC1) signaling pathway inhibitor, was employed to determine whether mechanical loading modulates heterotopic ossification in calcific tendinopathy through the mTORC1 signaling pathway. The data indicate that mechanical loading modulated HO of the tendon through the mTORC1 signaling pathway, and that low elongation mechanical loading attenuated HO, while high elongation mechanical loading accelerated HO in vivo. This study may improve the understanding of the effect of physical therapies used to treat calcific tendinopathy, so as to guide clinical treatment more effectively. Furthermore, rapamycin may be a potential drug for the treatment of calcific tendinopathy.

Topics: Achilles Tendon; Animals; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Gene Expression Regulation; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mechanotransduction, Cellular; Ossification, Heterotopic; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Sirolimus; Tendinopathy; Tenocytes; Tenotomy; Transcription Factors; Weight-Bearing

In nutrient restricted environments, the yeast endosulfines Igo1/2 are activated via TORC1 inhibition and function critically to initiate and coordinate the cellular stress response that promotes survival. We examined expression of αEnsa, the mammalian homolog of yeast endosulfines, in rat stroke. Prominent neuronal upregulation of αEnsa was identified in 3 patterns within the ischemic gradient: (1) neurons in GFAP-/HSF1+ cortex showed upregulation and near-complete nuclear translocation of αEnsa protein within hours of ischemic onset; (2) neurons in GFAP+/HSF1+ cortex showed upregulation in cytoplasm and nuclei that persisted for days; (3) neurons in GFAP+/HSF1- cortex showed delayed cytosolic-only upregulation that persisted for days. Findings were corroborated using in situ hybridization for ENSA mRNA. Rapamycin treatment was found to reduce infarct size and behavioral deficits and, in GFAP+/HSF1+ zones, enhance αEnsa neuronal nuclear translocation and mitigate cell death, relative to controls. Based on the conservation of TOR signaling across species, and on the finding that the Rim15-Igo1/2-PP2A module is triggered by substrate deprivation in eukaryotic yeast, we speculate that αEnsa is activated by substrate deprivation, functioning through the homologous MASTL-αEnsa/ARPP19-PP2A module to promote neuronal survival. In conjunction with recent studies suggesting a neuroprotective role, our data highlight a potential function for αEnsa within ischemic brain.

Topics: Animals; Brain; Brain Infarction; Cell Adhesion Molecules; Chaperonin 60; Disease Models, Animal; Gene Expression Regulation; Humans; Immunosuppressive Agents; Intercellular Signaling Peptides and Proteins; Male; Mitochondrial Proteins; Nerve Tissue Proteins; Neurons; Peptides; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; Sirolimus; Somatostatin; Stroke; Time Factors

Topics: Anemia, Aplastic; Animals; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Mice; Mice, Inbred C57BL; Pancytopenia; Sirolimus

Mitochondrial disorders affecting oxidative phosphorylation (OxPhos) are caused by mutations in both the nuclear and mitochondrial genomes. One promising candidate for treatment is the drug rapamycin, which has been shown to extend lifespan in multiple animal models, and which was previously shown to ameliorate mitochondrial disease in a knock-out mouse model lacking a nuclear-encoded gene specifying an OxPhos structural subunit (Ndufs4). In that model, relatively high-dose intraperitoneal rapamycin extended lifespan and improved markers of neurological disease, via an unknown mechanism. Here, we administered low-dose oral rapamycin to a knock-in (KI) mouse model of authentic mtDNA disease, specifically, progressive mtDNA depletion syndrome, resulting from a mutation in the mitochondrial nucleotide salvage enzyme thymidine kinase 2 (TK2). Importantly, low-dose oral rapamycin was sufficient to extend Tk2KI/KI mouse lifespan significantly, and did so in the absence of detectable improvements in mitochondrial dysfunction. We found no evidence that rapamycin increased survival by acting through canonical pathways, including mitochondrial autophagy. However, transcriptomics and metabolomics analyses uncovered systemic metabolic changes pointing to a potential 'rapamycin metabolic signature.' These changes also implied that rapamycin may have enabled the Tk2KI/KI mice to utilize alternative energy reserves, and possibly triggered indirect signaling events that modified mortality through developmental reprogramming. From a therapeutic standpoint, our results support the possibility that low-dose rapamycin, while not targeting the underlying mtDNA defect, could represent a crucial therapy for the treatment of mtDNA-driven, and some nuclear DNA-driven, mitochondrial diseases.

Topics: Animals; Autophagy; Disease Models, Animal; DNA, Mitochondrial; Dose-Response Relationship, Drug; Electron Transport Complex I; Female; Gene Knock-In Techniques; Male; Mice; Mitochondria; Mitochondrial Diseases; Mutation; Oxidative Phosphorylation; Signal Transduction; Sirolimus; Syndrome; Thymidine Kinase

To determine whether P7C3-A20 can inhibit the phosphorylation of the mammalian target of rapamycin (mTOR), depress neuroinflammation, and protect retinal ganglion cells (RGCs) of rats from optic nerve crush (ONC).. The left optic nerve was crushed, and 5.0 mg/kg/d of P7C3-A20, 1.0 mg/kg/d of rapamycin, or their vehicle was injected intraperitoneally for 3 consecutive days beginning 1 day before the ONC. The protective effects on the RGCs were determined by immunohistochemical staining for Tuj-1. The level of phosphorylated mTOR was determined by immunoblotting. The neuroinflammation in the optic nerve was determined by changes in the expression of CD68, TNF-α, MCP-1, and iNOS.. The density of Tuj-1-stained cells in the control was 2010 ± 81.5/mm2 and 1842 ± 80.4/mm2 on days 7 and 14 after the sham operation. These levels were lower at 995 ± 122/mm2 and 450 ± 52.4/mm2 on days 7 and 14 after the ONC, respectively. Rapamycin and P7C3-A20 preserved the density at significantly higher levels on both days (P < 0.05, Scheffe test). The level of phosphorylated mTOR increased by 1.56-fold above the control level on day 7. Rapamycin and P7C3 significantly lowered the level of phosphorylated mTOR to 0.89-fold and 0.67-fold of the control, respectively. There was an accumulation of CD68+ cells that were immunoreactive to TNF-α at the crush site. The expression of MCP-1 and iNOS was increased chiefly in the astrocytes around the lesion. These inflammatory events were suppressed by both rapamycin and P7C3.. P7C3-A20 can inhibit mTOR phosphorylation in the crushed optic nerve, which may suppress neuroinflammation and preserve the RGCs.

Topics: Animals; Anti-Inflammatory Agents; Biomarkers; Carbazoles; Disease Models, Animal; Male; Nerve Crush; Neuroprotective Agents; Optic Nerve; Optic Nerve Injuries; Phosphorylation; Rats; Rats, Wistar; Retinal Ganglion Cells; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia.. We found that germ-line-specific or smooth muscle-specific deletion of. Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.

Topics: Animals; Binding Sites; Cell Cycle Proteins; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Forkhead Transcription Factors; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Nuclear Proteins; Phenotype; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; Signal Transduction; Sirolimus; Time Factors; Trans-Activators; Transcription Factors; Transfection; Vascular System Injuries

A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.

Topics: Animals; Blast Injuries; Bone and Bones; Disease Models, Animal; Male; Ossification, Heterotopic; Osteogenesis; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; X-Ray Microtomography

Dry eye disease (DED) is a highly prevalent, ocular disorder characterized by an abnormal tear film and ocular surface. Recent experimental data has suggested that the underlying pathology of DED involves inflammation of the lacrimal functional unit (LFU), comprising the cornea, conjunctiva, lacrimal gland and interconnecting innervation. This inflammation of the LFU ultimately results in tissue deterioration and the symptoms of DED. Moreover, an increase of pathogenic lymphocyte infiltration and the secretion of pro-inflammatory cytokines are involved in the propagation of DED-associated inflammation. Studies have demonstrated that the adoptive transfer of regulatory T cells (Tregs) can mediate the inflammation caused by pathogenic lymphocytes. Thus, as an approach to treating the inflammation associated with DED, we hypothesized that it was possible to enrich the body's own endogenous Tregs by locally delivering a specific combination of Treg inducing factors through degradable polymer microspheres (TRI microspheres; TGF-β1, Rapamycin (Rapa), and IL-2). This local controlled release system is capable of shifting the balance of Treg/T effectors and, in turn, preventing key signs of dry eye disease such as aqueous tear secretion, conjunctival goblet cells, epithelial corneal integrity, and reduce the pro-inflammatory cytokine milieu in the tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Delayed-Action Preparations; Disease Models, Animal; Dry Eye Syndromes; Female; Inflammation; Interleukin-2; Lacrimal Apparatus; Mice, Inbred BALB C; Microspheres; Polymers; Sirolimus; T-Lymphocytes, Regulatory; Transforming Growth Factor beta1

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

Topics: Adenine; Animals; Apoptosis; Autophagy; Disease Models, Animal; Genotype; In Situ Nick-End Labeling; Mice; Motor Neurons; Muscular Atrophy, Spinal; Sirolimus

ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.. The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.. The activation of A. The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Animals; Cell Line; Cell Proliferation; CREB-Binding Protein; Cyclic AMP; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Extracellular Signal-Regulated MAP Kinases; Genetic Predisposition to Disease; Humans; Kidney; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Phosphorylation; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; TRPP Cation Channels

Achieving long-term cardiac allograft survival without continuous immunosuppression is highly desired in organ transplantation. Studies have shown that Salvia miltiorrhiza, an herb also known as danshen, improves microcirculation and is highly effective in treating coronary heart disease. Our objective is to determine whether tanshinol, an ingredient of danshen, improves cardiac allograft survival.. Fully vascularized heterotopic heart transplantation was performed using BALB/c mice as donors and C57BL/6 mice as recipients, which were then treated with tanshinol and rapamycin. CD4. We found that tanshinol significantly delayed cardiac allograft rejection. It promoted long-term allograft survival induced by rapamycin, a mammalian target-of-rapamycin (mTOR) inhibitor. Tanshinol increased CD4. Tanshinol suppresses cardiac allograft rejection by recruiting CD4

Topics: Allografts; Animals; Biomarkers; Biopsy, Needle; Blotting, Western; Caffeic Acids; Chemokine CCL22; Disease Models, Animal; Graft Rejection; Graft Survival; Heart Transplantation; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Random Allocation; Sensitivity and Specificity; Sirolimus; Tissue Donors; Transplant Recipients; Transplantation Immunology; Treatment Outcome

Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ~10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.

Topics: Administration, Oral; Adoptive Transfer; Animals; Antibodies; Antibodies, Monoclonal; Antigens, CD20; B-Lymphocytes; Coagulants; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Factor VIII; Hemophilia A; Immune Tolerance; Immunoglobulin G; Immunosuppressive Agents; Injections, Intravenous; Male; Mice, Inbred BALB C; Mice, Knockout; Sirolimus; T-Lymphocytes, Regulatory; Time Factors

mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Diphtheria Toxin; Disease Models, Animal; Flow Cytometry; Humans; Immunotherapy; Interleukin-2; Jurkat Cells; Lymphocyte Activation; Lymphoma, T-Cell; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Recombinant Fusion Proteins; Sirolimus; T-Lymphocytes

To investigate the role of mTOR signaling pathway in bone marrow mesenchymal stem cells (BMSCs) differentiation into osteoblast in degenerative scoliosis (DS). The rat model of DS was established. Thirty-two Sprague-Dawley (SD) rats were selected and divided into the normal control group, the positive control group (normal rats injected with rapamycin), the negative control group (DS rats injected with PBS) and the experiment group (DS rats injected with rapamycin). H&E staining was performed to observe the osteogenesis of scoliosis. The BMSCs were obtained and assigned into seven groups: the normal control group, the positive control group, the negative control group and 1.0/10.0/100.0/1000.0 nmol/L experiment groups. Flow cytometry was conducted to testify cell cycle. The mRNA and protein expressions of mTOR and osteoblastic differentiation markers were measured by qRT-PCR and western blotting. In vivo, compared with the negative control group, bone trabecular area and the number of differentiated bone cells were significantly increased in the experiment groups. In vitro, at 24 and 48 h after rapamycin treatment, compared with the negative control group, BMSCs at G0/G1 stage increased, but BMSCs at S stage decreased in the 1.0/10.0/100.0/1000.0 nmol/L experiment groups; the expressions of mTOR and p70-S6K1 proteins were reduced in the 1.0/10.0/100.0/1000.0 nmol/L experiment groups, while ALP activity, OC levels, calcium deposition, Co1-I protein expression and the mRNA expressions of OC and Co1-I were significantly increased. Suppression of mTOR signaling pathway by rapamycin could promote BMSCs differentiation into osteoblast in DS.

Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; In Vitro Techniques; Mesenchymal Stem Cells; Osteoblasts; Osteogenesis; Rats; Rats, Sprague-Dawley; Scoliosis; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The locomotor deficits in the group of diseases referred to as hereditary spastic paraplegia (HSP) reflect degeneration of upper motor neurons, but the mechanisms underlying this neurodegeneration are unknown. We established a Drosophila model for HSP, atlastin (atl), which encodes an ER fusion protein. Here, we show that neuronal atl loss causes degeneration of specific thoracic muscles that is preceded by other pathologies, including accumulation of aggregates containing polyubiquitin, increased generation of reactive oxygen species and activation of the JNK-Foxo stress response pathway. We show that inhibiting the Tor kinase, either genetically or by administering rapamycin, at least partially reversed many of these pathologies. atl loss from muscle also triggered muscle degeneration and rapamycin-sensitive locomotor deficits, as well as polyubiquitin aggregate accumulation. These results indicate that atl loss triggers muscle degeneration both cell autonomously and nonautonomously.

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Gene Knockdown Techniques; Larva; Longevity; Male; Motor Activity; Muscle Cells; Neurons; Phenotype; Polyubiquitin; Reactive Oxygen Species; Signal Transduction; Sirolimus; Spastic Paraplegia, Hereditary

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. It is one of the most common heritable human diseases with eventual development of renal failure; however, effective treatment is lacking. While inhibition of mechanistic target of rapamycin (mTOR) effectively slows cyst expansions in animal models, results from clinical studies are controversial, prompting further mechanistic studies of mTOR-based therapy. Here, we aim to establish autophagy, a downstream pathway of mTOR, as a new therapeutic target for PKD. We generated zebrafish mutants for pkd1 and noted cystic kidney and mTOR activation in pkd1a mutants, suggesting a conserved ADPKD model. Further assessment of the mutants revealed impaired autophagic flux, which was conserved in kidney epithelial cells derived from both Pkd1-null mice and ADPKD patients. We found that inhibition of autophagy by knocking down the core autophagy protein Atg5 promotes cystogenesis, while activation of autophagy using a specific inducer Beclin-1 peptide ameliorates cysts in the pkd1a model. Treatment with compound autophagy activators, including mTOR-dependent rapamycin as well as mTOR-independent carbamazepine and minoxidil, markedly attenuated cyst formation and restored kidney function. Finally, we showed that combination treatment with low doses of rapamycin and carbamazepine was able to attenuate cyst formation as effectively as a single treatment with a high dose of rapamycin alone. In summary, our results suggested a modifying effect of autophagy on ADPKD, established autophagy activation as a novel therapy for ADPKD, and presented zebrafish as an efficient vertebrate model for developing PKD therapeutic strategies.

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Carbamazepine; Cells, Cultured; Disease Models, Animal; Embryo, Nonmammalian; Epithelial Cells; Mice; Mice, Knockout; Polycystic Kidney, Autosomal Dominant; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels; Zebrafish

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

Topics: Adult; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Female; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Mutant Strains; Middle Aged; Multiprotein Complexes; Muscle Fibers, Skeletal; Muscle Relaxation; Myotonic Dystrophy; Myotonin-Protein Kinase; Ribonucleotides; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Neurodegeneration, along with inflammatory demyelination, is an important component of multiple sclerosis (MS) pathogenesis. Autophagy is known to play a pivotal role in neuronal homeostasis and is implicated in several neurodegenerative disorders. However, whether autophagy is involved in the mechanisms of neuronal damage during MS remains to be investigated. Experimental autoimmune encephalomyelitis (EAE), an in vivo model of MS, was induced in female C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein p35-55. After that, autophagic flux in the spinal cord of mice was evaluated by detection of LC3-II and Beclin1 protein expressions. EAE mice were then administered with rapamycin and 3-methyladenine (3-MA) for 10 days. Afterward, the changes in LC3-II, Beclin1, and p62 expression, number of infiltrated inflammatory cells, demyelinated lesion area, and neuronal damage, as well as clinical scores, were assessed. Further, apoptotic cell rate and apoptosis-related protein expressions were monitored. We observed an impaired autophagic flux and increased neuronal damage in the spinal cords of EAE mice. We also found that rapamycin, an autophagy inducer, mitigated EAE-induced autophagy decrease, inflammation, demyelination and neuronal injury, as well as the abnormal clinical score. In addition, rapamycin suppressed cell apoptosis, and decreased Bax/Bcl-2 ratio and cleaved caspase-3 expression. Conversely, the effect of autophagy inhibitor 3-MA on EAE mice resulted in completely opposite results. These results indicated that autophagy deficiency, at least in part, contributed to EAE-induced neuronal injury and that pharmacological modulation of autophagy might be a therapeutic strategy for MS.

Topics: Adenine; Animals; Apoptosis; Autophagy; Beclin-1; Caspase 3; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Inflammation; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Microtubule-Associated Proteins; Multiple Sclerosis; Neurons; Sirolimus; Spinal Cord

To evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome.. We developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity.. Lymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P < 0.0001) and 1.68-fold in lacrimal gland lysates (P = 0.003) relative to vehicle-treated mice. Rapamycin significantly altered the expression of several genes linked to Sjögren's syndrome pathogenesis, including major histocompatibility complex II, TNF-α, IFN-γ, and IL-12a, as well as Akt3, an effector of autophagy.. Our findings suggest that topical rapamycin reduces autoimmune-mediated lacrimal gland inflammation while improving ocular surface integrity and tear secretion, and thus has potential for treating Sjögren's syndrome-associated dry eye.

Topics: Animals; Cathepsins; Conjunctiva; Dacryocystitis; Disease Models, Animal; Follow-Up Studies; Gene Expression Regulation; Immunosuppressive Agents; Lacrimal Apparatus; Male; Mice; Mice, Inbred NOD; Ophthalmic Solutions; Real-Time Polymerase Chain Reaction; Sirolimus; Sjogren's Syndrome; Tears

The mammalian target of rapamycin (mTOR) is a kinase involved in a variety of physiological and pathological functions. However, the exact role of mTOR in excitotoxicity is poorly understood. Here, we investigated the effects of mTOR inhibition with rapamycin against neurodegeneration, and motor impairment, as well as inflammatory profile caused by an excitotoxic stimulus.. A single and unilateral striatal injection of quinolinic acid (QA) was used to induce excitotoxicity in mice. Rapamycin (250 nL of 0.2, 2, or 20 μM; intrastriatal route) was administered 15 min before QA injection. Forty-eight hours after QA administration, rotarod test was performed to evaluate motor coordination and balance. Fluoro-Jade C, Iba-1, and GFAP staining were used to evaluate neuronal cell death, microglia morphology, and astrocytes density, respectively, at this time point. Levels of cytokines and neurotrophic factors were measured by ELISA and Cytometric Bead Array 8 h after QA injection. Striatal synaptosomes were used to evaluate the release of glutamate.. We first demonstrated that rapamycin prevented the motor impairment induced by QA. Moreover, mTOR inhibition also reduced the neurodegeneration and the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α induced by excitotoxic stimulus. The lowest dose of rapamycin also increased the production of IL-10 and prevented the reduction of astrocyte density induced by QA. By using an in vitro approach, we demonstrated that rapamycin differently alters the release of glutamate from striatal synaptosomes induced by QA, reducing or enhancing the release of this neurotransmitter at low or high concentrations, respectively.. Taken together, these data demonstrated a protective effect of rapamycin against an excitotoxic stimulus. Therefore, this study provides new evidence of the detrimental role of mTOR in neurodegeneration, which might represent an important target for the treatment of neurodegenerative diseases.

Topics: Animals; Body Weight; Corpus Striatum; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Glutamic Acid; Male; Mice; Mice, Inbred C57BL; Movement Disorders; Nerve Degeneration; Neuroglia; Neurons; Neuroprotective Agents; Neurotoxicity Syndromes; Postural Balance; Potassium Chloride; Quinolinic Acid; Sirolimus; Synaptosomes

Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.

Topics: Animals; Biomarkers; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Disease Progression; Hypoglycemic Agents; Insulin Resistance; Liver; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Multiprotein Complexes; Protein Kinase Inhibitors; Proteinuria; Rats, Zucker; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Rapamycin protects mice against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons, which is an established model for Parkinson's disease. We demonstrated that rapamycin preserves astrocytic expression of glutamate transporters and glutamate reuptake. The protective effect was also observed in astrocyte cultures, indicating that rapamycin acts directly on astrocytes. In the MPTP model, rapamycin caused reduced expression of the E3 ubiquitin ligase Nedd4-2 (neuronal precursor cell expressed developmentally downregulated 4-2) and reduced colocalization of glutamate transporters with ubiquitin. Rapamycin increased interleukin-6 (IL-6) expression, which was associated with reduced expression of inflammatory cytokines, indicating anti-inflammatory properties of IL-6 in the MPTP model. NF-κB was shown to be a key mediator for rapamycin, whereas Janus kinase 2, signal transducer and activator of transcription 3, phosphoinositide 3-kinase, and Akt partially mediated rapamycin effects in astrocytes. These results demonstrate for the first time in a Parkinson's disease animal model that the neuroprotective effects of rapamycin are associated with glial and anti-inflammatory effects.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Transport System X-AG; Animals; Astrocytes; Cytokines; Disease Models, Animal; Down-Regulation; Glutamates; Inflammation; Interleukin-6; Janus Kinase 2; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; NF-kappa B; Parkinson Disease; Phosphatidylinositol 3-Kinases; Sirolimus; STAT3 Transcription Factor; Up-Regulation

BACKGROUND This study was proposed to compare the efficacy and safety of GTM-1, Rapamycin (Rap), and Carbamazepine (CBZ) in managing Alzheimer disease (AD). The impact of the above mentioned therapeutic drugs on autophagy was also investigated in our study. MATERIAL AND METHODS Firstly, 3×Tg AD mice were randomly allocated into 4 groups (each group with 10 mice), in which AD mice were separately treated with dimethylsulfoxide (DMSO, vehicle group), GTM-1 (6 mg/kg), Rap (1 mg/kg), and CBZ (100 mg/kg). Then spatial memory and learning ability of mice was tested using the Morris water maze. Routine blood tests were performed to evaluate the toxicity of these drugs. Amyloid-β42 (Aβ42) concentration was detected by ELISA and immunohistochemistry. Proteins related to autophagy were detected by Western blot. RESULTS GTM-1, Rap, and CBZ significantly improved the spatial memory of 3×Tg AD mice compared to that in the vehicle group (all P<0.05). Moreover, this study revealed that CBZ dosage was related to toxicity in mice. All of the above drugs significantly increased the expression of LC3-II and reduced Aβ42 levels in hippocampi of 3×Tg AD mice (all P<0.05). On the other hand, neither GTM-1 nor CBZ had significant influence on the expression of proteins on the mTOR pathway. CONCLUSIONS GTM-1 can alleviate the AD syndrome by activating autophagy in a manner that is dependent on the mTOR pathway and it therefore can be considered as an alternative to Rap.

Topics: Alzheimer Disease; Animals; Autophagy; Carbamazepine; Disease Models, Animal; Hippocampus; Male; Maze Learning; Mice; Neurons; Neuroprotective Agents; Plaque, Amyloid; Quinazolines; Random Allocation; Sirolimus; Spatial Memory

Topics: Animals; Anti-Inflammatory Agents; Aorta; Aortic Aneurysm, Abdominal; Cytokines; Dilatation, Pathologic; Disease Models, Animal; Disease Progression; Inflammation Mediators; Macrophages; Male; Matrix Metalloproteinases; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Pancreatic Elastase; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Vasoconstriction

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and activation of its signal pathway plays an important role in regulating protein growth and synthesis as well as cell proliferation and survival. In the present study, we examined the contribution of mTOR signal and its downstream products to brain injuries induced by intracerebral hemorrhage (ICH) in rats.. Western Blot analysis was employed to examine the protein expression of mTOR and its downstream pathway and Caspase-3. ELISA was used to measure pro-inflammatory cytokines (PICs) and vascular endothelial growth factor (VEGF). Additionally, neurological Severity Score and brain water content were used to indicate neurological function and brain edema.. The protein expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) pathways were amplified in ICH rats compared with controls. Blocking mTOR using rapamycin significantly attenuated upregulation of PICs, namely IL-1β, IL-6 and TNF-α, and Caspase-3 indicating cell apoptosis, and promoted the levels of VEGF and its subtype receptor VEGFR-2 in brain tissues. Moreover, the effects of rapamycin were linked to improvement of neurological deficits and increased brain water content observed in ICH rats.. Activation mTOR signal is engaged in pathophysiological process during ICH and blocking mTOR pathway plays a beneficial role in regulating neuronal tissues via PIC, apoptotic Caspase-3 and VEGF mechanisms. This has pharmacological implications to target specific mTOR and its downstream signal pathway for neuronal dysfunction and vulnerability related to ICH.

Topics: Animals; Brain; Brain Edema; Carrier Proteins; Caspase 3; Cerebral Hemorrhage; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Intracellular Signaling Peptides and Proteins; Male; Phosphoproteins; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.

Topics: Adolescent; Animals; Brain; Cell Line, Tumor; Child; Class I Phosphatidylinositol 3-Kinases; Cloning, Molecular; CRISPR-Cas Systems; Disease Models, Animal; Epilepsy; Female; HEK293 Cells; Humans; Male; Malformations of Cortical Development, Group I; Mice; Mutation; Neurons; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Saliva; Sequence Analysis, DNA; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

IgA nephropathy (IgAN) has been considered to be the most frequent form of primary glomerulonephritis that occurs worldwide with a variety of factors involved in its occurrence and development. The impact of autophagy in IgAN, however, remains partially unclear. This study was designed to investigate the effects of rapamycin in an IgAN model.. After establishing an IgAN rat model, SD rats were divided into 4 groups: control, control + rapamycin, IgAN, IgAN + rapamycin. Proteinuria and the pathological changes and the level of autophagy of kidney were texted. Identify the expression of phosphorylation and total mammalian target of rapamycin (mTOR) and s6k1 as well as cyclin D1 in the kidney of rats through Western blot and immunohistochemistry.. With rapamycin treatment, we observed a significant reduction in the progression of proteinuria as well as alleviation of pathological lesions in IgAN rats. Besides, autophagy was inhibited, while the mTOR/S6k1 pathway was activated and expression of cyclin D1 was increased in IgAN. Rapamycin treatment increased autophagy and decreased the expression of cyclin D1.. These results may suggest that mTOR-mediated autophagy inhibition may result in mesangial cell proliferation in IgAN.

Topics: Animals; Autophagy; Cell Proliferation; Cyclin D1; Disease Models, Animal; Glomerulonephritis, IGA; Humans; Immunohistochemistry; Immunosuppressive Agents; Mesangial Cells; Microscopy, Electron; Proteinuria; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Erythrocytes; Histone Demethylases; Humans; Mice; Mitochondria; Models, Biological; Reactive Oxygen Species; Rhodamines; Sirolimus; Spiro Compounds; Thiophenes; TOR Serine-Threonine Kinases

(+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly "mammalian") target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.

Topics: Animals; Apoptosis; Autophagy; Benzofurans; Benzylidene Compounds; Breast Neoplasms; Cell Proliferation; Crystallography, X-Ray; Disease Models, Animal; Female; Humans; Lichens; Mice; Mice, Nude; Models, Molecular; Molecular Conformation; Molecular Structure; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

"Cable-tie" type biodegradable stents with drug-eluting nanofiber were developed to treat rabbit denuded arteries in this study. Biodegradable stents were fabricated using poly-L-lactide film following being cut and rolled into a cable-tie type stent. Additionally, drug-eluting biodegradable nanofiber tubes were electrospun from a solution containing poly (lactic-co-glycolic acid), rapamycin, and hexafluoroisopropanol, and then mounted onto the stents. The fabricated rapamycin-eluting cable-tie stents exhibited excellent mechanical properties on evaluation of compression test and collapse pressure, and less than 8% weight loss following being immersed in phosphate-buffered saline for 16 weeks. Furthermore, the biodegradable stents delivered high rapamycin concentrations for over 4 weeks and achieved substantial reductions in intimal hyperplasia associated with elevated heme oxygenase-1 and calponin level on the denuded rabbit arteries during 6 months of follow-up. The drug-eluting cable-tie type stents developed in this study might have high potential impacts for the local drug delivery to treat various vascular diseases.

Topics: Absorbable Implants; Animals; Calcium-Binding Proteins; Calponins; Disease Models, Animal; Drug-Eluting Stents; Equipment Design; Heme Oxygenase-1; Male; Microfilament Proteins; Nanofibers; Polyesters; Rabbits; Sirolimus; Tunica Intima; Up-Regulation

Mesial temporal lobe epilepsy (MTLE) is the most common type of refractory epilepsy. It is often associated with hippocampal sclerosis, which is histopathologically characterized by selective neuron loss, mossy fiber sprouting, and synapse reconstruction, and is the primary cause of refractory epilepsy. Its mechanism has not been fully elucidated. Substantial evidence now supports that inflammatory pathways are activated in epilepsy foci. We have confirmed that the interleukin-1β (IL-1β) level is involved in the epileptogenesis of MTLE, and we further investigated how it works in its chronicity in this study.. The MTLE model was induced by pilocarpine, and Western blot and co-immunoprecipitation were used to detect proteins related to the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway in the hippocampi of MTLE rats and MTLE children. Meanwhile, primary hippocampal neurons were cultured and transfected by lentivirus, and the same methods were used to test the related protein expression; fluorescent dye FM4-64 was used to measure synaptic vesicle endocytosis (SVE) of neurons.. We revealed that mTOR is continuously activated in the rat MTLE model and children with MTLE, and it correlated with the IL-1β level. We further proved that IL-1β activates neurons via the PI3K/Akt/mTOR signaling pathway, accompanied by the upregulation of MAP2 and the enhancement of SVE in hippocampal neurons.. Our findings suggest that IL-1β can activate mTOR, followed by activated neurons, which is critical in the pathogenesis of MTLE chronicity. These findings contribute to the understanding of the pathogenesis of MTLE, and targeting inflammation modulators in MTLE may provide new pathways for therapy of refractory epilepsy.

Topics: Animals; Anticonvulsants; Cells, Cultured; Child; Diazepam; Disease Models, Animal; Enzyme Inhibitors; Epilepsy, Temporal Lobe; Female; Gene Expression Regulation; Hippocampus; Humans; Interleukin-1beta; Male; Muscarinic Agonists; Neurons; Pilocarpine; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate the effects of sirolimus and sunitinib on wound healing in experimental glaucoma filtering surgery (GFS).. Thirty-five male New Zealand pigmented rabbits were randomly assigned to five groups, each including seven rabbits: The rabbits in the control group were not operated on and did not receive any treatment. The rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days. The rabbits in the mitomycin-C (MMC) group underwent trabeculectomy, and a sponge soaked in 0.4 mg/mL MMC was applied intraoperatively to the scleral surgical site for three minutes. The rabbits in the sirolimus group underwent trabeculectomy and 30 ng/mL sirolimus-soaked sponge was applied intraoperatively to the scleral surgical site for three minutes. Sunitinib 0.5 mg/mL four drops in a day were applied in the sunitinib group for 14 days after surgery. On day 14 of the experiment, eyes were enucleated and histologically and immunohistochemically analyzed. Statistical analyses of the study were performed with Kruskal-Wallis variance analysis and Mann-Whitney U test.. The mean fibroblast and MNC numbers and the mean immunostaining intensities of transforming growth factor-β (TGF-β), fibroblast growth factor-β (FGF-β) and platelet derived growth factor (PDGF) in the MMC, sirolimus and sunitinib groups were statistically significantly lower than those of the sham group (p < 0.01). The mean fibroblast and MNC numbers and the mean immunostaining intensities of TGF-β, FGF-β and PDGF in the MMC, sirolimus and sunitinib groups were similar (p > 0.05).. Our study suggests that the applications of sirolimus and sunitinib effectively suppress the subconjunctival scarring after experimental GFS.

Topics: Alkylating Agents; Angiogenesis Inhibitors; Animals; Disease Models, Animal; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Immunosuppressive Agents; Indoles; Male; Mitomycin; Platelet-Derived Growth Factor; Pyrroles; Rabbits; Sirolimus; Sunitinib; Trabecular Meshwork; Trabeculectomy; Transforming Growth Factor beta; Wound Healing

We aimed to comprehensively evaluate poly-lactide polymer degradation and sirolimus release kinetics from a drug-eluting stent matrix in the in vivo setting using a nuclear magnetic resonance (NMR) method.. In 22 domestic swine, 18 biodegradable polymer-only coated stents (BPSs) and 36 biodegradable polymer-coated sirolimus-eluting stents (BP-SES) were implanted in coronary arteries with 115% overstretch. The animals were sacrificed at 1, 3, 7, 14, 28, and 56 days following baseline procedures. Vessel segments with BPS were harvested to evaluate polymer degradation with a NMR method, whereas BP-SES to analyze sirolimus tissue uptake and retention. Additionally, 8 BP-SES were implanted for histological analysis for 90 days of follow-up.. The NMR showed a gradual absorption of the polymer over the 6 consecutive time points, from 5.48 µg of the polymer on the stent at 1-day follow-up, through 4.33 µg at 3 days, 3.16 µg at 7 days, 2.42 µg at 14 days, 1.92 µg at 28 days to 1.24 µg in the last day of the study. The curve of polymer degradation corresponds well with the pharmacokinetic profile of sirolimus eluted from its surface and measured at identical time points. In histopathology, at 90 days, complete healing and biocompatibility were reported.. The utilization of NMR method for BP absorption kinetics evaluation is a useful tool, which may be widely adopted to test other biodegradable implants. Further, it may substantially improve their safety and efficacy by facilitating programmed polymer and drugs elution.

Topics: Absorbable Implants; Animals; Cardiovascular Agents; Coronary Restenosis; Disease Models, Animal; Drug-Eluting Stents; Female; Magnetic Resonance Spectroscopy; Male; Percutaneous Coronary Intervention; Polyesters; Prosthesis Design; Sirolimus; Sus scrofa

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway mediates many aspects of cell growth and regeneration and is upregulated after moderate to severe traumatic brain injury (TBI). The significance of this increased signaling event for recovery of brain function is presently unclear. We analyzed the time course and cell specificity of mTORC1 signal activation in the mouse hippocampus after moderate controlled cortical impact (CCI) and identified an early neuronal peak of activity that occurs within a few hours after injury. We suppressed this peak activity by a single injection of the mTORC1 inhibitor rapamycin 1 h after CCI and showed that this acute treatment significantly diminishes the extent of neuronal death, astrogliosis, and cognitive impairment 1-3 days after injury. Our findings suggest that the early neuronal peak of mTORC1 activity after TBI is deleterious to brain function, and that acute, early intervention with mTORC1 inhibitors after injury may represent an effective form of treatment to improve recovery in human patients.

Topics: Animals; Behavior, Animal; Brain Injuries; Cell Death; Cognition Disorders; Disease Models, Animal; Gliosis; Hippocampus; Maze Learning; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autophagy enables cells to digest endogenous/exogenous waste products, thus potentially prolonging the cellular lifespan. Early endothelial progenitor cells (eEPCs) protect mice from ischemic acute kidney injury (AKI). The mid-term prognosis in AKI critically depends on vascular rarefication and interstitial fibrosis with the latter partly being induced by mesenchymal transdifferentiation of endothelial cells (EndoMT). This study aimed to determine the impact of eEPC preconditioning with different autophagy inducing agents [suberoylanilide hydroxamic acid (SAHA)/temsirolimus] in ischemic AKI.. Male C57/Bl6 N mice were subjected to bilateral renal ischemia (40 min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48 h and 4 weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-β ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS-senescence-associated β-galactosidase, SA-β-Gal), and were evaluated 96 h later.. Cultured eEPCs showed reduced perinuclear density of LC3-II + vesicles and elevated levels of SA-β-Gal after treatment with TGF-β alone, indicating impaired autophagy and aggravated SIPS. These effects were completely abrogated by SAHA. Systemic administration of either SAHA or tems pretreated eEPCs resulted in elevated intrarenal endothelial p62 at 48 h and 4 weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4 weeks endothelial expression of mesenchymal alpha-smooth muscle actin (αSMA) was reduced in animals receiving untreated and SAHA-pretreated cells. In addition, SAHA-treated cells reduced fibrosis at week 4. Tems in contrast aggravated EndoMT. Postischemic renal function declined after renal ischemia and remained unaffected in all experimental cell treatment groups.. In ischemic AKI, intrarenal endothelial autophagy may be stabilized by systemic administration of pharmacologically preconditioned eEPCs. Early EPCs can reduce postischemic EndoMT and fibrosis in the mid-term. Autophagy induction in eEPCs either increases or decreases the mesenchymal properties of intrarenal endothelial cells, depending on the substance being used. Thus, endothelial autophagy induction in ischemic AKI, mediated by eEPCs is not a renoprotective event per se.

Topics: Actins; Acute Kidney Injury; Animals; Autophagy; beta-Galactosidase; Cells, Cultured; Disease Models, Animal; Endothelial Progenitor Cells; Epithelial-Mesenchymal Transition; Fibrosis; Hydroxamic Acids; Ischemia; Kidney; Male; Mice, Inbred C57BL; Microtubule-Associated Proteins; Phenotype; Sirolimus; Time Factors; Transforming Growth Factor beta; Vorinostat

Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP.. Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified.. Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction.. mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Brain; Cell Death; Cerebral Palsy; Disease Models, Animal; Hypoxia-Ischemia, Brain; Lipopolysaccharides; Mice, Inbred C57BL; Neuroimmunomodulation; Neurons; Neuroprotective Agents; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

B lymphocytes are generally considered to be activators of the immune response; however, recent findings have shown that a subtype of B lymphocytes, regulatory B lymphocytes, play a role in attenuating the immune response. Bronchiolitis obliterans remains the major limitation to modern-day lung transplantation. The role of regulatory B lymphocytes in bronchiolitis obliterans has not been elucidated. We hypothesized that regulatory B lymphocytes play a role in the attenuation of bronchiolitis obliterans.. We performed a standard heterotopic tracheal transplant model. Tracheas from Balb/c mice were transplanted into C57BL/6 recipients. Rapamycin treatment and dimethyl sulfoxide control groups were each treated for the first 14 days after the transplant. Tracheas were collected on days 7, 14, and 28 post-transplantation. Luminal obliteration was evaluated by hematoxylin-eosin staining and Picrosirius red staining. Immune cell infiltration and characteristics, and secretion of interleukin-10 and transforming growth factor-β1 were accessed by immunohistochemistry. Cytokines and transforming growth factor-β1 were measured using the Luminex assay (Bio-Rad, Hercules, Calif).. The results revealed that intraperitoneal injection of rapamycin for 14 days after tracheal transplantation significantly reduced luminal obliteration on day 28 when compared with the dimethyl sulfoxide control group (97.78% ± 3.63% vs 3.02% ± 2.14%, P < .001). Rapamycin treatment markedly induced regulatory B lymphocytes (B220(+)IgM(+)IgG(-)IL-10(+)TGF-β1(+)) cells when compared with dimethyl sulfoxide controls. Rapamycin treatment inhibited interleukin-1β, 6, 13, and 17 on days 7 and 14. Rapamycin also greatly increased interleukin-10 and transforming growth factor-β1 production in B cells and regulatory T lymphocytes infiltration on day 28.. Mammalian target of rapamycin inhibition decreases the development of bronchiolitis obliterans via inhibition of proinflammatory cytokines and increasing regulatory B lymphocytes cell infiltration, which subsequently produces anti-inflammatory cytokines and upregulates regulatory T lymphocyte cells.

Topics: Animals; Anti-Inflammatory Agents; B-Lymphocytes; Bronchiolitis Obliterans; Chemotaxis, Leukocyte; Cytokines; Disease Models, Animal; Inflammation Mediators; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Phenotype; Sirolimus; T-Lymphocytes, Regulatory; Time Factors; Trachea

Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein that modulates signal transduction at dopamine receptors, and also activates mammalian target of rapamycin complex 1 (mTORC1). Rhes binding to mTORC1 is hypothesized to play a role in motor disorders such as levodopa-induced dyskinesia. Here, we investigate the behavioral and in vivo neurocircuitry changes associated with genetic deletion of Rhes or inhibition of mTORC1 signaling in the mouse model of levodopa-induced dyskinesia. 6-Hydroxydopamine-hemilesioned Rhes knockout mice and wild-type littermates were chronically treated with levodopa. In parallel, 6-hydroxydopamine-hemilesioned naïve mice were chronically treated with levodopa or levodopa plus rapamycin, to block mTORC1 pathway activation. Dyskinetic movements were monitored during levodopa treatment along with motor activity on the rotarod. Finally, dyskinetic mice underwent microdialysis probe implantation in the dopamine-depleted striatum and ipsilateral substantia nigra reticulata, and GABA and glutamate levels were monitored upon acute challenge with levodopa. Both Rhes knockouts and rapamycin-treated mice developed less dyskinesia than controls, although only rapamycin-treated mice fully preserved rotarod performance on levodopa. Levodopa elevated nigral GABA and glutamate in controls but not in Rhes knockouts or rapamycin-treated mice. Levodopa also stimulated striatal glutamate in controls and Rhes knockouts but not in rapamycin-treated mice. We conclude that both genetic deletion of Rhes and pharmacological blockade of mTORC1 significantly attenuate dyskinesia development by reducing the sensitization of striato-nigral medium-sized spiny neurons to levodopa. However, mTORC1 blockade seems to provide a more favorable behavioral outcome and a wider effect on neurochemical correlates of dyskinesia.

Topics: Animals; Antiparkinson Agents; Corpus Striatum; Disease Models, Animal; Dyskinesia, Drug-Induced; Female; Glutamic Acid; GTP-Binding Proteins; Levodopa; Male; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Multiprotein Complexes; Neurons; Neuroprotective Agents; Sirolimus; Substantia Nigra; TOR Serine-Threonine Kinases

The current study was to examine the underlying mechanisms responsible for the role of mammalian target of rapamycin (mTOR) in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) as well as phosphatidylinositide 3-kinase (p-PI3K) pathways were amplified in the superficial dorsal horn of the spinal cord of bone cancer rats compared with control rats. Blocking spinal mTOR by using rapamycin significantly attenuated activities of PI3K signaling pathways as well as mechanical and thermal hyperalgesia. Additionally, rapamycin enhanced attenuations of protein kinase Cɛ (PKCɛ)/protein kinase A (PKA) induced by morphine and further extended analgesia of morphine via µ-opioid receptor (MOR). Our data for the first time revealed specific signaling pathways leading to bone cancer pain, including the activation of mTOR and PI3K and downstream PKCɛ/PKA, and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics.

Topics: Analgesics, Opioid; Animals; Blotting, Western; Bone Neoplasms; Disease Models, Animal; Drug Tolerance; Morphine; Pain; Rats; Rats, Wistar; Receptors, Opioid, mu; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.

Topics: Animals; Animals, Genetically Modified; Ataxin-3; Autophagy; Brain; Caenorhabditis elegans; Central Nervous System Agents; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Therapy, Combination; Lithium Compounds; Locomotion; Machado-Joseph Disease; Male; Mice, Inbred C57BL; Motor Activity; Neurotoxicity Syndromes; Sirolimus; TOR Serine-Threonine Kinases

Neurodegenerative diseases are characterized by increased levels of oxidative stress and an altered mammalian target of rapamycin (mTOR)/autophagy axis; however, the mutual relationship between these two events is controversial. Previous studies in Down's syndrome (DS) and Alzheimer's disease (AD) suggested that the accumulation of protein oxidative damage results from the increased free radical production, mainly related to metabolic alterations, mitochondrial degeneration and amyloid-β deposition, and aberrant activity of protein degradative systems.. This study analyzed mTOR signaling in Ts65Dn mice, a model of DS, at 6 and 12 months of age compared with euploid mice showing the early aberrant hyperphosphorylation of mTOR coupled with the reduction of autophagosome formation. Moreover, the evaluation of protein oxidation shows an increase in protein nitration and protein-bound 4-hydroxynonenal in 12-month-old Ts65Dn mice suggesting the potential involvement of altered autophagy in the buildup of protein oxidative damage. In addition, data obtained on cell culture support the protective role of autophagy in reducing protein oxidation.. Overall, this study provides further evidence for the role of mTOR hyperactivation and reduced autophagy in the accumulation of protein oxidative damage during DS and AD pathologies.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Disease Models, Animal; Down Syndrome; Hippocampus; Humans; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Oxidation-Reduction; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation.

Topics: Animals; Antibodies, Neutralizing; Disease Models, Animal; Factor VIII; Hemophilia A; Immune Tolerance; Immunosuppressive Agents; Mice; Nanoparticles; Sirolimus; Vaccines, Synthetic

Blood-brain barrier (BBB) leakage may play a pro-epileptogenic role after status epilepticus. In the accompanying contrast-enhanced magnetic resonance imaging (CE-MRI) study we showed that the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduced BBB leakage and seizure activity during the chronic epileptic phase. Given rapamycin's role in growth and immune response, the potential therapeutic effects of rapamycin after status epilepticus with emphasis on brain inflammation and brain vasculature were investigated.. Seven weeks after kainic acid-induced status epilepticus, rats were perfusion fixed and (immuno)histochemistry was performed using several glial and vascular markers. In addition, an in vitro model for the human BBB was used to determine the effects of rapamycin on transendothelial electrical resistance as a measure for BBB integrity.. (Immuno)histochemistry showed that local blood vessel density, activated microglia, and astrogliosis were reduced in rapamycin-treated rats compared to vehicle-treated rats. In vitro studies showed that rapamycin could attenuate TNFα-induced endothelial barrier breakdown.. These data suggest that rapamycin improves BBB function during the chronic epileptic phase by a reduction of local brain inflammation and blood vessel density that can contribute to a milder form of epilepsy.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Vessels; Blood-Brain Barrier; Brain; Disease Models, Animal; Electric Impedance; Excitatory Amino Acid Agonists; Gliosis; Immunosuppressive Agents; Kainic Acid; Lectins; Macrophages; Male; Mossy Fibers, Hippocampal; Rats; Rats, Sprague-Dawley; Sirolimus; Status Epilepticus; Tumor Necrosis Factor-alpha

The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this effect via restoration of blood-brain barrier (BBB) dysfunction, contrast-enhanced magnetic resonance imaging (CE-MRI) was used to determine BBB permeability throughout epileptogenesis.. Imaging was repeatedly performed until 6 weeks after kainic acid-induced status epilepticus in rapamycin (6 mg/kg for 6 weeks starting 4 h after SE) and vehicle-treated rats, using gadobutrol as contrast agent. Seizures were detected using video monitoring in the week following the last imaging session.. Gadobutrol leakage was widespread and extensive in both rapamycin and vehicle-treated epileptic rats during the acute phase, with the piriform cortex and amygdala as the most affected regions. Gadobutrol leakage was higher in rapamycin-treated rats 4 and 8 days after status epilepticus compared to vehicle-treated rats. However, during the chronic epileptic phase, gadobutrol leakage was lower in rapamycin-treated epileptic rats along with a decreased seizure frequency. This was confirmed by local fluorescein staining in the brains of the same rats. Total brain volume was reduced by this rapamycin treatment regimen.. The initial slow recovery of BBB function in rapamycin-treated epileptic rats indicates that rapamycin does not reduce seizure activity by a gradual recovery of BBB integrity. The reduced BBB leakage during the chronic phase, however, could contribute to the decreased seizure frequency in post-status epilepticus rats treated with rapamycin. Furthermore, the data show that CE-MRI (using step-down infusion with gadobutrol) can be used as biomarker for monitoring the effect of drug therapy in rats.

Topics: Animals; Anticonvulsants; Blood-Brain Barrier; Brain; Capillary Permeability; Disease Models, Animal; Electroencephalography; Excitatory Amino Acid Agonists; Follow-Up Studies; Kainic Acid; Magnetic Resonance Imaging; Male; Phospholipids; Rats; Rats, Sprague-Dawley; Sirolimus; Status Epilepticus; Sulfur Hexafluoride; Time Factors; Video Recording

In neurodegenerative diseases like AD, tau forms neurofibrillary tangles, composed of tau protein. In the AD brain, activated caspases cleave tau at the 421th Asp, generating a caspase-cleaved form of tau, TauC3. Although TauC3 is known to assemble rapidly into filaments in vitro, a role of TauC3 in vivo remains unclear. Here, we generated a transgenic mouse expressing human TauC3 using a neuron-specific promoter. In this mouse, we found that human TauC3 was expressed in the hippocampus and cortex. Interestingly, TauC3 mice showed drastic learning and spatial memory deficits and reduced synaptic density at a young age (2-3months). Notably, tau oligomers as well as tau aggregates were found in TauC3 mice showing memory deficits. Further, i.p. or i.c.v. injection with methylene blue or Congo red, inhibitors of tau aggregation in vitro, and i.p. injection with rapamycin significantly reduced the amounts of tau oligomers in the hippocampus, rescued spine density, and attenuated memory impairment in TauC3 mice. Together, these results suggest that TauC3 facilitates early memory impairment in transgenic mice accompanied with tau oligomer formation, providing insight into the role of TauC3 in the AD pathogenesis associated with tau oligomers and a useful AD model to test drug candidates.

Topics: Animals; Avoidance Learning; Brain; Caspases; Cell Line, Tumor; Dendritic Spines; Disease Models, Animal; Female; Humans; Male; Maze Learning; Memory Disorders; Mice, Transgenic; Neurons; Nootropic Agents; Protein Multimerization; Recognition, Psychology; Sirolimus; Spatial Memory; tau Proteins

Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

Topics: Activin Receptors, Type I; Adipose Tissue; Animals; Burns; Chondrogenesis; Disease Models, Animal; Gene Regulatory Networks; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Integrases; Luminescent Measurements; Mesenchymal Stem Cells; Mice, Knockout; Models, Biological; Mustard Compounds; Ossification, Heterotopic; Phenylpropionates; Receptor, Platelet-Derived Growth Factor alpha; RNA, Messenger; Signal Transduction; Sirolimus; SOX9 Transcription Factor; Tendons; Tenotomy; Up-Regulation; Wound Healing; Wounds and Injuries; X-Ray Microtomography

Activation of the mammalian target of rapamycin (mTOR) leads to cell growth and survival. We tested the hypothesis that inhibition of mTOR would increase infarct size and decrease microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1h and reperfusion for 2h with and without rapamycin (20mg/kg once daily for two days prior to ischemia). Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small-vessel arterial and venous oxygen saturations were determined microspectrophotometrically. The control ischemic-reperfused cortex had a similar blood flow and O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex. Rapamycin significantly increased cerebral O2 consumption and further reduced O2 supply/consumption balance in the reperfused area. This was associated with an increased cortical infarct size (13.5±0.8% control vs. 21.5±0.9% rapamycin). We also found that ischemia-reperfusion increased AKT and S6K1 phosphorylation, while rapamycin decreased this phosphorylation in both the control and ischemic-reperfused cortex. This suggests that mTOR is important for not only cell survival, but also for the control of oxygen balance after cerebral ischemia-reperfusion.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antipyrine; Blood Gas Analysis; Blood Pressure; Brain Ischemia; Carbon Isotopes; Cerebrovascular Circulation; Disease Models, Animal; Hemodynamics; Immunosuppressive Agents; Male; Oncogene Protein v-akt; Oxygen Consumption; Rats; Rats, Inbred F344; Reperfusion; Signal Transduction; Sirolimus; Time Factors

We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines.. Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo.. Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR.. Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Doxorubicin; Humans; Indoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Protein Kinase Inhibitors; Purines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

To determine whether intramural administration of rapamycin (RPM)-loaded polylactic-polyglycolic acid (PLGA) nanoparticles (NPs) can reduce intimal thickening and affect the mRNA expressions of matrix metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2 and p27(kipl) in a coronary injury-stenosis model of minipigs.. Twenty eight minipigs were randomly separated into four groups: saline group (n=7), blank PLGA NPs group (5.0 mg/ml)(n=7), RPM group (1.0 mg/ml)(n=7), and RPM-PLGA NPs(5.0 mg/ml)group (n=7), respectively. Different treatments were intracoronary locally delivered via a Dispatch™ catheter for 10 minutes. Serial angiography was performed pre-and post-modeling 30 days and the percent stenosis degree was assessed. Hematoxylin-Eosin (HE) staining, Weigert's resorcin fuchsin staining and picric acid-sirius red staining were used for morphometric analysis. Immunohistochemistry was performed to assess the levels of proliferating cell nuclear antigen (PCNA), MMP-2, and TIMP-2 at early and late time points, respectively. The expression of p27(kip1) mRNA was detected by in situ hybridization staining.. Data from 21 minipigs had been collected at the end of the experiment with 6, 4, 5, and 6 from the former mentioned 4 groups, respectively. For the instant injury index, there was no significant difference among the four groups. The percent stenosis degree of RPM-PLGA NPs group was significantly lower than that of the other three groups respectively (all P< 0.05). The neointima area, net external elastic lamina area to external elastic laminal area ratio, and proliferative index of RPM-PLGA NPs group were significantly less than those of the other three groups, with all the P values less than 0.05. The mean value of integral optical density of p27(kip1)mRNA expression of RPM-PLGA group was 0.35 ± 0.06, higher than that of blank PLGA NPs group (0.12 ± 0.05, P< 0.01), saline group (0.16 ± 0.03, P< 0.05), and RPM group (0.15 ± 0.03, P< 0.05), respectively. The MMP-2/TIMP-2 ratio and the positive expression index of PCNA in RPM-PLGA group were lower than that of the other groups (P< 0.05).. Locally delivered rapamycin-loaded PLGA NPs significantly reduces MMP-2/TIMP-2 ratio and PCNA expression, increases p27(kip1) mRNA expression and significantly relieves percent stenosis degree and shows excellent acute procedural results in the minipig interventional coronary artery oversized balloon injury model. The results from minipig model further support that this approach could be a potential clinical procedure for vascular proliferative disease.

Topics: Animals; Constriction, Pathologic; Disease Models, Animal; Lactic Acid; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Sirolimus; Swine; Swine, Miniature

Several studies suggest that glial scars pose as physical and chemical barriers that limit neurite regeneration after spinal cord injury (SCI). Evidences suggest that the activation of the PI3K/Akt/mTOR signaling pathway is involved in glial scar formation. Therefore, inhibition of the PI3K/Akt/mTOR pathway may beneficially attenuate glial scar formation after SCI. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates the PI3K/Akt/mTOR pathway. Therefore, we hypothesized that the overexpression of PTEN in the spinal cord will have beneficial effects after SCI. In the present study, we intrathecally injected a recombinant adenovirus carrying the pten gene (Ad-PTEN) to cause overexpression of PTEN in rats with contusion injured spinal cords. The results suggest overexpression of PTEN in spinal cord attenuated glial scar formation and led to improved locomotor function after SCI. Overexpression of PTEN following SCI attenuated gliosis, affected chondroitin sulfate proteoglycan expression, and improved axon regeneration into the lesion site. Furthermore, we suggest that the activation of the PI3K/Akt/mTOR pathway in astrocytes at 3 days after SCI may be involved in glial scar formation. Because delayed treatment with Ad-PTEN enhanced motor function recovery more significantly than immediate treatment with Ad-PTEN after SCI, the results suggest that the best strategy to attenuate glial scar formation could be to introduce 3 days after SCI. This study's findings thus have positive implications for patients who are unable to receive immediate medical attention after SCI.

Topics: Animals; Cicatrix; Disease Models, Animal; Female; Gliosis; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Locomotion; Nerve Tissue Proteins; Neuroglia; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; Rats; Rats, Wistar; Signal Transduction; Sirolimus; Spinal Cord Injuries; Time Factors

CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs) play critical roles in controlling the processes of tumors, infections, autoimmunity and graft rejection. Immunosuppressive drug rapamycin (RPM), targeting on the key cellular metabolism molecule mTOR, is currently used in clinics to treat patients with allo-grafts, autoimmune diseases and tumors. However, the effect of RPM on MDSCs has not been studied. RPM significantly decreases the cell number and the immunosuppressive ability on T cells of CD11b(+) Ly6C(high) monocytic MDSCs (M-MDSCs) in both allo-grafts-transplanted and tumor-bearing mice respectively. Mice with a myeloid-specific deletion of mTOR have poor M-MDSCs after grafting with allo-skin tissue or a tumor. Grafting of allo-skin or tumors significantly activates glycolysis pathways in myeloid precursor cells in bone marrow, which is inhibited by RPM or mTOR deletion. 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway, inhibits M-MDSC differentiation from precursors, while enhancing glycolysis by metformin significantly rescues the RPM-caused deficiency of M-MDSCs. Therefore, we offer evidence supporting that mTOR is an intrinsic factor essential for the differentiation and immunosuppressive function of M-MDSCs and that these metabolism-relevant medicines may impact MDSCs-mediated immunosuppression or immune tolerance induction, which is of considerable clinical importance in treating graft rejection, autoimmune diseases and cancers.

Topics: Allografts; Animals; Cell Count; Cell Differentiation; Disease Models, Animal; Energy Metabolism; Graft Rejection; Immunosuppression Therapy; Mice; Mice, Knockout; Myeloid-Derived Suppressor Cells; Neoplasms; Nitric Oxide Synthase Type II; Signal Transduction; Sirolimus; Skin Transplantation; T-Lymphocyte Subsets; TOR Serine-Threonine Kinases

To investigate the effect of rapamycin on TGFβ1 and MMP1 expression in a rabbit model of urethral stricture.. Twenty-four adult New Zealand male rabbits underwent an electrocoagulation of the bulbar urethra with a 13Fr pediatric resectoscope. Then rabbits were randomly divided into three groups: (1) normal control group: normal saline (NS), (2) the vehicle control group: dimethyl sulfoxide (DMSO), and (3) the treatment group: effective-dose rapamycin in DMSO (Ra), with 12, 6, and 6 rabbits in each group, respectively. Drugs were given by urethral irrigation daily for 4 weeks. Urethral tissue was harvested for histological and molecular analyses. TGFβ1 and MMP1 expression levels were evaluated by real-time quantitative PCR and immunohistochemistry.. Ten, six, and six rabbits were evaluated finally in Ra, DMSO, and NS group, respectively. Histological examination revealed the distribution of fibrosis and the degree of collagen deposition in the Ra group were smaller and slighter than the two control groups. Collagen content was significantly less in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). qRT-PCR analysis showed a higher expression of MMP1 mRNA in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). Immunohistochemistry showed the protein levels of MMP1 in the Ra group were significantly increased when compared with the DMSO group (P < 0.01) and the NS group (P < 0.01). On the other hand, no statistical difference could be found between every two groups in both mRNA and protein levels of TGFβ1.. Rapamycin enhances the expression of MMP1 in a rabbit model of urethral stricture, but has no direct effect on the expression of TGFβ1.

Topics: Animals; Antibiotics, Antineoplastic; Collagen; Disease Models, Animal; Fibrosis; Gene Expression; Male; Matrix Metalloproteinase 1; Protein Biosynthesis; Rabbits; RNA, Messenger; Sirolimus; Transforming Growth Factor beta1; Urethral Stricture

To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801.. Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA.. Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity.. The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors.

Topics: Animals; Axons; Cell Survival; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Gene Knockdown Techniques; Immunohistochemistry; Immunosuppressive Agents; Intravitreal Injections; Male; Nerve Crush; Nerve Growth Factors; Nerve Regeneration; Optic Nerve Injuries; Rats; Rats, Wistar; Repressor Proteins; Retinal Ganglion Cells; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transfection

Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.

Topics: Adenocarcinoma; Animals; beta Catenin; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Intestinal Mucosa; Kruppel-Like Transcription Factors; Mice; Mice, Nude; Neoplastic Stem Cells; Receptors, G-Protein-Coupled; Ribosomal Protein S6 Kinases; Sirolimus; Snail Family Transcription Factors; Stomach Neoplasms; TOR Serine-Threonine Kinases; Wnt Signaling Pathway; Zinc Finger Protein Gli2

DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.

Topics: Animals; Animals, Genetically Modified; Cerebral Cortex; Disease Models, Animal; Embryonic Development; Fibroblasts; Gene Knockout Techniques; Genotype; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neurons; Phosphorylation; Rats; Rats, Inbred F344; Rats, Wistar; Repressor Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

This study aimed to determine the expression of progranulin (PGRN) in hepatocellular carcinoma (HCC) cells in response to interleukin 6 (IL-6), a non-cellular component of the tumor microenvironment, and the molecular mechanism of PGRN oncogenic activity in hepatocarcinogenesis. Levels of IL-6 and PGRN were increased and positively correlated in HCC tissues. IL-6 dose- and time-dependently increased PGRN level in HCC cells. IL-6-driven PGRN expression was at least in part mediated by Erk/C/EBPβ signaling, and reduced expression of PGRN impaired IL-6-stimulated proliferation, migration and invasion of HepG2 cells. PGRN activated mammalian target of rapamycin (mTOR) signaling, as evidenced by increased phosphorylation of p70S6K, 4E-BP1, and Akt-Ser473/FoxO1. Inhibition of mTOR signaling with rapamycin, an mTOR signaling inhibitor, disturbed PGRN- or IL-6-mediated proliferation, migration and invasion of HCC cells in vitro. Persistent activation of mTOR signaling by knockdown of TSC2 restored PGRN-knockdown-attenuated pro-proliferation effects of IL-6 in HepG2 cells. In addition, rapamycin treatment in vivo in mice slowed tumor growth stimulated by recombinant human PGRN. Our findings provide a better understanding of the biological activities of the IL-6/PGRN/mTOR cascade in the carcinogenesis of HCC, which may suggest a novel target in the treatment of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-6; Liver Neoplasms; Mice; Progranulins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Th17 cells have been confirmed to increase neutrophils through cytokine secretions. ALI/ARDS are characterized as neutrophil infiltration in inflammation cases; however, there is conflicting information concerning the role of Th17 cells in ALI/ARDS, as well as their potential treatment value. We measured Th17-linear cytokines in the plasma of patients with sepsis-related ARDS. The consistently high levels of IL-17 and IL-22 in the nonsurvivors suggested that overreaction of the Th17-mediated immune response may be a risk factor for poor outcomes. Th17 linear cytokines were also increased in an LPS-induced murine model of acute lung injury, along with neutrophil accumulation. The mice that completely lacked IL-17 failed to accumulate and activate neutrophils. Lung inflammation was obviously attenuated in the IL-17(-)/(-) mice. Meanwhile, the neutrophil count was markedly increased in the healthy WT mice challenged with recombinant IL-22 and IL-17. Rapamycin attenuated lung injury by inhibiting the differentiation of Th17 cells through RORγt and STAT3 dysfunction. Furthermore, we demonstrated that SOCS3 and Gfi1, which were responsible for the molecular suppression of RORγt and STAT3, were up-regulated by rapamycin. These results point toward a pivotal view to treatment of ALI through weakening the proliferation of Th17 cells with rapamycin.

Topics: Acute Lung Injury; Animals; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Interleukin-17; Interleukin-22; Interleukins; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Nuclear Receptor Subfamily 1, Group F, Member 3; Sirolimus; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Th17 Cells; Transcription Factors

Adiponectin (APN), an adipose-derived adipokine, alleviates lipopolysaccharide (LPS)-induced injury in multiple organs including hearts although the underlying mechanism in endotoxemia remains elusive. This study was designed to examine the role of adiponectin in LPS-induced cardiac anomalies and inflammation as well as the underlying mechanism with a focus on autophagy - a conserved machinery for bulk degradation of intracellular components.. Wild-type (WT) and APN(-/-) mice were challenged with LPS (4mg/kg) or saline for 6h. Echocardiography, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated. Markers of autophagy, apoptosis and inflammation including LC3B, p62, Beclin1, AMPK, mTOR, ULK, Caspase 3, Bcl-2, Bax, TLR4, TRAF6, MyD88, IL-1B, TNFα, HMGB1, JNK and IκB were examined using Western blot or RT-PCR. Our results showed that LPS challenge reduced fractional shortening, compromised cardiomyocyte contractile capacity, intracellular Ca(2+) handling properties, apoptosis and inflammation, which were accentuated by adiponectin ablation. Adiponectin ablation unmasked the LPS-induced cardiac remodeling (left ventricular end systolic diameter) and prolongation of cell shortening. The detrimental effects of adiponectin ablation were associated with dampened autophagy in response to LPS through an AMPK-mTOR-ULK1-dependent mechanism. In vivo administration of AMPK activator AICAR or the autophagy inducer rapamycin effectively attenuated or obliterated LPS-induced and adiponectin deficiency-accentuated responses without affecting TLR4, TRAF6 and MyD88.. The findings suggest that AMPK and autophagy may play a permissive role in the adiponectin deficiency-exacerbated cardiac dysfunction, apoptosis and inflammation under LPS challenge possibly at the post-TLR4 receptor level.

Topics: Adiponectin; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Death; Disease Models, Animal; Endotoxemia; Lipopolysaccharides; Male; Mice; Mice, Knockout; Myocarditis; Myocardium; Myocytes, Cardiac; Ribonucleotides; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Dysfunction

Alzheimer's disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aβ plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aβ plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aβ plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aβ plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aβ plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

Topics: Amyloid beta-Peptides; Animals; Apolipoprotein E3; Apolipoprotein E4; Astrocytes; Autophagy; Brain; Cells, Cultured; Central Nervous System Agents; Chloroquine; Disease Models, Animal; Humans; Mice, Transgenic; Plaque, Amyloid; Sirolimus; Time Factors

Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post-status epilepticus (SE) rat model.. Effects of mTOR inhibitor rapamycin were tested using the following three treatment protocols: (1) "stop-treatment"-post-SE treatment (6 mg/kg/day) was discontinued after 3 weeks; rats were monitored for 5 more weeks thereafter, (2) "pretreatment"-rapamycin (3 mg/kg/day) was applied during 3 days preceding SE; and (3) "chronic phase-treatment"-5 days rapamycin treatment (3 mg/kg/day) in the chronic phase. We also tested curcumin, an alternative mTOR inhibitor with antiinflammatory and antioxidant effects, using chronic phase treatment. Seizures were continuously monitored using video-electroencephalography (EEG) recordings; mossy fiber sprouting, cell death, and inflammation were studied using immunohistochemistry. Blood was withdrawn regularly to assess rapamycin and curcumin levels with high performance liquid chromatography (HPLC).. Stop-treatment led to a strong reduction of seizures during the 3-week treatment and a gradual reappearance of seizures during the following 5 weeks. Three days pretreatment did not prevent seizure development, whereas 5-day rapamycin treatment in the chronic phase reduced seizure frequency. Washout of rapamycin was slow and associated with a gradual reappearance of seizures. Rapamycin treatment (both 3 and 6 mg/kg) led to body growth reduction. Curcumin treatment did not reduce seizure frequency or lead to a decrease in body weight.. The present study indicates that rapamycin cannot prevent epilepsy in the electrical stimulation post-SE rat model but has seizure-suppressing properties as long as rapamycin blood levels are sufficiently high. Oral curcumin treatment had no effect on chronic seizures, possibly because it did not reach the brain at adequate levels.

Topics: Analysis of Variance; Animals; Anticonvulsants; Body Weight; Curcumin; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Electric Stimulation; Electroencephalography; Hippocampus; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Status Epilepticus; Time Factors; Treatment Outcome

Everolimus inhibits mTOR kinase activity and its downstream targets by acting on mTORC1 and has anti-tumorigenic activity in ovarian cancer. Clinical and epidemiologic data find that obesity is associated with worse outcomes in ovarian cancer. In addition, obesity leads to hyperactivation of the mTOR pathway in epithelial tissues, suggesting that mTOR inhibitors may be a logical choice for treatment in obesity-driven cancers. However, it remains unclear if obesity impacts the effect of everolimus on tumor growth in ovarian cancer. The present study was aimed at evaluating the effects of everolimus on cytotoxicity, cell metabolism, apoptosis, cell cycle, cell stress and invasion in human ovarian cancer cells. A genetically engineered mouse model of serous ovarian cancer fed a high fat diet or low fat diet allowed further investigation into the inter-relationship between everolimus and obesity in vivo. Everolimus significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, reduced invasion and caused cellular stress via inhibition of mTOR pathways in vitro. Hypoglycemic conditions enhanced the sensitivity of cells to everolimus through the disruption of glycolysis. Moreover, everolimus was found to inhibit ovarian tumor growth in both obese and lean mice. This reduction coincided with a decrease in expression of Ki-67 and phosphorylated-S6, as well as an increase in cleaved caspase 3 and phosphorylated-AKT. Metabolite profiling revealed that everolimus was able to alter tumor metabolism through different metabolic pathways in the obese and lean mice. Our findings support that everolimus may be a promising therapeutic agent for obesity-driven ovarian cancers.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinogenesis; Carcinoma, Ovarian Epithelial; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Disease Models, Animal; Female; Humans; Mice; Neoplasms, Glandular and Epithelial; Obesity; Ovarian Neoplasms; Phosphorylation; Prognosis; Signal Transduction; Sirolimus; Tumor Cells, Cultured

Epilepsy is a frequent comorbidity in patients with focal cortical dysplasia (FCD). Recent studies utilizing massive sequencing data identified subsets of genes that are associated with epilepsy and FCD. AKT and mTOR-related signals have been recently implicated in the pathogenic processes of epilepsy and FCD. To clarify the functional roles of the AKT-mTOR pathway in the hippocampal neurons, we generated conditional knockout mice harboring the deletion of Pten (Pten-cKO) in Proopiomelanocortin-expressing neurons. The Pten-cKO mice developed normally until 8 weeks of age, then presented generalized seizures at 8-10 weeks of age. Video-monitored electroencephalograms detected paroxysmal discharges emerging from the cerebral cortex and hippocampus. These mice showed progressive hypertrophy of the dentate gyrus (DG) with increased expressions of excitatory synaptic markers (Psd95, Shank3 and Homer). In contrast, the expression of inhibitory neurons (Gad67) was decreased at 6-8 weeks of age. Immunofluorescence studies revealed the abnormal sprouting of mossy fibers in the DG of the Pten-cKO mice prior to the onset of seizures. The treatment of these mice with an mTOR inhibitor rapamycin successfully prevented the development of seizures and reversed these molecular phenotypes. These data indicate that the mTOR pathway regulates hippocampal excitability in the postnatal brain.

Topics: Animals; Craniofacial Abnormalities; Dentate Gyrus; Disease Models, Animal; Electroencephalography; Epilepsies, Partial; Epilepsy; Hippocampus; Humans; Malformations of Cortical Development; Mice; Mice, Knockout; Neurons; Pro-Opiomelanocortin; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Postsurgical adhesions can occur after laparotomy and can cause morbidity. Local delivery of sirolimus prevented adhesion formation in various experiments. We analyzed the impact of orally dosed mammalian target of rapamycin inhibitors on abdominal adhesion formation and wound tensile strength in an experimental model.. Wistar albino rats were divided into sirolimus, everolimus, and control groups (n = 6 per group). Experimental animals underwent midline laparotomy and adhesion induction procedure which included cecum abrasion and mesh implantation. Animals were administered oral sirolimus (4 mg/kg), everolimus (3 mg/kg), or placebo starting on postoperative day 1. Treatments were given until postoperative day 7. At postoperative day 21, adhesions were scored. Meshes were resected with the attached abdominal wall and cecal segment and stained with Sirius red for collagen density analysis. Midline scars were excised for tensile strength measurement. Effects of sirolimus and everolimus on fibroblast proliferation were also assessed.. Mean adhesion score of the everolimus group (7.83 ± 1.17) was significantly lower compared to sirolimus (11.00 ± 0.63) and control (11.66 ± 0.51) groups. Mean collagen density of the everolimus group (33.5 ± 7.8) was significantly lower compared to sirolimus (50.7 ± 9.69) and control (53.8 ± 12.4) groups. Mean tensile strength of the control group (26.41 ± 2.10) was significantly higher compared to sirolimus (17.89 ± 1.9) and everolimus (21.37 ± 1.25) groups. It was significantly lower in sirolimus group than everolimus group. Both sirolimus and everolimus treated media inhibited fibroblast proliferation significantly compared to media alone.. Everolimus effectively reduced adhesions. Nevertheless, it also reduced wound tensile strength: an effect which seemed to be due to inhibition of fibroblast proliferation.

Topics: 3T3 Cells; Abdomen; Administration, Oral; Animals; Cecum; Cell Proliferation; Collagen; Disease Models, Animal; Everolimus; Fibroblasts; Immunosuppressive Agents; Male; Mice; Rats; Rats, Wistar; Sirolimus; Tensile Strength; Tissue Adhesions; TOR Serine-Threonine Kinases; Wounds and Injuries

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the Akt/mammalian target of rapamycin (MTOR) pathway. Mutations in PTEN have been found in a subset of individuals with autism and macrocephaly. Further, focal cortical dysplasia (FCD) has been observed in patients with PTEN mutations prompting us to examine the role of Pten in neuronal migration. The dentate gyrus of Pten(Flox/Flox) mice was injected with Cre- and non-Cre-expressing retroviral particles, which integrate into the dividing genome to birthdate cells. Control and Pten knockout (KO) cell position in the granule cell layer was quantified over time to reveal that Pten KO neurons exhibit an aberrant migratory phenotype beginning at 7.5days-post retroviral injection (DPI). We then assessed whether rapamycin, a mTor inhibitor, could prevent or reverse aberrant migration of granule cells. The preventative group received daily intraperitoneal (IP) injections of rapamycin from 3 to 14 DPI, before discrepancies in cell position have been established, while the reversal group received rapamycin afterward, from 14 to 24 DPI. We found that rapamycin prevented and reversed somal hypertrophy. However, rapamycin prevented, but did not reverse aberrant migration in Pten KO cells. We also find that altered migration occurs through mTorC1 and not mTorC2 activity. Together, these findings suggest a temporal window by which rapamycin can treat aberrant migration, and may have implications for the use of rapamycin to treat PTEN-mutation associated disorders.. Mutations in phosphatase and tensin homolog (PTEN) have been linked to a subset of individuals with autism and macrocephaly, as well as Cowden Syndrome and focal cortical dysplasia. Pten loss leads to neuronal hypertrophy, but the role of Pten in neuronal migration is unclear. Here we have shown that loss of Pten leads to aberrant migration, which can be prevented but not reversed by treatment with rapamycin, a mTor inhibitor. These results are important to consider as clinical trials are developed to examine rapamycin as a therapeutic for autism with PTEN mutations. Our findings show that some abnormalities cannot be reversed, and suggest the potential need for genetic screening and preventative treatment.

Topics: Animals; Autistic Disorder; Brain; Cell Movement; Disease Models, Animal; Mice, Knockout; Mutation; Neurons; Phenotype; PTEN Phosphohydrolase; Signal Transduction; Sirolimus

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

Topics: Animals; Carrier Proteins; Cation Transport Proteins; Cell Movement; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Immunosuppressive Agents; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mossy Fibers, Hippocampal; Neurogenesis; Neurons; Pilocarpine; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Status Epilepticus; Weight Gain; Zinc Finger Protein GLI1

Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.

Topics: Animals; Apoptosis; Cell Hypoxia; Cell Nucleus; Disease Models, Animal; Enzyme Inhibitors; Glucosamine; Glycerol-3-Phosphate O-Acyltransferase; Glycosylation; Mice; Mouse Embryonic Stem Cells; Phosphorylation; RNA Interference; RNA, Small Interfering; Sirolimus; Skin Diseases; Sp1 Transcription Factor; TOR Serine-Threonine Kinases; Up-Regulation

Eccentric contractions may cause immediate and long-term reductions in muscle strength that can be recovered through increased protein synthesis rates. The purpose of this study was to determine whether the mechanistic target-of-rapamycin complex 1 (mTORC1), a vital controller of protein synthesis rates, is required for return of muscle strength after injury.. Isometric muscle strength was assessed before, immediately after, and then 3, 7, and 14 days after a single bout of 150 eccentric contractions in mice that received daily injections of saline or rapamycin.. The bout of eccentric contractions increased the phosphorylation of mTORC1 (1.8-fold) and p70s6k1 (13.8-fold), mTORC1's downstream effector, 3 days post-injury. Rapamycin blocked mTORC1 and p70s6k1 phosphorylation and attenuated recovery of muscle strength (∼20%) at 7 and 14 days.. mTORC1 signaling is instrumental in the return of muscle strength after a single bout of eccentric contractions in mice. Muscle Nerve 54: 914-924, 2016.

Topics: Animals; Disease Models, Animal; Electric Stimulation; Female; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Skeletal; Muscular Diseases; Recovery of Function; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Torque

The cascade of events leading to post-traumatic epilepsy (PTE) after traumatic brain injury (TBI) remains unclear. Altered inhibition in the hippocampal formation and dentate gyrus is a hallmark of several neurological disorders, including TBI and PTE. Inhibitory synaptic signaling in the hippocampus is predominately driven by γ-aminobutyric acid (GABA) neurotransmission, and is prominently mediated by postsynaptic type A GABA receptors (GABAAR's). Subsets of these receptors involved in tonic inhibition of neuronal membranes serve a fundamental role in maintenance of inhibitory state, and GABAAR-mediated tonic inhibition is altered functionally in animal models of both TBI and epilepsy. In this study, we assessed the effect of mTOR inhibition on hippocampal hilar inhibitory interneuron loss and synaptic and tonic GABAergic inhibition of dentate gyrus granule cells (DGCs) after controlled cortical impact (CCI) to determine if mTOR activation after TBI modulates GABAAR function. Hilar inhibitory interneuron density was significantly reduced 72h after CCI injury in the dorsal two-thirds of the hemisphere ipsilateral to injury compared with the contralateral hemisphere and sham controls. Rapamycin treatment did not alter this reduction in cell density. Synaptic and tonic current measurements made in DGCs at both 1-2 and 8-13weeks post-injury indicated reduced synaptic inhibition and THIP-induced tonic current density in DGCs ipsilateral to CCI injury at both time points post-injury, with no change in resting tonic GABAAR-mediated currents. Rapamycin treatment did not alter the reduced synaptic inhibition observed in ipsilateral DGCs 1-2weeks post-CCI injury, but further reduced synaptic inhibition of ipsilateral DGCs at 8-13weeks post-injury. The reduction in THIP-induced tonic current after injury, however, was prevented by rapamycin treatment at both time points. Rapamycin treatment thus differentially modifies CCI-induced changes in synaptic and tonic GABAAR-mediated currents in DGCs.

Topics: Anesthetics; Animals; Brain Injuries; Dentate Gyrus; Disease Models, Animal; Functional Laterality; GABA Agents; GABAergic Neurons; Glutamate Decarboxylase; Green Fluorescent Proteins; Immunosuppressive Agents; In Vitro Techniques; Interneurons; Isoxazoles; Male; Membrane Potentials; Mice; Mice, Transgenic; Patch-Clamp Techniques; Ribosomal Protein S6 Kinases; Sirolimus; Time Factors

Mechanistic target of rapamycin (mTOR) has been shown to play an important role in red blood cell physiology, with inhibition of mTOR signalling leading to alterations in erythropoiesis. To determine if mTOR inhibition would improve anaemia in sickle cell disease (SCD), mice with SCD were treated with the dual mTORC1/2 inhibitor, INK128. One week after daily oral drug treatment, erythrocyte count, haemoglobin, and haematocrit were all significantly increased while reticulocyte counts were reduced. These parameters remained stable during 3 weeks of treatment. Similar effects were observed following oral treatment with the mTORC1 inhibitor, sirolimus. Sirolimus treatment prolonged the lifespan of sickle cell erythrocytes in circulation, reduced spleen size, and reduced renal and hepatic iron accumulation in SCD mice. Following middle cerebral artery occlusion, stroke size was reduced in SCD mice treated with sirolimus. In conclusion, mTOR inhibition is protective against anaemia and organ damage in a murine model of SCD.

Topics: Anemia; Anemia, Sickle Cell; Animals; Benzoxazoles; Disease Models, Animal; Erythrocytes; Hematocrit; Hemoglobins; Mice; Pyrimidines; Sirolimus; Splenomegaly; TOR Serine-Threonine Kinases

We have shown that bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) from SLE patients exhibit senescent behavior and are involved in the pathogenesis of SLE. The aim of this study was to investigate the effects of rapamycin (RAPA) on the senescences and immunoregulatory ability of MSCs of MRL/lpr mice and SLE patients and the underlying mechanisms. Cell morphology, senescence associated β-galactosidase (SA-β-gal) staining, F-actin staining were used to detect the senescence of cells. BM-MSCs and purified CD4+ T cells were co-cultured indirectly. Flow cytometry was used to inspect the proportion of regulatory T (Treg) /T helper type 17 (Th17). We used small interfering RNA (siRNA) to interfere the expression of mTOR, and detect the effects by RT-PCR, WB and immunofluorescence. Finally, 1x106 of SLE BM-MSCs treated with RAPA were transplanted to cure the 8 MRL/lpr mice aged 16 weeks for 12 weeks. We demonstrated that RAPA alleviated the clinical symptoms of lupus nephritis and prolonged survival in MRL/lpr mice. RAPA reversed the senescent phenotype and improved immunoregulation of MSCs from MRL/lpr mice and SLE patients through inhibition of the mTOR signaling pathway. Marked therapeutic effects were observed in MRL/lpr mice following transplantation of BM-MSCs from SLE patients pretreated with RAPA.

Topics: Animals; beta-Galactosidase; Cell Shape; Cells, Cultured; Cellular Senescence; Disease Models, Animal; Female; Humans; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Lupus Nephritis; Mesenchymal Stem Cells; Mice; Mice, Inbred MRL lpr; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Ciliopathies; Disease Models, Animal; Elafin; Epithelial Cells; Germ-Line Mutation; Humans; Kidney; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphoric Monoester Hydrolases; Polycystic Kidney Diseases; Proto-Oncogene Proteins c-akt; Sequence Deletion; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The guideline for the management of new-onset diabetes after transplantation recommends metformin (MET) as a first-line drug, and addition of a second-line drug is needed to better control of hyperglycemia. We tested the effect of addition of a dipeptidyl peptidase IV (DPP IV) inhibitor to MET on sirolimus (SRL)-induced diabetes mellitus (DM). In animal model of SRL-induced DM, MET treatment improved pancreatic islet function (blood glucose level and insulin secretion) and attenuated oxidative stress and apoptotic cell death. Addition of a DPP IV inhibitor to MET improved these parameters more than MET alone. An in vitro study showed that SRL treatment increased pancreas beta cell death and production of reactive oxygen species (ROS), and pretreatment of ROS inhibitor, or p38MAPK inhibitor effectively decreased SRL-induced islet cell death. Exendin-4 (EXD), a substrate of DPP IV or MET significantly improved cell viability and decreased ROS production compared with SRL treatment, and combined treatment with the 2 drugs improved both parameters. At the subcellular level, impaired mitochondrial respiration by SRL were partially improved by MET or EXD and much improved further after addition of EXD to MET. Our data suggest that addition of a DPP IV inhibitor to MET decreases SRL-induced oxidative stress and improves mitochondrial respiration. This finding provides a rationale for the combined use of a DPP IV inhibitor and MET in treating SRL-induced DM.

Topics: Animals; Apoptosis; Cell Survival; Diabetes Mellitus, Experimental; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Drug Therapy, Combination; Exenatide; Hyperglycemia; Islets of Langerhans; Male; Metformin; Mitochondria; Oxidative Stress; Peptides; Piperidones; Pyrimidines; Rats, Sprague-Dawley; Reactive Oxygen Species; Sirolimus; Venoms

The constitutive hyper-activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways has frequently been associated with acute myeloid leukemia (AML). While many inhibitors targeting these pathways have been developed, the anti-leukemic effect was not as robust as expected. As part of the molecular link between PI3K/Akt and mTOR kinase, the role of Rheb1 in AML remains unexplored. Our study aims to explore the role of Rheb1 in AML and estimate whether Rheb1 could be a potential target of AML treatment.. The expressions of Rheb1 and other indicated genes were analyzed using real-time PCR. AML mouse model was established by retrovirus transduction. Leukemia cell properties and related signaling pathways were dissected by in vitro and in vivo studies. The transcriptional changes were analyzed via gene chip analysis. Molecular reagents including mTOR inhibitor and mTOR activator were used to evaluate the function of related signaling pathway in the mouse model.. We observed that Rheb1 is overexpressed in AML patients and the change of Rheb1 level in AML patients is associated with their median survival. Using a Rheb1-deficient MLL-AF9 murine AML model, we revealed that Rheb1 deletion prolonged the survival of AML mice by weakening LSC function. In addition, Rheb1 deletion arrested cell cycle progression and enhanced apoptosis of AML cells. Furthermore, while Rheb1 deletion reduced mTORC1 activity in AML cells, additional rapamycin treatment further decreased mTORC1 activity and increased the apoptosis of Rheb1 (Δ/Δ) AML cells. The mTOR activator 3BDO partially rescued mTORC1 signaling and inhibited apoptosis in Rheb1 (Δ/Δ) AML cells.. Our data suggest that Rheb1 promotes AML progression through mTORC1 signaling pathway and combinational drug treatments targeting Rheb1 and mTOR might have a better therapeutic effect on leukemia.

Topics: Acute Disease; Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Cell Cycle Checkpoints; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation, Leukemic; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C57BL; Mice, Knockout; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Neuropeptides; Oncogene Proteins, Fusion; Ras Homolog Enriched in Brain Protein; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Type 1 diabetes (T1D) is one of the most common autoimmune diseases, affecting nearly 20 million people worldwide. The pathogenesis of T1D is characterized by the progressive destruction of insulin-producing pancreatic β-cells by autoreactive T cells. The significant role of immunomodulation in preserving residual insulin-producing β-cells in newly diagnosed T1D has not been confirmed yet. However, a combination of treatments acting via distinct mechanisms is widely considered to be the most promising future therapeutic approach in this respect. Rapamycin and gamma-aminobutyric acid (GABA) administration alone showed no effects on late-stage disease. By contrast, combined rapamycin/GABA treatment effectively suppressed autoimmune responses to islet cells and improved islet function in recent-onset diabetes. In particular, after the onset of hyperglycemia, those treated with the rapamycin/GABA combination showed significant amelioration of diabetes amelioration compared to those treated with either rapamycin or GABA alone. This protective effect of the rapamycin/GABA combination treatment in nonobese diabetic (NOD) mice was exerted through two distinct mechanisms. Rapamycin induced regulatory T cells and consequently suppressed targeted autoimmune responses to islet cells, which may be relevant to the reduced insulitis observed in rapamycin-treated NOD mice. By contrast, treatment with GABA improved islet function in diabetic NOD mice. We believe that our observations are highly relevant to establishing clinical strategies for the prevention and treatment of T1D in future.

Topics: Animals; Blotting, Western; Diabetes Mellitus, Type 1; Disease Models, Animal; Drug Therapy, Combination; Female; Flow Cytometry; gamma-Aminobutyric Acid; Islets of Langerhans; Mice; Mice, Inbred NOD; Real-Time Polymerase Chain Reaction; Sirolimus; T-Lymphocytes, Regulatory

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).

Topics: Animals; cdc42 GTP-Binding Protein; Disease Models, Animal; Epithelial-Mesenchymal Transition; Epithelium; Fibronectins; Gene Expression Regulation; Glucose; Humans; Peritoneal Fibrosis; Peritoneum; rac1 GTP-Binding Protein; Rats; rhoA GTP-Binding Protein; Sirolimus

The formation of intraarticular adhesion is a common complication after total knee arthroplasty or anterior cruciate ligament reconstruction. Previously, little research was reported regarding whether the local application of rapamycin (RAPA) could reduce intraarticular adhesion following knee surgery. In our present study, we determined the therapeutic effect of RAPA by local application on the reduction of intraarticular adhesion following knee surgery in rabbits.. In this study, we built the model of knee surgery according to a previous study. The decorticated areas of the cortical bone were exposed and covered with cotton pads soaked with different concentrations of RAPA or physiological saline for 10 min. All of the rabbits were euthanized 4 weeks after the surgery. Macroscopic evaluation of the hydroxyproline content, the histological morphological analysis and collagen density and fibroblast density were used to evaluate the effect of RAPA on reducing intraarticular adhesion.. The results shown that RAPA could significantly inhibit the proliferation of fibroblasts and reduce collagen synthesis; in the rabbit model of knee surgery, there were weak scar tissues around the decorticated areas in the 0.2 mg/ml RAPA group; moderate scar tissues were found in the 0.1 mg/ml RAPA group. However, severe fibrous adhesions were found in the 0.05 mg/ml RAPA group and the control group. The hydroxyproline content and the fibroblast density in the 0.2 mg/ml and 0.1 mg/ml RAPA groups were significantly less than those of the control group.. We concluded that the local application of RAPA could reduce intraarticular adhesion after knee surgery in the rabbit model; this effect was mediated by inhibition of fibroblast proliferation and collagen synthesis, which may provide a new method for reducing intraarticular adhesion after clinical knee surgery.

Topics: Administration, Topical; Animals; Cell Proliferation; Collagen; Disease Models, Animal; Drug Evaluation, Preclinical; Fibroblasts; Immunosuppressive Agents; Knee Joint; Male; Rabbits; Sirolimus; Tissue Adhesions

Autophagy plays an important role in spinal cord ischemia reperfusion (I/R) injury, but its neuroprotective or neurodegenerative role remains controversial. The extent and persistence of autophagy activation may be the critical factor to explain the opposing effects. In this study, the different roles and action mechanisms of autophagy in the early and later stages after I/R injury were investigated in rats. Thespinal cord I/R injury was induced by 14-min occlusion of the aortic arch, after which rats were treated with autophagic inhibitor (3-methyladenine, 3-MA) or agonist (rapamycin) immediately or 48h following the injury. Autophagy markers, microtubule-associated protein light chain 3-II (LC3-II) and Beclin 1 increased and peaked at the early stage (8h) and the later stage (72h) after spinal cord I/R injury. Beclin 1 was mostly expressed in neurons, but was also expressed to an extent in astrocytes, microglia and vascular endothelial cells. 8h after injury, rats treated with 3-MA showed a decrease in the hind-limb Basso-Beattie-Bresnahan (BBB) motor function scores, surviving motor neurons, and B-cell lymphoma-2 (Bcl-2) expression, and increase in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, Bcl-2-associated X protein (Bax), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) expression, and activation of microglia, while those treated with rapamycin showed opposing effects. However, 72h after injury, rats treated with 3-MA improved the BBB scores, and the surviving motor neurons, and reduced the autophagic cell death, while those treated with rapamycin had adverse effects. These findings provide the first evidence that early activated autophagy alleviates spinal cord I/R injury via inhibiting apoptosis and inflammation; however later excessively elevated autophagy aggravates I/R injury through inducing autophagic cell death.

Topics: Adenine; Animals; Apoptosis; Autophagy; Central Nervous System Agents; Disease Models, Animal; Male; Motor Activity; Neuroimmunomodulation; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Spinal Cord; Spinal Cord Ischemia

The study aimed to compare the impact of allogeneic bone marrow cells (BMCs) infusion through the inferior vena cava (IVC) and portal vein (PV) combined with rapamycin on allogeneic islet grafts in diabetic rats.. Recipient diabetic Wistar rats were infused with islets from Sprague-Dawley rats through the PV. PKH26-labeled BMCs of Sprague-Dawley rats were infused to recipients through the PV or IVC, followed by administration of rapamycin for 4 days. Blood glucose level was measured to evaluate the survival time of the islets. Lymphocytes separated from blood, BMCs, thymus, liver, spleen and lymph node were analyzed by flow cytometry. The peripheral blood smear, BMCs smear and frozen sections of tissues were observed by a fluorescence microscope.. The survival time of the islets was significantly prolonged by the BMCs infusion combined with rapamycin. The rats receiving BMCs infusion through the PV induced a significantly longer survival time of the islets, and increased mixed chimeras of allogeneic BMCs in the thymus, liver, spleen and lymph node compared with the rats receiving BMCs infusion through the IVC. The amount of the mixed chimeras on day 14 was lower than that on day 7 after islet transplantation. Furthermore, PV transplantation had significantly more mixed chimera than IVC transplantation in all analyzed organs or tissues.. BMCs infusion combined with rapamycin prolongs the islets survival and induces mixed chimeras of BMCs. PV infusion of BMCs might be a more effective strategy than IVC infusion of BMCs.

Topics: Animals; Blood Glucose; Bone Marrow Cells; Bone Marrow Transplantation; Diabetes Mellitus, Experimental; Disease Models, Animal; Graft Survival; Islets of Langerhans Transplantation; Male; Portal Vein; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sirolimus; Tissue Distribution; Transplantation, Homologous; Vena Cava, Inferior

Phenotype-guided re-profiling of approved drug molecules presents an accelerated route to developing anticancer therapeutics by bypassing the target-identification bottleneck of target-based approaches and by sampling drugs already in the clinic. Further, combinations incorporating targeted therapies can be screened for both efficacy and toxicity. Previously we have developed an oncogenic-RAS-driven zebrafish melanoma model that we now describe display melanocyte hyperplasia while still embryos. Having devised a rapid method for quantifying melanocyte burden, we show that this phenotype can be chemically suppressed by incubating V12RAS transgenic embryos with potent and selective small molecule inhibitors of either MEK or PI3K/mTOR. Moreover, we demonstrate that combining MEK inhibitors (MEKi) with dual PI3K/mTOR inhibitors (PI3K/mTORi) resulted in a super-additive suppression of melanocyte hyperplasia. The robustness and simplicity of our novel screening assay inspired us to perform a modest screen of FDA approved compounds for their ability to potentiate MEKi PD184352 or PI3K/mTORi NVPBEZ235 suppression of V12RAS-driven melanocyte hyperplasia. Through this route, we confirmed Rapamycin as a compound that could synergize with MEKi and even more so with PI3K/mTORi to suppress melanoma development, including suppressing the growth of cultured human melanoma cells. Further, we discovered two additional compounds-Disulfiram and Tanshinone-that also co-operate with MEKi to suppress the growth of transformed zebrafish melanocytes and showed activity toward cultured human melanoma cells. In conclusion, we provide proof-of-concept that our phenotype-guided screen could be used to identify compounds that affect melanoma development and prompt further evaluation of Disulfiram and Tanshinone as possible partners for combination therapy.

Topics: Abietanes; Animals; Animals, Genetically Modified; Apoptosis; Benzamides; Cell Line, Tumor; Disease Models, Animal; Disulfiram; Drug Repositioning; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Melanins; Melanocytes; Melanoma; Oligonucleotides, Antisense; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Skin Neoplasms; Zebrafish

Focal cortical dysplasia (FCD), a local malformation of cortical development, is the most common cause of pharmacoresistant epilepsy associated with life-long neurocognitive impairments. It remains unclear whether neuronal misplacement is required for seizure activity. Here we show that dyslamination and white matter heterotopia are not necessary for seizure generation in a murine model of type II FCDs. These experimental FCDs generated by increasing mTOR activity in layer 2/3 neurons of the medial prefrontal cortex are associated with tonic-clonic seizures and a normal survival rate. Preventing all FCD-related defects, including neuronal misplacement and dysmorphogenesis, with rapamycin treatments from birth eliminates seizures, but seizures recur after rapamycin withdrawal. In addition, bypassing neuronal misplacement and heterotopia using inducible vectors do not prevent seizure occurrence. Collectively, data obtained using our new experimental FCD-associated epilepsy suggest that life-long treatment to reduce neuronal dysmorphogenesis is required to suppress seizures in individuals with FCD.

Topics: Animals; Cell Movement; Cognitive Dysfunction; Disease Models, Animal; Female; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Male; Malformations of Cortical Development; Mice; Neurons; Prefrontal Cortex; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; White Matter

Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation.. We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features.. Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing.. These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.

Topics: Animals; Biomechanical Phenomena; Disease Models, Animal; Down-Regulation; Dystroglycans; Electric Stimulation; Female; Genetic Predisposition to Disease; Glycosylation; Male; Mice, Knockout; Muscle Contraction; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Animal; Myogenic Regulatory Factor 5; Phenotype; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Torque; Transferases

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.

Topics: Animals; Animals, Newborn; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Female; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Associated Glycoprotein; Nerve Regeneration; Neurotrophin 3; Nogo Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Somatosensory Disorders; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

Topics: Animals; Biomarkers; Carbon Tetrachloride; Cell Communication; Coculture Techniques; Cytokines; Disease Models, Animal; Hepatic Stellate Cells; Immunomodulation; Liver Cirrhosis; Liver Function Tests; Lymphocyte Count; Male; Mice; Sirolimus; T-Lymphocytes, Regulatory; Th17 Cells

Several drugs targeting the pathogenesis of aortic aneurysm have shown efficacy in model systems but not in clinical trials, potentially owing to the lack of targeted drug delivery. Here, we designed a novel drug delivery system using nanoparticles to target the disrupted aortic aneurysm micro-structure. We generated poly(ethylene glycol)-shelled nanoparticles incorporating rapamycin that exhibited uniform diameter and long-term stability. When injected intravenously into a rat model in which abdominal aortic aneurysm (AAA) had been induced by infusing elastase, labeled rapamycin nanoparticles specifically accumulated in the AAA. Microscopic analysis revealed that rapamycin nanoparticles were mainly distributed in the media and adventitia where the wall structures were damaged. Co-localization of rapamycin nanoparticles with macrophages was also noted. Rapamycin nanoparticles injected during the process of AAA formation evinced significant suppression of AAA formation and mural inflammation at 7 days after elastase infusion, as compared with rapamycin treatment alone. Correspondingly, the activities of matrix metalloproteinases and the expression of inflammatory cytokines were significantly suppressed by rapamycin nanoparticle treatment. Our findings suggest that the nanoparticle-based delivery system achieves specific delivery of rapamycin to the rat AAA and might contribute to establishing a drug therapy approach targeting aortic aneurysm.

Topics: Animals; Aortic Aneurysm; Aortic Aneurysm, Abdominal; Cytokines; Disease Models, Animal; Drug Delivery Systems; Gelatinases; Inflammation Mediators; Microscopy, Fluorescence; Molecular Imaging; Nanoparticles; Rats; Sirolimus; Tissue Distribution

Used in combination with immunomodulatory therapies, remyelinating therapies are a viable therapeutic approach for treating individuals with multiple sclerosis. Studies of postmortem MS brains identified greater remyelination in demyelinated cerebral cortex than in demyelinated brain white matter and implicated reactive astrocytes as an inhibitor of white matter remyelination. An animal model that recapitulates these phenotypes would benefit the development of remyelination therapeutics. We have used a modified cuprizone protocol that causes a consistent and robust demyelination of mouse white matter and cerebral cortex. Spontaneous remyelination occurred significantly faster in the cerebral cortex than in white matter and reactive astrocytes were more abundant in white matter lesions. Remyelination of white matter and cerebral cortex was therapeutically enhanced by daily injections of thyroid hormone triiodothyronine (T3). In summary, we describe an in vivo demyelination/remyelination paradigm that can be powered to determine efficacy of therapies that enhance white matter and cortical remyelination.

Topics: Animals; Axons; Brain; Calcium-Binding Proteins; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Glial Fibrillary Acidic Protein; Gliosis; Immunosuppressive Agents; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Monoamine Oxidase Inhibitors; Myelin Proteolipid Protein; Regeneration; Sirolimus; Time Factors; Triiodothyronine; White Matter

Here, we aimed to study the role and underlying mechanism of mTOR in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Experiment 1, the time course of mTOR activation in the cortex following SAH. Experiment 2, the role of mTOR in SAH-induced EBI. Adult SD rats were divided into four groups: sham group (n=18), SAH+vehicle group (n=18), SAH+rapamycin group (n=18), SAH+AZD8055 group (n=18). Experiment 3, we incubated enriched microglia with OxyHb. Rapamycin and AZD8055 were also used to demonstrate the mTOR's role on microglial polarization in vitro. The phosphorylation levels of mTOR and its substrates were significantly increased and peaked at 24h after SAH. Rapamycin or AZD8055 markedly decreased the phosphorylation levels of mTOR and its substrates and the activation of microglia in vivo, and promoted the microglial polarization from M1 phenotype to M2 phenotype. In addition, administration of rapamycin and AZD8055 following SAH significantly ameliorated EBI, including neuronal apoptosis, neuronal necrosis, brain edema and blood-brain barrier permeability. Our findings suggested that the rapamycin and AZD8055 could attenuate the development of EBI in this SAH model, possibly through inhibiting the activation of microglia by mTOR pathway.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Brain Edema; Capillary Permeability; Cell Polarity; Cells, Cultured; Disease Models, Animal; Microglia; Morpholines; Necrosis; Neurons; Neuroprotective Agents; Phosphorylation; Random Allocation; Rats, Sprague-Dawley; Sirolimus; Subarachnoid Hemorrhage; TOR Serine-Threonine Kinases

Impairment of immune defenses can contribute to severe influenza infections. Rapamycin is an immunosuppressive drug often used to prevent transplant rejection and is currently undergoing clinical trials for treating cancers and autoimmune diseases. We investigated whether rapamycin has deleterious effects during lethal influenza viral infections. We treated mice with two concentrations of rapamycin and infected them with A/Puerto Rico/8/1934 (A/PR8), followed by a heterosubtypic A/Hong Kong/1/68 (A/HK68) challenge. Our data show similar morbidity, mortality, and lung viral titer with both rapamycin treatment doses compared to untreated controls, with a delay in morbidity onset in rapamycin high dose recipients during primary infection. Rapamycin treatment at high dose also led to increase in percent cytokine producing T cells in the spleen. However, all infected animals had similar serum antibody responses against A/PR8. Post-A/HK68 challenge, rapamycin had no impeding effect on morbidity or mortality and had similar serum antibody levels against A/PR8 and A/HK68. We conclude that rapamycin treatment does not adversely affect morbidity, mortality, or antibody production during lethal influenza infections.

Topics: Animals; Antibody Formation; Cytokines; Disease Models, Animal; Immunosuppressive Agents; Influenza A virus; Lung; Mice; Orthomyxoviridae Infections; Sirolimus; Spleen; Survival Analysis; T-Lymphocytes; Viral Load

SET is a multifunctional protein, but when present in the cytoplasm, acts as a powerful inhibitor of phosphatase 2A. We previously observed that in CA1 of Down syndrome (DS) patients, the level of SET is increased, and SET is translocated to the cytoplasm and associated with the hyperphosphorylation of tau at ser202/thr205. The presence of SET in the cytoplasm in DS brains may play a role in the progression of the disease. Here, we show that in CA1 of 3-month-old Ts65Dn mice modeling DS, SET level is increased, and SET is translocated to the cytoplasm and associated with tau hyperphosphorylations at ser202/thr205 and with amyloid precursor protein caspase cleaved as observed in Alzheimer disease brains. Tau hyperphosphorylation at ser356 and activation of other phosphatase 2A targets such as the mammalian target of rapamycin and adenosine monophosphate protein kinases were also observed, suggesting deleterious mechanisms. We propose Ts65Dn mice as a model for therapeutic approaches focused on SET overexpression and its cytoplasmic translocation to slow down disease progression.

Topics: Amyloid beta-Protein Precursor; Animals; Brain; CA1 Region, Hippocampal; Cytoplasm; Disease Models, Animal; DNA-Binding Proteins; Down Syndrome; Gene Expression; Histone Chaperones; Male; Mice; Mice, Inbred Strains; Molecular Targeted Therapy; Oncogene Proteins; Phosphorylation; Protein Phosphatase 2; Protein Transport; Serine; Sirolimus; tau Proteins; Threonine

Atorvastatin is a cholesterol-lowering statin that has been shown to exert several pleiotropic effects in the nervous system as a neuroprotective and antidepressant-like agent. Antidepressant-like effect of atorvastatin in mice is mediated by glutamatergic and serotoninergic receptors, although the precise intracellular signaling pathways involved are unknown. PI3K/Akt/GSK-3β/mTOR signaling pathway has been associated to neurobiology of depression and seems to be modulated by some pharmacological antidepressant strategies. The present study investigated the participation of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of an acute atorvastatin treatment in mice. Atorvastatin sub-effective (0.01 mg/kg) or effective (0.1 mg/kg) doses in the tail suspension test (TST) was administered orally alone or in combination with PI3K, GSK-3β or mTOR inhibitors. The administration of PI3K inhibitor, LY294002 (10 nmol/site, i.c.v) completely prevented the antidepressant-like effect of atorvastatin (0.1 mg/kg, p.o.). The participation of GSK-3β in the antidepressant-like effect of atorvastatin was demonstrated by co-administration of a sub-effective dose of atorvastatin (0.01 mg/kg, p.o.) with AR-A014418 (0.01 μg/site, i.c.v., a selective GSK-3β inhibitor) or with lithium chloride (10 mg/kg, p.o., a non-selective GSK-3β inhibitor). The mTOR inhibitor, rapamycin (0.2 nmol/site, i.c.v.) was also able to prevent atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. These behavioral findings were supported by neurochemical observations, as atorvastatin treatment increased the immunocontent of the phosphorylated isoforms of Akt, GSK-3β and mTOR in the hippocampus of mice. Taken together, our results suggest an involvement of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of atorvastatin in mice.

Topics: Analysis of Variance; Animals; Antidepressive Agents; Atorvastatin; Depression; Disease Models, Animal; Enzyme Inhibitors; Exploratory Behavior; Glycogen Synthase Kinase 3 beta; Hindlimb Suspension; Immunosuppressive Agents; Male; Mice; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Interventions that target fundamental aging processes have the potential to transform human health and health care. A variety of candidate drugs have emerged from basic and translational research that may target aging processes. Some of these drugs are already in clinical use for other purposes, such as metformin and rapamycin. However, designing clinical trials to test interventions that target the aging process poses a unique set of challenges. This paper summarizes the outcomes of an international meeting co-ordinated by the NIH-funded Geroscience Network to further the goal of developing a translational pipeline to move candidate compounds through clinical trials and ultimately into use. We review the evidence that some drugs already in clinical use may target fundamental aging processes. We discuss the design principles of clinical trials to test such interventions in humans, including study populations, interventions, and outcomes. As examples, we offer several scenarios for potential clinical trials centered on the concepts of health span (delayed multimorbidity and functional decline) and resilience (response to or recovery from an acute health stress). Finally, we describe how this discussion helped inform the design of the proposed Targeting Aging with Metformin study.

Topics: Acarbose; Aging; Animals; Anti-Bacterial Agents; Biomedical Research; Clinical Trials as Topic; Congresses as Topic; Disease Models, Animal; Drug Evaluation; Glycoside Hydrolase Inhibitors; Humans; Hypoglycemic Agents; Metformin; Research Design; Sirolimus

It has been demonstrated that mTOR/p70S6K pathway was abnormally activated in many cancers and rapamycin and its analogs can restrain tumor growth through inhibiting this pathway, but some tumors including esophageal squamous cell carcinoma (ESCC) appear to be insensitive to rapamycin in recent studies. In the present study, we explored the measures to improve the sensitivity of ESCC cells to rapamycin and identified the clinical significance of the expression of phosphorylated p70S6K (p-p70S6K). The results showed that, after downregulating the expression of p70S6K and p-p70S6K by p70S6K siRNA, the inhibitory effects of rapamycin on cell proliferation, cell cycle, and tumor growth were significantly enhanced in vitro and in vivo. Furthermore, p-p70S6K had strong positive expression in ESCC tissues and its expression was closely related to lymph node metastasis and the TNM staging. These results indicated that p-p70S6K may participate in the invasion and metastasis in the development of ESCC and downregulation of the expression of p-p70S6K could improve the sensitivity of cells to rapamycin in ESCC.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Down-Regulation; Drug Resistance, Neoplasm; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression; Humans; Lymphatic Metastasis; Male; Mice; Neoplasm Staging; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Sirolimus; Xenograft Model Antitumor Assays

Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.

Topics: Amino Acids; Animals; ATP-Binding Cassette Transporters; Autophagy; Disease Models, Animal; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice, Inbred C57BL; Multiprotein Complexes; Peroxisomal Biogenesis Factor 2; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Protein-Energy Malnutrition; Proteins; Proteolysis; Rats; Receptors, Cytoplasmic and Nuclear; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Ubiquitination

The therapeutic efficacy of rapamycin conjugated monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles (rapamycin micelles) was evaluated in a rat experimental autoimmune uveitis (EAU) model. Rapamycin micelles exhibited spherical morphology and had a mean particle size of 40nm and a zeta-potential of -0.89mv. The water solubility of rapamycin improved by more than 1000-fold in a micellar formulation. Intravitreal injection of MPEG-PCL micelles did not result in vitreous hemorrhage or retinal detachment. Fluorescence microscopy demonstrated that labeled micelles localized to the retinal pigment epithelium for at least 14 days following injection and the drug concentration of rapamycin micelles in the retinal tissue was significantly higher than unconjugated rapamycin over this period. At the optimal concentration of rapamycin micelles (9μg/eye), clinical signs of EAU were abolished via the downregulation of the Th1 and Th17 response. There were no significant difference in T cell proliferation and delayed-type hypersensitivity between the treatment and control groups, suggesting that the therapeutic effect of rapamycin manifested locally in the eye and not systemically. These results indicate that intravitreal injection of rapamycin micelles is a promising therapy for controlling sterile intraocular inflammation.

Topics: Animals; Autoimmune Diseases; Cell Proliferation; Ciliary Body; Cytokines; Disease Models, Animal; Immunosuppressive Agents; Intravitreal Injections; Micelles; Polyesters; Polyethylene Glycols; Rats, Sprague-Dawley; Retina; Sirolimus; T-Lymphocytes; Uveitis; Vitreous Body

Rapamycin is an immunosuppressive agent that is added to drug eluting stents. It prevents restenosis, but it also impairs reendothelialization. Nicorandil is a hybrid agent with adenosine triphosphated (ATP)-sensitive K+ (KATP) channel opener and nitrate properties. It prevents oxidative stress and cell apoptosis induced by rapamycin in endothelial cells in vitro. However, whether nicorandil promotes reendothelialization after angioplasty delayed by rapamycin remains to be determined. Balloon injury model was established in SD rats. Nicorandil increased reendothelialization impaired by rapamycin, and it decreased xanthine oxidase (XO)-generated reactive oxygen species (ROS) induced by rapamycin. In addition, eNOS expression inhibited by rapamycin was increased by nicorandil in vivo. In vitro, rapamycin-impeded cardiac microvascular endothelial cells (CMECs) migration, proliferation and rapamycin-induced ROS production were reversed by nicorandil. Knockdown of XO partially inhibited rapamycin-induced ROS production and cell apoptosis in CMECs, and it promoted CMECs migration and proliferation suppressed by rapamycin. Knockdown of Akt partially prevents eNOS upregulation promoted by nicorandil. The beneficial effect of nicorandil is exhibited by inhibiting XO and up-regulating Akt pathway. Nicorandil combined with rapamycin in effect rescue the deficiencies of rapamycin alone in arterial healing after angioplasty.

Topics: Animals; Apoptosis; Carotid Artery Injuries; Cell Movement; Cell Proliferation; Disease Models, Animal; Endothelium, Vascular; Nicorandil; Nitric Oxide Synthase Type III; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species; Sirolimus; Xanthine Oxidase

The disease-modifying effects of target of rapamycin complex 1 (TORC1) inhibitors during different stages of polycystic kidney disease (PKD) are not well defined. In this study, male Lewis Polycystic Kidney Disease (LPK) rats (a genetic ortholog of human NPHP9, phenotypically characterised by diffuse distal nephron cystic growth) and Lewis controls received either vehicle (V) or sirolimus (S, 0.2 mg/kg by intraperitoneal injection 5 days per week) during the early (postnatal weeks 3 to 10) or late stages of disease (weeks 10 to 20). In early-stage disease, sirolimus reduced kidney enlargement (by 63%), slowed the rate of increase in total kidney volume (TKV) in serial MRI by 78.2% (LPK+V: 132.3±59.7 vs. LPK+S: 28.8±12.0% per week) but only partly reduced the percentage renal cyst area (by 19%) and did not affect the decline in endogenous creatinine clearance (CrCl) in LPK rats. In late-stage disease, sirolimus reduced kidney enlargement (by 22%) and the rate of increase in TKV by 71.8% (LPK+V: 13.1±6.6 vs. LPK+S: 3.7±3.7% per week) but the percentage renal cyst area was unaltered, and the CrCl only marginally better. Sirolimus reduced renal TORC1 activation but not TORC2, NF-κB DNA binding activity, CCL2 or TNFα expression, and abnormalities in cilia ultrastructure, hypertension and cardiac disease were also not improved. Thus, the relative treatment efficacy of TORC1 inhibition on kidney enlargement was consistent at all disease stages, but the absolute effect was determined by the timing of drug initiation. Furthermore, cystic microarchitecture, renal function and cardiac disease remain abnormal with TORC1 inhibition, indicating that additional approaches to normalise cellular dedifferentiation, inflammation and hypertension are required to completely arrest the progression of PKDs.

Topics: Animals; Cardiovascular Diseases; Chemokine CCL2; Cilia; Creatinine; Disease Models, Animal; Gene Expression; Kidney; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Myocardium; NF-kappa B; Polycystic Kidney Diseases; Rats; Rats, Inbred Lew; Severity of Illness Index; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Our lab has recently shown that genetic inhibition of the mechanistic target of rapamycin (TOR) rescues the short lifespan of yeast mutants with defective mitochondrial function, and that pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. Here, we probe the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, we identify a fat storage defect in the ND2 mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism.

Topics: Animals; Animals, Genetically Modified; Autophagy; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Electron Transport Complex I; Lipid Metabolism; Longevity; Mitochondrial Diseases; Mutation; Oxidative Stress; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Intracerebral hemorrhage (ICH) results in a devastating brain disorder with high mortality and poor prognosis and effective therapeutic intervention for the disease remains a challenge at present. The present study investigated the neuroprotective effects of rapamycin on ICH-induced brain damage and the possible involvement of activated microglia. ICH was induced in rats by injection of type IV collagenase into striatum. Different dose of rapamycin was systemically administrated by intraperitoneal injection beginning at 1 h after ICH induction. Western blot analysis showed that ICH led to a long-lasting increase of phosphorylated mTOR and this hyperactivation of mTOR was reduced by systemic administration of rapamycin. Rapamycin treatment significantly improved the sensorimotor deficits induced by ICH, and attenuated ICH-induced brain edema formation as well as lesion volume. Nissl and Fluoro-Jade C staining demonstrated that administration with rapamycin remarkably decreased neuronal death surrounding the hematoma at 7 d after ICH insult. ELISA and real-time quantitative PCR demonstrated that rapamycin inhibited ICH-induced excessive expression of TNF-α and IL-1β in ipsilateral hemisphere. Furthermore, activation of microglia induced by ICH was significantly suppressed by rapamycin administration. These data indicated that treatment of rapamycin following ICH decreased the brain injuries and neuronal death at the peri-hematoma striatum, and increased neurological function, which associated with reduced the levels of proinflammatory cytokines and activated microglia. The results provide novel insight into the neuroprotective therapeutic strategy of rapamycin for ICH insult, which possibly involving the regulation of microglial activation.

Topics: Animals; Blotting, Western; Brain; Brain Edema; Cerebral Hemorrhage; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression; Immunohistochemistry; Interleukin-1beta; Male; Microglia; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Alzheimer's disease (AD) is the most common type of progressive neurodegenerative disorder, and is responsible for the most common form of dementia in the elderly. Inflammation occurs in the brains of patients with AD, and is critical for disease progression. In the present study, the effects of rapamycin (RAPA) on neuroinflammation lipopolysaccharide (LPS)-induced were investigated. SH‑SY5Y human neuroblastoma cells were treated with 20 µg/ml LPS and 0.1, 1 or 10 nmol/l RAPA, and were analyzed at various time points (6, 12 and 24 h). The mRNA expression levels of interleukin (IL) 1β, IL6 and hypoxia‑inducible factor 1α (HIF1α) were determined using reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of phosphorylated (p‑)S6, p‑nuclear factor κB (NFκB), p‑inhibitor of NFκB kinase subunit β (IKKβ) and p‑tau protein were measured by western blot analysis. p‑IKKβ, p‑NFκB, p‑S6 and p‑tau were significantly decreased at 6, 12 and 24 h when cells were treated with ≥0.1 nmol/ml RAPA. In addition, female Sprague Dawley rats were intracranially injected with a single dose of 100 µg/kg LPS in the absence or presence of 1 mg/kg RAPA pretreatment. Brain tissues were subjected to immunohistochemical analysis 6‑24 h later, which revealed that the expression levels of HIF1α and p‑S6 in rat cerebral cortex were increased following LPS injection; however, this increase was abrogated by RAPA treatment. RAPA may therefore be considered a potential therapeutic agent for the early or emergency treatment of neuroinflammation.

Topics: Animals; Biomarkers; Cell Line, Tumor; Cerebral Cortex; Cytokines; Disease Models, Animal; Female; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; I-kappa B Kinase; Inflammation; Inflammation Mediators; Lipopolysaccharides; Nervous System Diseases; Neurons; NF-kappa B; Phosphorylation; Rats; Ribosomal Protein S6 Kinases; Sirolimus; tau Proteins

Bladder disorders associated with interstitial cystitis are frequently characterized by increased contractility and pain. The purposes of this study were to examine (1) the effects of blocking mammalian target of rapamycin (mTOR) on the exaggerated bladder activity and pain evoked by cystitis and (2) the underlying mechanisms responsible for the role of mTOR in regulating cystic sensory activity.. The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4 E-binding protein 4 (p-4 E-BP1), as well as phosphatidylinositide 3-kinase (p-PI3K) pathway were amplified in cyclophosphamide rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated bladder hyperactivity and pain. In addition, blocking PI3K signal pathway attenuated activities of mTOR, which was accompanied with decreasing bladder hyperactivity and pain. Inhibition of either mTOR or PI3K blunted the enhanced spinal substance P and calcitonin gene-related peptide in cyclophosphamide rats.. The data for the first time revealed specific signaling pathways leading to cyclophosphamide-induced bladder hyperactivity and pain, including the activation of mTOR and PI3K. Inhibition of these pathways alleviates cystic pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of overactive bladder and pain often observed in cystitis.

Topics: Animals; Calcitonin Gene-Related Peptide; Chromones; Cyclophosphamide; Cystitis; Disease Models, Animal; Enzyme Inhibitors; Female; Hypersensitivity; Immunosuppressive Agents; Morpholines; Pain; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Urinary Bladder

Hepatic stellate cells are of mesenchymal cell type located in the space of Disse. Upon liver injury, HSCs transactivate into myofibroblasts with increase in expression of fibrillar collagen, especially collagen I and III, leading to liver fibrosis. Previous studies have shown mTOR signaling is activated during liver fibrosis. However, there is no direct evidence in vivo. The aim of this study is to examine the effects of conditional deletion of TSC1 in mesenchymal on pathogenesis of liver fibrosis. Crossing mice bearing the floxed TSC1 gene with mice harboring Col1α2-Cre-ER(T) successfully generated progeny with a conditional knockout of TSC1 (TSC1 CKO) in collagen I expressing mesenchymal cells. TSC1 CKO and WT mice were subjected to CCl

Topics: Animals; Carbon Tetrachloride; Disease Models, Animal; Gene Deletion; Liver; Liver Cirrhosis; Mice; Mice, Knockout; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Glucocerebrosidase (GBA1) mutations are associated with Gaucher disease (GD), an autosomal recessive disorder caused by functional deficiency of glucocerebrosidase (GBA), a lysosomal enzyme that hydrolyzes glucosylceramide to ceramide and glucose. Neuronopathic forms of GD can be associated with rapid neurological decline (Type II) or manifest as a chronic form (Type III) with a wide spectrum of neurological signs. Furthermore, there is now a well-established link between GBA1 mutations and Parkinson's disease (PD), with heterozygote mutations in GBA1 considered the commonest genetic defect in PD. Here we describe a novel Drosophila model of GD that lacks the two fly GBA1 orthologs. This knock-out model recapitulates the main features of GD at the cellular level with severe lysosomal defects and accumulation of glucosylceramide in the fly brain. We also demonstrate a block in autophagy flux in association with reduced lifespan, age-dependent locomotor deficits and accumulation of autophagy substrates in dGBA-deficient fly brains. Furthermore, mechanistic target of rapamycin (mTOR) signaling is downregulated in dGBA knock-out flies, with a concomitant upregulation of Mitf gene expression, the fly ortholog of mammalian TFEB, likely as a compensatory response to the autophagy block. Moreover, the mTOR inhibitor rapamycin is able to partially ameliorate the lifespan, locomotor, and oxidative stress phenotypes. Together, our results demonstrate that this dGBA1-deficient fly model is a useful platform for the further study of the role of lysosomal-autophagic impairment and the potential therapeutic benefits of rapamycin in neuronopathic GD. These results also have important implications for the role of autophagy and mTOR signaling in GBA1-associated PD SIGNIFICANCE STATEMENT: We developed a Drosophila model of neuronopathic GD by knocking-out the fly orthologs of the GBA1 gene, demonstrating abnormal lysosomal pathology in the fly brain. Functioning lysosomes are required for autophagosome-lysosomal fusion in the autophagy pathway. We show in vivo that autophagy is impaired in dGBA-deficient fly brains. In response, mechanistic target of rapamycin (mTOR) activity is downregulated in dGBA-deficient flies and rapamycin ameliorates the lifespan, locomotor, and oxidative stress phenotypes. dGBA knock-out flies also display an upregulation of the Drosophila ortholog of mammalian TFEB, Mitf, a response that is unable to overcome the autophagy block. Together, our res

Topics: Animals; Animals, Genetically Modified; Autophagy; Disease Models, Animal; Drosophila; Gaucher Disease; Gene Knockout Techniques; Glucosylceramidase; Lysosomes; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Numerous studies suggest that rapamycin treatment promotes insulin resistance, implying that rapamycin could have negative effects on patients with, or at risk for, type 2 diabetes (T2D). New evidence, however, indicates that rapamycin treatment produces some

Topics: Adiposity; Aging; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Humans; Hypoglycemic Agents; Insulin; Insulin Resistance; Male; Mice; Sirolimus; TOR Serine-Threonine Kinases

Novel sirolimus eluting stents (SES) have shown non-inferior clinical outcomes when compared to everolimus eluting stents (EES), however only limited preclinical data have been published. Therefore, we evaluate vascular response of a new generation biodegradable polymer SES (BP-SES: Alex Plus, Balton) and fluoropolymer EES (EES: Xience Pro, Abbott) in the porcine coronary restenosis model.. A total of 40 stents were implanted with 120% overstretch in coronaries of 17 domestic swine: 16 BP-SES, 16 EES and 8 bare metal controls (BMS). Following 28 and 90 days, coronary angiography and optical coherence tomography (OCT) was performed, animals sacrificed and stented segments harvested for pathological evaluation.. At 28 days neointimal thickness in OCT was lowest in the BP-SES when compared to EES and BMS (0.18 ± 0.1 vs. 0.39 ± 0.1 vs. 0.34 ± 0.2 mm, respectively; p = 0.04). There was no difference in the proportion of malapposed or uncovered struts, although protruding covered struts were more common in BP-SES (14.8 ± 10% vs. 4.1 ± 4% vs. 3.7 ± 6%; p = 0.03). In pathology, the lowest neointimal thickness was confirmed in BP-SES (p < 0.05). The inflammation score was significantly lower in BP-SES and EES when compared to BMS (0.24 ± 0.1 vs. 0.4 ± 0.1 vs. 0.77 ± 0.4; p < 0.01) whilst EES and BP-SES had higher fibrin scores than BMS (1.2 ± 0.4 vs. 1.3 ± 0.3 vs. 0.17 ± 0.2; p < 0.01). At 90 days neointimal coverage and thickness in OCT was comparable between groups and healing in histopathology was complete.. New generation, BP-SES show similar vascular healing and biocompatibility profile with marginally higher degree of restenosis inhibition, when compared to fluoropolymer EES in the porcine coronary restenosis model.

Topics: Absorbable Implants; Animals; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Female; Immunosuppressive Agents; Male; Percutaneous Coronary Intervention; Polymers; Prosthesis Design; Sirolimus; Swine; Tomography, Optical Coherence

Bladder deterioration after partial outlet obstruction (pBOO) occurs commonly and has significant clinical implications. Our previous animal model results described the progression of pBOO to hypertrophy and fibrosis. We wished to determine if the pathologic process of pBOO can be altered with rationally chosen oral medications.. Female Sprague-Dawley rats underwent controlled surgically induced pBOO. Rats were maintained for a period of 16 weeks at which point urodynamics were performed, and organs harvested. Rats were divided into four groups, each receiving different daily treatment: control (saline), oxybutynin (3 mg/kg), rapamycin (2 mg/kg), and tadalafil (2 mg/kg). Outcomes were assessed after 4,8,12, or 16 weeks. Measures included animal health, urodynamics, histology, mass spectrometry for collagen content, and rtPCR for inflammatory mediators.. Rapamycin treated animals exhibited significant mortality at later time points. Oxybutinin and tadalafil treated bladders demonstrated significant improvements in bladder capacity and compliance, with less detrusor hypertrophy than controls. Tadalafil also resulted in a significant down-regulation of HIF-1α, while decorin, biglycan, and TGF-β were upregulated in treated animals. Tadalafil treated bladders measured lower collagen content towards the end of the study, indicating an antifibrotic effect.. Our study has effectively demonstrated that deleterious changes secondary to pBOO can be altered pharmacologically. Oxybutinin and tadalafil seem to have a time-dependent protective effect on the detrusor muscle, although with different mechanisms of action. Tadalafil treatment in this setting appears to have an antifibrotic effect. This work has the potential to seed important clinical studies and improve clinical practice.

Topics: Administration, Oral; Animals; Carbolines; Disease Models, Animal; Female; Mandelic Acids; Muscarinic Antagonists; Phosphodiesterase 5 Inhibitors; Rats; Rats, Sprague-Dawley; Sirolimus; Tadalafil; Urinary Bladder Neck Obstruction; Urodynamics

Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo.. Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling.. mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage.. This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

Topics: Aged; Aged, 80 and over; AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Autophagy-Related Protein-1 Homolog; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; Dogs; Gene Silencing; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Male; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Osteoarthritis; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction.. The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry.. The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells.. Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.

Topics: Adenine; Animals; Autophagy; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Foscarnet; Humans; Hyperphosphatemia; Male; Microtubule-Associated Proteins; Phosphates; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Wistar; Renal Insufficiency, Chronic; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection

Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate the numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 days after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 hours later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2,280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 days after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy.

Topics: Animals; Dendrites; Dentate Gyrus; Disease Models, Animal; Epilepsy, Temporal Lobe; Male; Mice; Microscopy, Electron; Neuroprotective Agents; Pilocarpine; Sirolimus; Status Epilepticus; Synapses

Changes in ion channel expression are implicated in the etiology of epilepsy. However, the molecular leading to long-term aberrant expression of ion channels are not well understood. The mechanistic/mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates activity-dependent protein synthesis in neurons. mTOR is overactive in epilepsy, suggesting that excessive protein synthesis may contribute to the neuronal pathology. In contrast, we found that mTOR activity and the microRNA miR-129-5p reduce the expression of the voltage-gated potassium channel Kv1.1 in an animal model of temporal lobe epilepsy (TLE). When mTOR activity is low, Kv1.1 expression is high and the frequency of behavioral seizures is low. However, as behavioral seizure activity rises, mTOR activity increases and Kv1.1 protein levels drop. In CA1 pyramidal neurons, the reduction in Kv1.1 lowers the threshold for action potential firing. Interestingly, blocking mTOR activity with rapamycin reduces behavioral seizures and temporarily keeps Kv1.1 levels elevated. Overtime, seizure activity increases and Kv1.1 protein decreases in all animals, even those treated with rapamycin. Notably, the concentration of miR-129-5p, the negative regulator of Kv1.1 mRNA translation, increases by 21days post-status epilepticus (SE), sustaining Kv1.1 mRNA translational repression. Our results suggest that following kainic-acid induced status epilepticus there are two phases of Kv1.1 repression: (1) an initial mTOR-dependent repression of Kv1.1 that is followed by (2) a miR-129-5p persistent reduction of Kv1.1.

Topics: Action Potentials; Animals; Disease Models, Animal; ELAV Proteins; Excitatory Amino Acid Agonists; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Kainic Acid; Kv1.1 Potassium Channel; Male; MicroRNAs; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sirolimus; Status Epilepticus; Synaptic Transmission; Time Factors; TOR Serine-Threonine Kinases

Epilepsy and autism spectrum disorder (ASD) are common comorbidities of one another. Despite the prevalent correlation between the two disorders, few studies have been able to elucidate a mechanistic link. We demonstrate that forebrain specific Tsc1 deletion in mice causes epilepsy and autism-like behaviors, concomitant with disruption of 5-HT neurotransmission. We find that epileptiform activity propagates to the raphe nuclei, resulting in seizure-dependent hyperactivation of mTOR in 5-HT neurons. To dissect whether mTOR hyperactivity in 5-HT neurons alone was sufficient to recapitulate an autism-like phenotype we utilized Tsc1flox/flox;Slc6a4-cre mice, in which mTOR is restrictively hyperactivated in 5-HT neurons. Tsc1flox/flox;Slc6a4-cre mice displayed alterations of the 5-HT system and autism-like behaviors, without causing epilepsy. Rapamycin treatment in these mice was sufficient to rescue the phenotype. We conclude that the spread of seizure activity to the brainstem is capable of promoting hyperactivation of mTOR in the raphe nuclei, which in turn promotes autism-like behaviors. Thus our study provides a novel mechanism describing how epilepsy can contribute to the development of autism-like behaviors, suggesting new therapeutic strategies for autism.

Topics: Animals; Autistic Disorder; Behavior, Animal; Disease Models, Animal; Epilepsy; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Protein Kinase Inhibitors; Raphe Nuclei; Serotonin; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Although rapamycin (RPM) have been studied extensively in ischemia models, its functional mechanisms remains to be defined.. We determined how RPM impacted the pathogenesis of ischemia-reperfusion injury (IRI) in a murine liver partial warm ischemia model, with emphasis on its regulation of hepatocyte death.. Rapamycin protected livers from IRI in the presence of fully developed liver inflammatory immune response. Rapamycin enhanced liver autophagy induction at the reperfusion stage. Dual mammalian (mechanistic) target of rapamycin (mTOR)1/2 inhibitor Torin 1, despite its ability to induced autophagy, failed to protect livers from IRI. The treatment with RPM, but not Torin 1, resulted in the enhanced activation of the mTORC2-Akt signaling pathway activation in livers after reperfusion. Inactivation of Akt by Triciribine abolished the liver protective effect of RPM. The differential cytoprotective effect of RPM and Torin 1 was confirmed in vitro in hepatocyte cultures. Rapamycin, but not Trin 1, protected hepatocytes from stress and tumor necrosis factor-α induced cell death; and inhibition of autophagy by chloroquine or Akt by Triciribine abolished RPM-mediated cytoprotection.. Rapamycin protected livers from IRI by both autophagy and mTORC2-Akt activation mechanisms.

Topics: Animals; Autophagy; Cell Line; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Activation; Hepatocytes; Liver; Liver Diseases; Male; Mechanistic Target of Rapamycin Complex 2; Mice, Inbred C57BL; Multiprotein Complexes; Naphthyridines; Protective Agents; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Warm Ischemia

IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN.

Topics: Animals; Blotting, Western; Cell Cycle; Cell Proliferation; Disease Models, Animal; Flow Cytometry; Fluorescent Antibody Technique; Glomerulonephritis, IGA; Immunohistochemistry; Immunosuppressive Agents; Male; Mesangial Cells; Rats; Rats, Sprague-Dawley; Sirolimus

G protein-regulated cell function is crucial for cardiomyocytes, and any deregulation of its gene expression or protein modification can lead to pathological cardiac hypertrophy. Herein, we report that protein prenylation, a lipidic modification of G proteins that facilitates their association with the cell membrane, might control the process of cardiomyocyte hypertrophy. We found that geranylgeranyl diphosphate synthase (GGPPS), a key enzyme involved in protein prenylation, played a critical role in postnatal heart growth by regulating cardiomyocyte size. Cardiac-specific knockout of GGPPS in mice led to spontaneous cardiac hypertrophy, beginning from week 4, accompanied by the persistent enlargement of cardiomyocytes. This hypertrophic effect occurred by altered prenylation of G proteins. Evaluation of the prenylation, membrane association and hydrophobicity showed that Rheb was hyperactivated and increased mTORC1 signalling pathway after GGPPS deletion. Protein farnesylation or mTORC1 inhibition blocked GGPPS knockdown-induced mTORC1 activation and suppressed the larger neonatal rat ventricle myocyte size and cardiomyocyte hypertrophy in vivo, demonstrating a central role of the FPP-Rheb-mTORC1 axis for GGPPS deficiency-induced cardiomyocyte hypertrophy. The sustained cardiomyocyte hypertrophy progressively provoked cardiac decompensation and dysfunction, ultimately causing heart failure and adult death. Importantly, GGPPS was down-regulated in the hypertrophic hearts of mice subjected to transverse aortic constriction (TAC) and in failing human hearts. Moreover, HPLC-MS/MS detection revealed that the myocardial farnesyl diphosphate (FPP):geranylgeranyl diphosphate (GGPP) ratio was enhanced after pressure overload. Our observations conclude that the alteration of protein prenylation promotes cardiomyocyte hypertrophic growth, which acts as a potential cause for pathogenesis of heart failure and may provide a new molecular target for hypertrophic heart disease clinical therapy.

Topics: Animals; Cardiomegaly; Cell Line; Disease Models, Animal; Disease Progression; Farnesyltranstransferase; Female; Heart Failure; Male; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C57BL; Mice, Knockout; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Myocytes, Cardiac; Neuropeptides; Protein Kinase Inhibitors; Protein Prenylation; Ras Homolog Enriched in Brain Protein; Rats; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Ventricular Function, Left

Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes.

Topics: Adaptor Proteins, Signal Transducing; Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Frontotemporal Lobar Degeneration; Gene Knockdown Techniques; Locomotion; Phenotype; Sequestosome-1 Protein; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish; Zebrafish Proteins

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that exists in two complexes (mTORC1 and mTORC2) and integrates extracellular and intracellular signals to act as a master regulator of cell growth, survival, and metabolism. The PI3K/AKT/mTOR prosurvival pathway is often dysregulated in multiple sarcoma subtypes. First-generation allosteric inhibitors of mTORC1 (rapalogues) have been extensively tested with great preclinical promise, but have had limited clinical utility. Here, we report that MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2 and has potent in vitro and in vivo antitumor activity in multiple sarcoma subtypes. In vitro, MLN0128 inhibits mTORC1/2 targets in a concentration-dependent fashion and shows striking antiproliferative effect in rhabdomyosarcoma (RMS), Ewing sarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, osteosarcoma, and liposarcoma. Unlike rapamycin, MLN0128 inhibits phosphorylation of 4EBP1 and NDRG1 as well as prevents the reactivation of pAKT that occurs via negative feedback release with mTORC1 inhibition alone. In xenograft models, MLN0128 treatment results in suppression of tumor growth with two dosing schedules (1 mg/kg daily and 3 mg/kg b.i.d. t.i.w.). At the 3 mg/kg dosing schedule, MLN0128 treatment results in significantly better tumor growth suppression than rapamycin in RMS and Ewing sarcoma models. In addition, MLN0128 induces apoptosis in models of RMS both in vitro and in vivo. Results from our study strongly suggest that MLN0128 treatment should be explored further as potential therapy for sarcoma.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Benzoxazoles; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Down-Regulation; Female; Humans; Inhibitory Concentration 50; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice, Nude; Multiprotein Complexes; Osteosarcoma; Phosphorylation; Protein Kinase Inhibitors; Pyrimidines; Sarcoma; Sirolimus; Substrate Specificity; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

1. Paraquat (PQ) is an organic nitrogen heterocyclic herbicide that is widely used in agriculture throughout the world. Numerous studies have reported PQ intoxication on humans. 2. In this study, we established a rat lung injury model induced by PQ and evaluated the intervention effect of rapamycin on the model, exploring the pathogenesis of PQ on lung injury as well as therapeutic effects of rapamycin on PQ-induced lung injury. 3. A rat lung injury model was established by gavage of PQ, and rapamycin was used to treat the model animals with PQ-induced lung injury. Different physiological indices were measured through Western blot and real-time polymerase chain reaction to evaluate the effect of rapamycin on the PQ-induced lung injury. 4. The analyses showed that application of rapamycin could significantly reduce the lung injury damage caused by PQ, with lung tissue wet-dry weight ratio, pathological features, compositions in serum, protein in bronchoalveolar lavage fluid and other indices being significantly improved after the injection of rapamycin. 5. It was inferred that the use of rapamycin could improve the PQ-induced lung injury through inhibiting the activity of mTOR. And we expected the use of rapamycin to be a potential treatment method for the PQ intoxication in future.

Topics: Acute Lung Injury; Animals; Disease Models, Animal; Female; Herbicides; Male; Paraquat; Rats; Rats, Wistar; Sirolimus

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Factor VIII; Female; Hemocyanins; Hemophilia A; Humans; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Humoral; Immunosuppression Therapy; Immunosuppressive Agents; Lactic Acid; Mice; Mice, Inbred BALB C; Nanocapsules; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Proteins; Recombinant Proteins; Sirolimus

Ingestion of paraquat (PQ), a widely used herbicide, can cause severe toxicity in humans, leading to a poor survival rate and prognosis. One of the main causes of death by PQ is PQ-induced pulmonary fibrosis, for which there are no effective therapies. The aim of this study was to evaluate the effects of rapamycin (RAPA) on inhibiting PQ-induced pulmonary fibrosis in mice and to explore its possible mechanisms.. Male C57BL/6J mice were exposed to either saline (control group) or PQ (10 mg/kg body weight, intraperitoneally; test group). The test group was divided into four subgroups: a PQ group (PQ-exposed, non-treated), a PQ+RAPA group (PQ-exposed, treated with RAPA at 1 mg/kg intragastrically), a PQ+MP group (PQ-exposed, treated with methylprednisolone (MP) at 30 mg/kg intraperitoneally), and a PQ+MP+RAPA group (PQ-exposed, treated with MP at 30 mg/kg intraperitoneally and with RAPA at 1 mg/kg intragastrically). The survival rate and body weight of all the mice were recorded every day. Three mice in each group were sacrificed at 14 d and the rest at 28 d after intoxication. Lung tissues were excised and stained with hematoxylin-eosin (H&E) and Masson's trichrome stain for histopathological analysis. The hydroxyproline (HYP) content in lung tissues was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in lung tissues was detected by immunohistochemical staining and Western blotting.. A mice model of PQ-induced pulmonary fibrosis was established. Histological examination of lung tissues showed that RAPA treatment moderated the pathological changes of pulmonary fibrosis, including alveolar collapse and interstitial collagen deposition. HYP content in lung tissues increased soon after PQ intoxication but had decreased significantly by the 28th day after RAPA treatment. Immunohistochemical staining and Western blotting showed that RAPA treatment significantly down-regulated the enhanced levels of TGF-β1 and α-SMA in lung tissues caused by PQ exposure. However, RAPA treatment alone could not significantly ameliorate the lower survival rate and weight loss of treated mice. MP treatment enhanced the survival rate, but had no significant effects on attenuating PQ-induced pulmonary fibrosis or reducing the expression of TGF-β1 and α-SMA.. This study demonstrates that RAPA treatment effectively suppresses PQ-induced alveolar collapse and collagen deposition in lung tissues through reducing the expression of TGF-β1 and α-SMA. Thus, RAPA has potential value in the treatment of PQ-induced pulmonary fibrosis.

Topics: Actins; Animals; Body Weight; Collagen; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Herbicides; Immunohistochemistry; Immunosuppressive Agents; Lung; Male; Methylprednisolone; Mice; Mice, Inbred C57BL; Paraquat; Prognosis; Pulmonary Fibrosis; Sirolimus; Transforming Growth Factor beta1; Treatment Outcome

The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells.. In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable.. EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

Topics: Actins; Animals; Biomarkers; Bone Morphogenetic Protein Receptors, Type II; Cell Movement; Cell Transdifferentiation; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Gene Expression Profiling; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Mesoderm; Monocrotaline; Mutation; Rats; RNA, Messenger; Sirolimus; Vascular Remodeling; Vimentin

Recent studies in animal models have suggested that the mammalian target of rapamycin (mTOR) signaling pathway is involved in several features of mesio-temporal lobe epilepsy (MTLE), and that its inhibition could have therapeutic interests. However, it remains controversial whether mTOR activation is the cause or the consequence of MTLE. We previously showed in a mouse model of MTLE associated with hippocampal sclerosis that increased neuronal excitability and brain-derived neurotrophic factor (BDNF) overexpression contribute to the development of morphological features of this form of epilepsy. Here, we addressed whether mTOR activation promotes MTLE epileptogenesis via increasing neuronal excitability and/or BDNF expression or rather mediates neuroplasticity associated with hippocampal sclerosis. In mice injected intrahippocampally with kainate (1 nmol), we showed a biphasic increase of phospho-S6 (p-S6) ribosomal protein expression, the downstream product of the mTOR signaling pathway, in the dispersed granule cell layer (GCL) of the dentate gyrus with a second phase lasting up to 6 months. Chronic treatment with rapamycin suppressed p-S6 expression, granule cell dispersion and mossy fiber sprouting, but did not reduce cell loss, BDNF overexpression, glutamic acid decarboxylase (GAD)67 expression or the development of hippocampal paroxysmal discharges. Neuronal inhibition by midazolam (2 × 10 mg/kg, i.p.) abolished the increased expression of p-S6 in the dispersed GCL. Our data suggest that activation of the mTOR signaling pathway results from the increased neuronal excitation that develops in the GCL and may contribute to MTLE morphological changes. However, these data do not support the role of this pathway in the development of MTLE or its inhibition as a therapy for this form of epilepsy.

Topics: Animals; Astrocytes; Brain-Derived Neurotrophic Factor; Central Nervous System Agents; Disease Models, Animal; Epilepsy, Temporal Lobe; Gliosis; Hippocampus; Kainic Acid; Male; Mice, Inbred C57BL; Midazolam; Neural Inhibition; Neurons; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system.. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization.. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls.. Porous silicon-based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina.

Topics: Animals; Cells, Cultured; Delayed-Action Preparations; Disease Models, Animal; Drug Delivery Systems; Follow-Up Studies; Humans; Immunosuppressive Agents; Intravitreal Injections; Macular Degeneration; Particle Size; Porosity; Rabbits; Silicon; Sirolimus; Surface Properties

Electroacupuncture (EA) pretreatment has been reported to induce tolerance against cerebral ischemia/reperfusion (I/R) injury; however, the mechanisms underlying the beneficial effects of EA remain to be elucidated. Increasing evidence has suggested that excess activation of autophagy is important in I/R injury. The present study aimed to investigate the hypothesis that EA pretreatment‑induced tolerance to cerebral I/R injury was mediated by inhibition of the autophagy pathway. Rats were treated with EA at the acupoint 'Baihui (GV20)' 30 min/day, for five consecutive days prior to the induction of focal cerebral ischemia for 120 min by middle cerebral artery occlusion. Levels of autophagy, cerebral apoptosis, infarct volumes, brain water content and motor deficit were evaluated 12 h following I/R. The autophagy inducer rapamycin was used to investigate the role of autophagy in mediating neuroprotective effects. The results showed that the number of autophagosomes and the expression of the marker proteins of autophagy, including microtubule‑associated protein 1A light chain 3 (LC3)‑II and Beclin 1 were significantly increased 12 h post‑I/R. EA pretreatment decreased the expression of autophagy markers and the number of autophagosomes in the ischemic cortex. In addition, EA pretreatment inhibited neuronal apoptosis, reduced infarct volume and water content, as well as improved neurological outcome of rats following I/R. Furthermore, the reduced expression of LC3‑II and Beclin 1 and the neuroprotective effects were reversed by the autophagy inducer rapamycin. In conclusion, the results of the present study demonstrated that EA pretreatment protected the brain against I/R injury via inhibition of the autophagy process.

Topics: Acupuncture Points; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain; Disease Models, Animal; Electroacupuncture; Male; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

Much evidence demonstrated that autophagy played an important role in neural inflammation response after ischemia stroke. However, the specific effect of microglia autophagy in cerebral ischemia is still unknown. In the current study, we constructed focal cerebral ischemia model by permanent middle cerebral artery occlusion (pMCAO) in mice. We detected microglia autophagy and inflammation response in vivo, and observed infarct brain areas, edema formation, and neurological deficits of mice. We found that pMCAO induced microglia autophagy and inflammatory response. The suppression of autophagy using either pharmacologic inhibitor (3-MA) not only decreased the microglia autophagy and inflammatory response, but also significantly decreased infarct size, edema formation and neurological deficits in vivo. Taken together, these results suggested that cerebral ischemia induced microglia autophagy contributed to ischemic neural inflammation and injury. In addition, our findings also provided novel therapeutic strategy for ischemic stroke.

Topics: Adenine; Animals; Autophagy; Brain; Cell Hypoxia; Cerebral Arteries; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Interleukin-1beta; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Microglia; Sirolimus; Stroke; Tumor Necrosis Factor-alpha

Hyperactivation of the mechanistic target of rapamycin (mTOR; also known as mammalian target of rapamycin) pathway has been demonstrated in human cortical dysplasia (CD) as well as in animal models of epilepsy. Although inhibition of mTOR signaling early in epileptogenesis suppressed epileptiform activity in the neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD, the effects of mTOR inhibition after epilepsy is fully established were not previously examined in this model. Here, we investigated whether mTOR inhibition suppresses epileptiform activity and other neuropathological correlates in adult NS-Pten KO mice with severe and well-established epilepsy.. The progression of epileptiform activity, mTOR pathway dysregulation, and associated neuropathology with age in NS-Pten KO mice were evaluated using video-electroencephalography (EEG) recordings, Western blotting, and immunohistochemistry. A cohort of NS-Pten KO mice was treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) starting at postnatal week 9 and video-EEG monitored for epileptiform activity. Western blotting and immunohistochemistry were performed to evaluate the effects of rapamycin on the associated pathology.. Epileptiform activity worsened with age in NS-Pten KO mice, with parallel increases in the extent of hippocampal mTOR complex 1 and 2 (mTORC1 and mTORC2, respectively) dysregulation and progressive astrogliosis and microgliosis. Rapamycin treatment suppressed epileptiform activity, improved baseline EEG activity, and increased survival in severely epileptic NS-Pten KO mice. At the molecular level, rapamycin treatment was associated with a reduction in both mTORC1 and mTORC2 signaling and decreased astrogliosis and microgliosis.. These findings reveal a wide temporal window for successful therapeutic intervention with rapamycin in the NS-Pten KO mouse model, and they support mTOR inhibition as a candidate therapy for established, late-stage epilepsy associated with CD and genetic dysregulation of the mTOR pathway.

Topics: Animals; Disease Models, Animal; Epilepsy; Female; Male; Malformations of Cortical Development; Mice; Mice, Knockout; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases

Hypoxia-inducible factor-1 (HIF-1) is one of the most promising pharmacological targets for all types of cancer, including ovarian cancer. Ovarian clear cell carcinoma (OCCC) has poor prognosis because of its insensitivity to chemotherapy. To elucidate the characteristics of this troublesome cancer, we examined HIF-1α expression under normoxia or hypoxia in various ovarian cancer cell lines. HIF-1α was highly expressed under normoxia only in RMG-1, an OCCC cell line. To examine whether HIF-1 is involved in the tumorigenesis of RMG-1 cells, we established HIF-1α-silenced cells, RMG-1HKD. The proliferation rate of RMG-1HKD cells was faster than that of RMG-1 cells. Furthermore, the activity of MEK/ERK in the Ras pathway increased in RMG-1HKD cells, whereas that of mTOR in the PI3K pathway did not change. Activation of the Ras pathway was attributable to the increase in phosphorylated MEK via PP2A inactivation. To confirm the crosstalk between the PI3K and Ras pathways in vivo, RMG-1 or RMG-1HKD cells were transplanted into the skin of nude mice with rapamycin (an inhibitor of mTOR), PD98059 (an inhibitor of MEK), or both. RMG-1HKD cells showed higher sensitivity to PD98059 than that observed in RMD-1 cells, whereas the combination therapy resulted in synergistic inhibition of both cells. These findings suggest that inhibition of HIF-1, a downstream target of mTOR in the PI3K pathway, activates the Ras pathway on account of the increase in MEK phosphorylation via PP2A inactivation, and the crosstalk between the 2 pathways could be applied in the combination therapy for HIF-1-overexpressing cancers such as OCCC.

Topics: Adenocarcinoma, Clear Cell; Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mitogen-Activated Protein Kinases; Ovarian Neoplasms; Oxygen Consumption; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Protein Phosphatase 2; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The aged population suffers increased morbidity and higher mortality in response to episodes of acute kidney injury (AKI). Aging is associated with telomere shortening, and both telomerase reverse transcriptase (TerT) and RNA (TerC) are essential to maintain telomere length. To define a role of telomerase deficiency in susceptibility to AKI, we used ischemia/reperfusion injury in wild-type mice or mice with either TerC or TerT deletion. Injury induced similar renal impairment at day 1 in each genotype, as assessed by azotemia, proteinuria, acute tubular injury score, and apoptotic tubular epithelial cell index. However, either TerC or TerT knockout significantly delayed recovery compared with wild-type mice. Electron microscopy showed increased autophagosome formation in renal tubular epithelial cells in wild-type mice but a significant delay of their development in TerC and TerT knockout mice. There were also impeded increases in the expression of the autophagosome marker LC3 II, prolonged accumulation of the autophagosome protein P62, an increase of the cell cycle regulator p16, and greater activation of the mammalian target of rapamycin (mTOR) pathway. The mTORC1 inhibitor, rapamycin, partially restored the ischemia/reperfusion-induced autophagy response, without a significant effect on either p16 induction or tubule epithelial cell proliferation. Thus, muting the maintenance of normal telomere length in mice impaired recovery from AKI, owing to an increase in tubule cell senescence and impairment of mTOR-mediated autophagy.

Topics: Acute Kidney Injury; Aging; Animals; Apoptosis; Autophagy; Blood Urea Nitrogen; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Epithelial Cells; Kidney; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Regeneration; Reperfusion Injury; RNA; Signal Transduction; Sirolimus; Telomerase; Telomere Shortening; TOR Serine-Threonine Kinases

1. This study investigated the alteration of carboxylesterases in type 2 diabetes. We found that the carboxylesterase 1d (Ces1d) and carboxylesterase 1e (Ces1e) expression and the capacity of hydrolytic activity of liver and intestine decreased, whereas the Akt/mTOR/HIF-1α/ Stra13 (DEC1) signaling was activated in T2D mice. Consistently, high insulin could give rise to the same results in the high-glucose DMEM condition, which mimicked T2D, in primary mouse hepatocytes. 2. Perifosine or rapamycin almost abolished the decrease of the Ces1d and Ces1e expression and the hydrolytic activity induced by the insulin in the primary mouse hepatocytes. 3. The responsiveness of human hepatoma (HepG2) cells to high insulin in high-glucose condition was similar to that of primary mouse hepatocytes in terms of the altered expression of carboxylesterases. 4. The knockdown of HIF-1α or DEC1 with shRNA construct abrogated the decrease of the CES1 and CES2 expression induced by the insulin in high glucose condition in HepG2 cells. 5. Taken together, the decreased carboxylesterases expression and hydrolytic activity in T2D mice are through the Akt/mTOR/HIF-1α/Stra13 (DEC1) pathway.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Blood Glucose; Carboxylic Ester Hydrolases; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Knockdown Techniques; Glucose; Hep G2 Cells; Hepatocytes; Homeodomain Proteins; Humans; Hydrolysis; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Intestines; Liver; Male; Mice, Inbred C57BL; Overweight; Phosphorylcholine; Proto-Oncogene Proteins c-akt; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Sirolimus (SRL) is widely used to prevent restenosis after percutaneous coronary intervention. However, its beneficial effect is hampered by complications of thrombosis. Several studies imply that reactive oxygen species (ROS) play a critical role in endothelial dysfunction and thrombus formation. The present study investigated the protective effect of nicorandil (NIC), an anti-angina agent, on SRL-associated thrombosis. In human coronary artery endothelial cells (HCAECs), SRL stimulated ROS production, which was prevented by co-treatment with NIC. The preventive effect of NIC on ROS was abolished by 5-hydroxydecanoate but not by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. NIC also inhibited SRL-induced up-regulation of NADPH oxidase subunit p22(phox) mRNA. Co-treatment with NIC and SRL significantly up-regulated superoxide dismutase 2. NIC treatment significantly improved SRL-induced decrease in viability of HCAECs. The functional relevance of the preventive effects of NIC on SRL-induced ROS production and impairment of endothelial viability was investigated in a mouse model of thrombosis. Pretreatment with NIC inhibited the SRL-induced acceleration of FeCl3-initiated thrombus formation and ROS production in the testicular arteries of mice. In conclusion, NIC prevented SRL-induced thrombus formation, presumably due to the reduction of ROS and to endothelial protection. The therapeutic efficacy of NIC could represent an additional option in the prevention of SRL-related thrombosis.

Topics: Animals; Anti-Arrhythmia Agents; Arteries; Cell Survival; Cells, Cultured; Coronary Vessels; Disease Models, Animal; Endothelial Cells; Humans; Male; Mice, Inbred ICR; NADPH Oxidases; Nicorandil; Reactive Oxygen Species; Sirolimus; Superoxide Dismutase; Testis; Thrombosis; Up-Regulation

Type 2 corticotropin-releasing factor receptor (CRFR2) is expressed in skeletal muscle and stimulation of the receptor has been shown to inhibit the effect of insulin on glucose uptake in muscle cells. Currently, little is known about the mechanisms underlying this process. In this study, we first showed that both in vivo and in vitro CRFR2 expression in muscle was closely correlated with insulin sensitivity, with elevated receptor levels observed in insulin resistant muscle cells. Stimulation of CRFR2 by urocortin 2 (Ucn 2), a CRFR2-selective ligand, in C2C12 myotubes greatly attenuated insulin-induced glucose uptake. The inhibitory effect of CRFR2 signaling required cAMP production and is involved the mammalian target of rapamycine pathway, as rapamycin reversed the inhibitory effect of CRFR2 stimulation on insulin-induced glucose uptake. Moreover, stimulation of CRFR2 failed to inhibit glucose uptake in muscle cells induced by platelet-derived growth factor, which, similar to insulin, signals through Akt-mediated pathway but is independently of insulin receptor substrate (IRS) proteins to promote glucose uptake. This result argues that CRFR2 signaling modulates insulin's action likely at the levels of IRS. Consistent with this notion, Ucn 2 reduced insulin-induced tyrosine phosphorylation of IRS-1, and treatment with rapamycin reversed the inhibitory effect of Ucn 2 on IRS-1 and Akt phosphorylation. In conclusion, the inhibitory effect of CRFR2 signaling on insulin action is mediated by cAMP in a mammalian target of rapamycine-dependent manner, and IRS-1 is a key nodal point where CRFR2 signaling modulates insulin-stimulated glucose uptake in muscle cells.

Topics: Adenylyl Cyclases; Animals; Cell Differentiation; Cell Line; Deoxyglucose; Disease Models, Animal; Gene Knockdown Techniques; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Male; Mice, Obese; Models, Biological; Muscle Cells; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphorylation; Phosphotyrosine; Physical Conditioning, Animal; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urocortins

Focal adhesion kinase (FAK) controls cell growth and survival downstream of integrin-matrix receptors. Upon adhesion loss or FAK inhibition, FAK can translocate to the nucleus. The nucleolus is a non-membrane nuclear structure that regulates ribosome biogenesis and cell proliferation. Nucleostemin (NS), a nucleolar-localized protein, modulates cell cycle progression, stemness, and three-dimensional tumor spheroid formation. The signaling pathways that regulate NS levels in tumors remain undefined.. Human breast carcinoma cells were evaluated for growth in culture (adherent and anchorage-independent spheroid) and as orthotopic tumors. FAK signaling was evaluated by pharmacological FAK inhibitor addition (PF-271, IC50~0.1 μM) and by small hairpin RNA (shRNA) knockdown followed by re-expression of FAK wildtype (WT) or a kinase-dead (KD, K454R) FAK point mutant. Immunoblotting was used to evaluate FAK, NS, nucleolar phosphoprotein B23, and nucleolin levels. Total and phosphospecific antibody imunoblotting were used to detect changes in FAK, Akt kinase (Akt also known as protein kinase B), and 4E-binding protein 1 (4E-BP1) phosphorylation, a translation repressor protein and target of the mammalian target of rapamycin (mTOR) complex. Immunohistochemical, co-immunoprecipitation, and cellular fractionation analyses were used to evaluate FAK association with nucleoli.. Pharmacological (0.1 μM PF-271) or genetic inhibition of FAK activity prevents MDA-MB-231 and 4T1L breast carcinoma growth as spheroids and as orthotopic tumors. FAK inhibition triggers proteasome-mediated decreased NS levels but no changes in other nucleolar proteins such as B23 (nucleophosmin) or nucleolin. Active FAK was associated with purified nucleoli of anchorage-independent cells and present within nucleoli of human invasive ductal carcinoma tumor samples. FAK co-immunoprecipitated with B23 that binds NS and a complex between FAK, NS, Akt, and mTOR was detected. Constitutively-active Akt kinase promoted tumor spheroid growth, stabilized NS levels, and promoted pS65 4E-BP1 phosphorylation in the presence of inhibited FAK. Rapamycin lowered NS levels and inhibited pS65 4E-BP1 phosphorylation in cells with activated Akt-mTOR signaling.. FAK signaling occurs in the nucleolus, active FAK protects NS, and Akt-mTOR pathway regulates NS protein stability needed for breast carcinoma spheroid and tumor growth.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Female; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Proteins; Humans; Mice; Nuclear Proteins; Nucleophosmin; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sirolimus; Spheroids, Cellular; Tumor Burden; Tumor Cells, Cultured; Tumor Stem Cell Assay

Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

Topics: Animals; Apoptosis; Autophagy; Cell Cycle Proteins; Cell Line; Chloroquine; Disease Models, Animal; DNA-Binding Proteins; Eye Proteins; Frontotemporal Dementia; Gene Knock-In Techniques; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Transport Proteins; Mice; Muscle, Skeletal; Myoblasts; Osteitis Deformans; Peptides; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin

Cones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 & mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 & mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal.

Topics: Animals; Cell Survival; Diabetic Retinopathy; Disease Models, Animal; Electroretinography; Eye Proteins; Immunosuppressive Agents; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Retinal Cone Photoreceptor Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Many cancer research studies have extensively examined the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) pathway. There are only few reports that suggest that PTEN might affect pain; however, there is still a lack of evidence to show the role of PTEN for modulating pain. Here, we report a role for PTEN in a rodent model of neuropathic pain.. We found that chronic constriction injury (CCI) surgery in rats could elicit downregulation of spinal PTEN as well as upregulation of phosphorylated PTEN (phospho-PTEN) and phosphorylated mammalian target of rapamycin (phospho-mTOR). After examining such changes in endogenous PTEN in neuropathic rats, we explored the effects of modulating the spinal PTEN pathway on nociceptive behaviors. The normal rats exhibited mechanical allodynia after intrathecal (i.t.) injection of adenovirus-mediated PTEN antisense oligonucleotide (Ad-antisense PTEN). These data indicate the importance of downregulation of spinal PTEN for nociception. Moreover, upregulation of spinal PTEN by i.t. adenovirus-mediated PTEN (Ad-PTEN) significantly prevented CCI-induced development of nociceptive sensitization, thermal hyperalgesia, mechanical allodynia, cold allodynia, and weight-bearing deficits in neuropathic rats. Furthermore, upregulation of spinal PTEN by i.t. Ad-PTEN significantly attenuated CCI-induced microglia and astrocyte activation, upregulation of tumor necrosis factor-α (TNF-α) and phospho-mTOR, and downregulation of PTEN in neuropathic rats 14 days post injury.. These findings demonstrate that PTEN plays a key, beneficial role in a rodent model of neuropathic pain.

Topics: Animals; CD11b Antigen; Disease Models, Animal; Gene Expression Regulation; Green Fluorescent Proteins; Hyperalgesia; Male; Pain Threshold; PTEN Phosphohydrolase; Rats; Rats, Wistar; Sciatica; Sirolimus; Spinal Cord; Time Factors; Transduction, Genetic

It has been reported that cerebral ischemia/reperfusion (I/R) injury can activate autophagy. However, the role of autophagy in cerebral I/R injury remains controversy. Two major proteins, mTOR and Beclin-1, govern the formation of autophagosomes to regulate autophagy activity. However, the cross-talking between Beclin-1 and mTOR in cerebral I/R injury remains elusive. In this study, global cerebral I/R injury animal model and focal cerebral I/R injury animal model were induced to test the variation of Beclin-1 level in vivo. To further confirm the variation of Beclin-1 level and investigate the cross-talking between Beclin-1 and mammalian target of rapamycin (mTOR) in I/R injury, we used cobalt chloride (CoCl2) to develop an I/R injury cell model in HT22 cell line. Our data showed that the levels of Beclin-1 and phosphorylated mammalian target of rapamycin (p-mTOR) were clearly induced by I/R injury in vitro. And the time course studies suggested that the Beclin-1 and mTOR may have coordinated regulation in ischemia stages but not in reperfusion stages. Moreover, inhibitor of mTOR could prevent Beclin-1 decreasing, but this prevention may play opposite roles in different stages of I/R injury. We conclude that this study represents a major advance in our understanding of the cross-talking of two key proteins, Beclin-1 and mTOR, in autophagy and the role of autophagy in cerebral I/R injury.

Topics: Analysis of Variance; Animals; Antimutagenic Agents; Apoptosis Regulatory Proteins; Beclin-1; Brain Ischemia; Cell Line; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Male; Mice; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus

The mTOR pathway is a master regulator of cellular growth and metabolism. The biosynthetic and energetic demand of rapidly proliferating cells such as cancer cells is met by metabolic adaptations such as an increased glycolytic rate known as the Warburg effect. Herein, we characterize the anti-tumor effect of rapamycin in a mouse model of T-cell lymphoma and examine the metabolic effects in vitro. The murine T-cell lymphoma line, MBL2, and human cutaneous T-cell lymphoma (CTCL) lines, HH and Hut78, were used in syngeneic or standard NSG mouse models to demonstrate a marked suppression of tumor growth by rapamycin accompanied by inhibition of mTORC1/2. Analysis of the metabolic phenotype showed a substantial reduction in the glycolytic rate and glucose utilization in rapamycin-treated lymphoma cells. This was associated with reduced expression of glucose transporters and glycolytic enzymes in cultured cells and xenograft tumors. As a result of the decrease in glycolytic state, rapamycin-treated cells displayed reduced sensitivity to low-glucose conditions but continued to rely on mitochondrial oxidative phosphorylation (OXPHOS) with sensitivity to inhibition of OXPHOS. Taken together, we demonstrate that rapamycin suppresses growth of T-cell lymphoma tumors and leads to a reduction in aerobic glycolysis counteracting the Warburg effect of cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glycolysis; Heterografts; Humans; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Oxidative Phosphorylation; Phenotype; Sensitivity and Specificity; Sirolimus; TOR Serine-Threonine Kinases

Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future.

Topics: Acute Kidney Injury; Adenine; Animals; Apoptosis; Autophagy; Blood Urea Nitrogen; Caspase 3; Creatinine; Disease Models, Animal; In Situ Nick-End Labeling; Male; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus

Activated mammalian target of rapamycin (P-mTOR) has been shown to maintain the sensitivity of subsets of small-diameter primary afferent A-nociceptors. Local or systemic inhibition of the mTOR complex 1 (mTORC1) pathway reduced punctate mechanical and cold sensitivity in neuropathic pain and therefore offered a new approach to chronic pain control. In this study, we have investigated the effects of the rapamycin analog temsirolimus (CCI-779) on itch. Bouts of scratching induced by the histamine-dependent pruritogenic compound 48/80 and histamine-independent pruritogens, chloroquine and SLIGRL-NH2, injected intradermally were significantly reduced by local (intradermal) or systemic (intraperitoneal, i.p.) pretreatment with CCI-779. We also investigated the action of metformin, a drug taken to control type 2 diabetes and recently shown to inhibit mTORC1 in vivo. Although the response to nonhistaminergic stimuli was reduced at all of the time points tested, scratching to compound 48/80 was modified by metformin only when the drug was injected 24 hours before this pruritogen. We also examined the colocalization of P-mTOR with gastrin-releasing peptide, a putative marker for some itch-sensitive primary afferents, and found that P-mTOR was coexpressed in less than 5% of gastrin-releasing peptide-positive fibers in the mouse skin. Taken together, the data highlight the role that P-mTOR-positive A-fibers play in itch signaling and underline the importance of the mTORC1 pathway in the regulation of homeostatic primary afferent functions such as pain and itch. The actions of the antidiabetic drug metformin in ameliorating nonhistamine-mediated itch also suggest a new therapeutic route for the control of this category of pruritus.

Topics: Animals; Disease Models, Animal; Gastrin-Releasing Peptide; Histamine; Hypoglycemic Agents; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Neuralgia; Phosphoproteins; Protein Kinase Inhibitors; Pruritus; Signal Transduction; Sirolimus; Skin; TOR Serine-Threonine Kinases; Treatment Outcome

In this study, we compared the effect of oral administration of metformin (MET) and rapamycin (RAPA) alone or in combination on prostate cancer development and progression in HiMyc mice. MET (250 mg/kg body weight in the drinking water), RAPA (2.24 mg/kg body weight microencapsulated in the diet), and the combination inhibited progression of prostatic intraepithelial neoplasia lesions to adenocarcinomas in the ventral prostate (VP). RAPA and the combination were more effective than MET at the doses used. Inhibition of prostate cancer progression in HiMyc mice by RAPA was associated with a significant reduction in mTORC1 signaling that was further potentiated by the combination of MET and RAPA. In contrast, treatment with MET alone enhanced AMPK activation, but had little or no effect on mTORC1 signaling pathways in the VP of HiMyc mice. Further analyses revealed a significant effect of all treatments on prostate tissue inflammation as assessed by analysis of the expression of cytokines, the presence of inflammatory cells and NFκB signaling. MET at the dose used appeared to reduce prostate cancer progression primarily by reducing tissue inflammation whereas RAPA and the combination appeared to inhibit prostate cancer progression in this mouse model via the combined effects on both mTORC1 signaling as well as on tissue inflammation. Overall, these data support the hypothesis that blocking mTORC1 signaling and/or tissue inflammation can effectively inhibit prostate cancer progression in a relevant mouse model of human prostate cancer. Furthermore, combinatorial approaches that target both pathways may be highly effective for prevention of prostate cancer progression in men.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Cell Proliferation; Disease Models, Animal; Disease Progression; Immunohistochemistry; Male; Metformin; Mice; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Sirolimus

Here we showed that pAMPKα and PTEN were down-regulated and p-mTOR, p-S6, p-4EBP1, MMP7, and DCN1 were up-regulated in human gastric cancer tissue samples as compared to that in the noncancerous tissues. Metformin inhibited tumor growth in mice. Also it enhanced cisplatin- or rapamycin-induced reduction of tumor growth as compared with treatment of either drug alone. In addition to activation of AMPK and suppression of the mTOR pathway, a series of increased and decreased genes expression were induced by metformin, including PTEN, MMP7, and FN1. We suggest that metformin could potentially be used for the treatment of gastric cancer especially in combination with cisplatin or rapamycin.

Topics: Adult; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Cell Cycle; Cisplatin; Disease Models, Animal; Female; Flow Cytometry; Humans; Immunohistochemistry; Male; Metformin; Mice; Middle Aged; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Signal Transduction; Sirolimus; Stomach Neoplasms; Transcriptome; Xenograft Model Antitumor Assays

Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.

Topics: Animals; Apoptosis; Blotting, Western; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Disease Models, Animal; Immunosuppressive Agents; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Reperfusion Injury; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

Current treatments for major depressive disorder (MDD) have a time lag and are ineffective for a large number of patients. Development of novel pharmacological therapies requires a comprehensive understanding of the molecular events that contribute to MDD pathophysiology. Recent evidence points toward aberrant activity of synaptic proteins as a critical contributing factor. In the present studies, we used viral-mediated gene transfer to target a key mediator of activity-dependent synaptic protein synthesis downstream of mechanistic target of rapamycin complex 1 (mTORC1) known as p70 S6 kinase 1 (S6K1). Targeted delivery of two mutants of S6K1, constitutively active or dominant-negative, to the medial prefrontal cortex (mPFC) of rats allowed control of the mTORC1/S6K1 translational pathway. Our results demonstrate that increased expression of S6K1 in the mPFC produces antidepressant effects in the forced swim test without altering locomotor activity. Moreover, expression of active S6K1 in the mPFC blocked the anhedonia caused by chronic stress, resulting in a state of stress resilience. This antidepressant response was associated with increased neuronal complexity caused by enhanced S6K1 activity. Conversely, expression of dominant-negative S6K1 in the mPFC resulted in prodepressive behavior in the forced swim test and was sufficient to cause anhedonia in the absence of chronic stress exposure. Together, these data demonstrate a critical role for S6K1 activity in depressive behaviors, and suggest that pathways downstream of mTORC1 may underlie the pathophysiology and treatment of MDD.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Depressive Disorder, Major; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Ketamine; Male; Maze Learning; Neurons; Phenotype; Phosphorylation; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Swimming

Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI. In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.

Topics: Animals; Astrocytes; CD11b Antigen; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; ELAV-Like Protein 3; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Humans; Immunosuppressive Agents; Inflammation; Ki-67 Antigen; Male; Mice; Mice, Inbred C57BL; Neurons; Rats; Sirolimus; Spinal Cord Injuries; Time Factors

Benign prostatic hyperplasia (BPH) is the most common age-related disease in men. Here we tested the efficacy of Rapatar, a micellar nanoformulation of rapamycin, in two rat models of BPH: testosterone-induced and sulpiride-induced hyperplasia in ventral lobes and lateral/dorsal lobes, respectively. We found that Rapatar prevented hypertrophic and hyperplastic abnormalities and degenerative alterations in both BPH models. Rapatar normalized weight of the lateral lobes in sulpiride-induced BPH, the most relevant animal model of human BPH. Unlike Finasteride, a standard therapy of BPH, Rapatar reduced inflammation caused by sulpiride. No obvious side effects of Rapatar were detected. Our data provide a rationale for clinical trials of Rapatar in patients suffering from BPH.

Topics: Aging; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Drug Administration Schedule; Finasteride; Male; Nanomedicine; Prostate; Prostatic Hyperplasia; Rats; Rats, Wistar; Sirolimus; Sulpiride; Testosterone; TOR Serine-Threonine Kinases

To evaluate the effects of intrathecal injection of rapamycin on pain threshold and spinal cord glial activation in rats with neuropathic pain.. Healthy 30 male Sprague Dawley (SD) rats were randomly divided into six groups (n = 5 in each group): (1) control group without any treatments; (2) chronic constriction injury (CCI) group; (3) Early-rapamycin group with intrathecal injection of rapamycin 4 hours after CCI days; (4) Early-vehicle group with intrathecal injection of DMSO; (5) Late-rapamycin group with intrathecal injection of rapamycin 7 days after CCI; (6) Late-vehicle group with intrathecal injection of DMSO 7 days after CCI. Rapamycin or DMSO was injected for 3 consecutive days. Mechanical and thermal threshold were tested before and after the CCI operation. Lumbar segment of spinal cords was tested for glial fibrillary acidic protein (GFAP) by immunohistochemistry on 14th day after operation.. Mechanical and thermal hyperalgesia emerged on fourth day were maintained till fourteenth day after operation. After intrathecal injection of rapamycin 4 hours or 7 days after CCI, mechanical and thermal threshold significantly increased compared to injection of DMSO. The area of GFAP positive and the mean density of GFAP positive area in the dorsal horn of the ipsilateral side greatly increased in rapamycin-treated groups.. Intrathecal injection of rapamycin may attenuate CCI-induced hyperalgesia and inhibit the activation of astrocyte.

Topics: Analgesics; Animals; Astrocytes; Chronic Disease; Constriction, Pathologic; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hot Temperature; Hyperalgesia; Injections, Spinal; Male; Neuralgia; Pain Threshold; Random Allocation; Rats, Sprague-Dawley; Sciatic Nerve; Sirolimus; Spinal Cord; Touch

The risk of developing neurodegenerative disorders such as Alzheimer's disease (AD) increases dramatically with age. Understanding the underlying mechanisms of brain aging is crucial for developing preventative and/or therapeutic approaches for age-associated neurological diseases. Recently, it has been suggested that epigenetic factors, such as histone modifications, maybe be involved in brain aging and age-related neurodegenerations. In this study, we investigated 14 histone modifications in brains of a cohort of young (3 months), old (22 months), and old age-matched dietary restricted (DR) and rapamycin treated BALB/c mice. Results showed that 7 out of all measured histone markers were changed drastically with age. Intriguingly, histone methylations in brain tissues, including H3K27me3, H3R2me2, H3K79me3 and H4K20me2 tend to disappear with age but can be partially restored by both DR and rapamycin treatment. However, both DR and rapamycin treatment also have a significant impact on several other histone modifications such as H3K27ac, H4K16ac, H4R3me2, and H3K56ac, which do not change as animal ages. This study provides the first evidence that a broad spectrum of histone modifications may be involved in brain aging. Besides, this study suggests that both DR and rapamycin may slow aging process in mouse brain via these underlying epigenetic mechanisms.

Topics: Age Factors; Animals; Brain; Diet; Disease Models, Animal; Female; Histone Code; Histones; Mice; Mice, Inbred BALB C; Sirolimus

Liver biopsy, the gold standard for assessing liver fibrosis, suffers from limitations due to sampling error and invasiveness. There is therefore a critical need for methods to non-invasively quantify fibrosis throughout the entire liver. The goal of this study was to use molecular Magnetic Resonance Imaging (MRI) of Type I collagen to non-invasively image liver fibrosis and assess response to rapamycin therapy.. Liver fibrosis was induced in rats by bile duct ligation (BDL). MRI was performed 4, 10, or 18 days following BDL. Some BDL rats were treated daily with rapamycin starting on day 4 and imaged on day 18. A three-dimensional (3D) inversion recovery MRI sequence was used to quantify the change in liver longitudinal relaxation rate (ΔR1) induced by the collagen-targeted probe EP-3533. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for Sirius Red staining and hydroxyproline content.. ΔR1 increased significantly with time following BDL compared to controls in agreement with ex vivo measures of increasing fibrosis. Receiver operating characteristic curve analysis demonstrated the ability of ΔR1 to detect liver fibrosis and distinguish intermediate and late stages of fibrosis. EP-3533 MRI correctly characterized the response to rapamycin in 11 out of 12 treated rats compared to the standard of collagen proportional area (CPA). 3D MRI enabled characterization of disease heterogeneity throughout the whole liver.. EP-3533 allowed for staging of liver fibrosis, assessment of response to rapamycin therapy, and demonstrated the ability to detect heterogeneity in liver fibrosis.

Topics: Animals; Bile Ducts; Disease Models, Animal; Elasticity Imaging Techniques; Ligation; Liver Cirrhosis, Experimental; Magnetic Resonance Imaging; Male; Rats; ROC Curve; Sirolimus

Autism spectrum disorder (ASD) is multifactorial, with both genetic as well as environmental factors working in concert to develop the autistic phenotype. Immunological disturbances in autistic individuals have been reported and a role for food allergy has been suggested in ASD. Single gene mutations in mammalian target of rapamycin (mTOR) signaling pathway are associated with the development of ASD and enhanced mTOR signaling plays a central role in directing immune responses towards allergy as well. Therefore, the mTOR pathway may be a pivotal link between the immune disturbances and behavioral deficits observed in ASD. In this study it was investigated whether the mTOR pathway plays a role in food allergy-induced behavioral and immunological deficits. Mice were orally sensitized and challenged with whey protein. Meanwhile, cow's milk allergic (CMA) mice received daily treatment of rapamycin. The validity of the CMA model was confirmed by showing increased allergic immune responses. CMA mice showed reduced social interaction and increased repetitive self-grooming behavior. Enhanced mTORC1 activity was found in the brain and ileum of CMA mice. Inhibition of mTORC1 activity by rapamycin improved the behavioral and immunological deficits of CMA mice. This effect was associated with increase of Treg associated transcription factors in the ileum of CMA mice. These findings indicate that mTOR activation may be central to both the intestinal, immunological, and psychiatric ASD-like symptoms seen in CMA mice. It remains to be investigated whether mTOR can be seen as a therapeutic target in cow's milk allergic children suffering from ASD-like symptoms.

Topics: Animals; Brain; Cattle; Chemokine CCL2; Compulsive Behavior; Diet; Disease Models, Animal; Grooming; Ileum; Immunosuppressive Agents; Male; Mechanistic Target of Rapamycin Complex 1; Mice, Inbred C3H; Milk Hypersensitivity; Multiprotein Complexes; RNA, Messenger; Signal Transduction; Sirolimus; Social Behavior; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2(kd/kd) mice with complexes I-III/II-III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse array of human diseases whose pathogenesis involves RC dysfunction.

Topics: Animals; Autophagy; Cell Survival; Cycloheximide; Cytosol; Disease Models, Animal; Electron Transport; Endoplasmic Reticulum Stress; Enzyme Activation; Gene Expression Profiling; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Mitochondria; Mitochondrial Diseases; Models, Biological; Multiprotein Complexes; Phosphorylation; Probucol; Protein Biosynthesis; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome

Tuberous sclerosis (TSC) is associated with autism spectrum disorders and has been linked to metabolic dysfunction and unrestrained signaling of the mammalian target of rapamycin (mTOR). Inhibition of mTOR by rapamycin can mitigate some of the phenotypic abnormalities associated with TSC and autism, but whether this is due to the mTOR-related function in energy metabolism remains to be elucidated. In young Eker rats, an animal model of TSC and autism, which harbors a germ line heterozygous Tsc2 mutation, we previously reported that cerebral oxygen consumption was pronouncedly elevated. Young (4 weeks) male control Long-Evans and Eker rats were divided into control and rapamycin-treated (20 mg/kg once daily for 2 days) animals. Cerebral regional blood flow ((14)C-iodoantipyrine) and O2 consumption (cryomicrospectrophotometry) were determined in isoflurane-anesthetized rats. We found significantly increased basal O2 consumption in the cortex (8.7 ± 1.5 ml O2/min/100 g Eker vs. 2.7 ± 0.2 control), hippocampus, pons and cerebellum. Regional cerebral blood flow and cerebral O2 extractions were also elevated in all brain regions. Rapamycin had no significant effect on O2 consumption in any brain region of the control rats, but significantly reduced consumption in the cortex (4.1 ± 0.3) and all other examined regions of the Eker rats. Phosphorylation of mTOR and S6K1 was similar in the two groups and equally reduced by rapamycin. Thus, a rapamycin-sensitive, mTOR-dependent but S6K1-independent, signal led to enhanced oxidative metabolism in the Eker brain. We found decreased Akt phosphorylation in Eker but not Long-Evans rat brains, suggesting that this may be related to the increased cerebral O2 consumption in the Eker rat. Our findings suggest that rapamycin targeting of Akt to restore normal cerebral metabolism could have therapeutic potential in tuberous sclerosis and autism.

Topics: Animals; Autism Spectrum Disorder; Brain; Cerebrovascular Circulation; Disease Models, Animal; Heterozygote; Male; Nerve Tissue Proteins; Organ Specificity; Oxygen; Oxygen Consumption; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Rats; Rats, Long-Evans; Rats, Mutant Strains; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The mTORC1 complex integrates different inputs from intracellular and extracellular signals to control various cellular processes. Therefore, any disruption in the mTORC1 pathway could promote different neurological disorders. mTORC1 overactivation has been verified in different genetic and acquired epilepsy animal models. Therefore, inhibitors of this complex could have both antiepileptogenic and antiseizure effects. In our study, we investigated the effects of rapamycin pretreatment on pentylenetetrazole (PTZ)-induced seizures in zebrafish. Our results have shown that the latency to reach the tonic-clonic stage (stage III) of progressive behavioral alterations shown during PTZ-induced seizures was prolonged in larval (7days post fertilization, 7dpf), juvenile (45days post fertilization, 45dpf) and adult (6-8months) zebrafish after pretreatment with rapamycin. Furthermore, rapamycin pretreatment did not alter the locomotor activity in zebrafish. Therefore, the results obtained in our study indicate that rapamycin pretreatment is an important mechanism to control the progress of seizures in zebrafish throughout different developmental stages (larval, juvenile, and adult). Taken as a whole, our data support that rapamycin has immediate antiseizure effects and could be a potential alternative therapy for seizure control in epilepsy.

Topics: Animals; Anticonvulsants; Convulsants; Disease Models, Animal; Larva; Mechanistic Target of Rapamycin Complex 1; Motor Activity; Multiprotein Complexes; Pentylenetetrazole; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish; Zebrafish Proteins

Gentamicin may cause acute kidney injury. The pathogenesis of gentamicin nephrotoxicity is unclear. Autophagy is a highly conserved physiological process involved in removing damaged or aged biological macromolecules and organelles from the cytoplasm. The role of autophagy in the pathogenesis of gentamicin nephrotoxicity is unclear. The miniature pigs are more similar to humans than are those of rodents, and thus they are more suitable as human disease models. Here we established the first gentamicin nephrotoxicity model in miniature pigs, investigated the role of autophagy in gentamicin-induced acute kidney injury, and determined the prevention potential of rapamycin against gentamicin-induced oxidative stress and renal dysfunction. At 0, 1, 3, 5, 7 and 10 days after gentamicin administration, changes in autophagy, oxidative damage, apoptosis and inflammation were assessed in the model group. Compared to the 0-day group, gentamicin administration caused marked nephrotoxicity in the 10-day group. In the kidneys of the 10-day group, the level of autophagy decreased, and oxidative damage and apoptosis were aggravated. After rapamycin intervention, autophagy activity was activated, renal damage in proximal tubules was markedly alleviated, and interstitium infiltration of inflammatory cells was decreased. These results suggest that rapamycin may ameliorate gentamicin-induced nephrotoxicity by enhancing autophagy.

Topics: Acute Kidney Injury; Animals; Anti-Bacterial Agents; Apoptosis; Autophagy; Disease Models, Animal; Gentamicins; Inflammation; Kidney Tubules, Proximal; Microscopy, Electron, Transmission; Mitochondria; Oxidation-Reduction; Oxidative Stress; Protective Agents; Sirolimus; Swine; Swine, Miniature

The mammalian target of rapamycin (mTOR) signalling pathway regulates immune responses, and promotes cell growth and differentiation. Inhibition of mTOR with rapamycin modulates allergic asthma, while the underlying molecular mechanisms remain elusive. Here, we demonstrate that rapamycin, effectively inhibits eosinophil differentiation, contributing to its overall protective role in allergic airway inflammation.. Rapamycin was administered in a mouse model of ovalbumin-induced allergic airway inflammation, and the eosinophil differentiation was analysed in vivo and in vitro.. Rapamycin significantly attenuated allergic airway inflammation and markedly decreased the amount of eosinophils in local airways, peripheral blood and bone marrow, independently of levels of interleukin-5 (IL-5). In vitro colony forming unit assay and liquid culture demonstrated that rapamycin directly inhibited IL-5-induced eosinophil differentiation. In addition, rapamycin reduced the production of IL-6 and IL-13 by eosinophils. Rapamycin was also capable of reducing the eosinophil levels in IL-5 transgenic NJ.1638 mice, again regardless of the constitutive high levels of IL-5. Interestingly, rapamycin inhibition of eosinophil differentiation in turn resulted in an accumulation of eosinophil lineage-committed progenitors in bone marrow.. Altogether these results clearly demonstrate a direct inhibitory role of rapamycin in eosinophil differentiation and function, and reemphasize the importance of rapamycin and possibly, mTOR, in allergic airway disease.

Topics: Animals; Asthma; Cell Differentiation; Disease Models, Animal; Eosinophils; Hypersensitivity; Immunosuppressive Agents; Inflammation; Interleukins; Leukocyte Count; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Serine Proteinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Aortic Diseases; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Genetic Predisposition to Disease; Glucuronidase; Humans; Kidney Failure, Chronic; Klotho Proteins; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Phenotype; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Vascular Calcification

To explore the impact of triple anti-tumor therapy based on thymosin α1 (Tα1) combined with Huaier granule(HG) and sirolimus on the level of serum alpha-fetoprotein (AFP) in rat models of liver cancer.. Ninety Sprague-Dawley rats were randomly divided into triple anti-tumor therapy group, Tα1 group, HG group, sirolimus group, positive control and blank control groups, with 15 rats in each group. Except the blank control group, the rats in the other groups were induced using diethylnitrosamine (DEN) to establish liver cancer models. After DEN treatment, the triple therapy group underwent 0.8 mg/kg Tα1 subcutaneous injection (from once a day for two weeks to twice a week since the third week), 0.35 g/kg HG gavage (three times a day) and 1 mg/kg sirolimus gavage (once a day). The dose of the rest single drug groups were the same with that of the triple therapy group. The positive control and blank control groups were not treated with the drugs. The treatment lasted 20 weeks. Then, the behavior of the rats were observed at the different time points, and the level of serum AFP in the rats were detected at 6, 16, 18, 20 weeks, respectively.. The typical symptoms of liver cancer were seen in the DEN-induced rats at 16 weeks. Since the tenth week, 6 rats died one after another. Pathological section of rat liver tissue suggested that the rat models were established successfully. According to the incidence rate of liver cancer and the survival rate at 20 weeks, the triple anti-tumor therapy was significantly superior to the single drug treatments. In addition, the triple anti-tumor therapy significantly reduced the level of serum AFP in the rats.. The triple anti-tumor therapy can significantly prolong the survival time of rats with liver cancer, decrease the cancer incidence rate and the level of serum AFP.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Combined Chemotherapy Protocols; Behavior, Animal; Carcinoma, Hepatocellular; Disease Models, Animal; Liver; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Survival Analysis; Thymalfasin; Thymosin

The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-β signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.

Topics: Animals; Antibiotics, Antineoplastic; Carcinogenesis; Carcinoma, Renal Cell; Cysts; Disease Models, Animal; Hyperplasia; Kidney Neoplasms; Kidney Tubules, Proximal; Mice; Mice, Knockout; Proto-Oncogene Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Suppressor Proteins

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig-to-nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen-free (DPF) miniature pigs and infused intraportally into streptozotocin-induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti-thymocyte globulin (ATG) induction and maintenance with anti-CD154 monoclonal antibody and low-dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90-110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow-up period showed excellent glucose disposal capacity and porcine C-peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Topics: Analysis of Variance; Animals; Biopsy, Needle; Diabetes Mellitus, Type 1; Disease Models, Animal; Enzyme-Linked Immunospot Assay; Female; Flow Cytometry; Graft Rejection; Graft Survival; Immunocompromised Host; Immunohistochemistry; Islets of Langerhans Transplantation; Macaca mulatta; Male; Sirolimus; Statistics, Nonparametric; Swine; Transplantation Conditioning; Transplantation, Heterologous

Inhibitors of the mechanistic target of rapamycin (mTOR) pathway have antiepileptogenic effects in preventing epilepsy and pathologic and molecular mechanisms of epileptogenesis in mouse models of tuberous sclerosis complex (TSC). However, long-term treatment with mTOR inhibitors may be required to maintain efficacy and potentially has chronic side effects, such as immunosuppression. Attempts to minimize drug exposure will facilitate translational efforts to develop mTOR inhibitors as antiepileptogenic agents for patients with TSC. In this study, we tested intermittent dosing paradigms of mTOR inhibitors for antiepileptogenic properties in a TSC mouse model.. Western blot analysis of phosphorylation of S6 protein was used to assess the dose- and time-dependence of mTOR inhibition by rapamycin in control mice and conditional knockout mice with inactivation of the Tsc1 gene in glial fibrillary acidic protein (GFAP)-expressing cells (Tsc1(GFAP)CKO mice). Based on the Western blot studies, different dosing paradigms of rapamycin starting at postnatal day 21 were tested for their ability to prevent epilepsy or pathologic abnormalities in Tsc1(GFAP)CKO mice: 4 days of rapamycin only (4-∞), 4 days on-24 days off (4-24), and 4 days on-10 days off (4-10).. mTOR activity was inhibited by rapamycin in a dose-dependent fashion and recovered to baseline by about 10 days after the last rapamycin dose. The 4-10 and 4-24 dosing paradigms almost completely prevented epilepsy and the 4-10 paradigm inhibited glial proliferation and megalencephaly in Tsc1(GFAP)CKO mice.. Intermittent dosing of rapamycin, with drug holidays of more than 3 weeks, maintains significant antiepileptogenic properties in mouse models of TSC. These findings have important translational applications in developing mTOR inhibitors as antiepileptogenic agents in TSC patients by minimizing drug exposure and potential side effects.

Topics: Animals; Anticonvulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Male; Mice; Mice, Knockout; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Tuberous Sclerosis

The metabolic sensor mTOR broadly regulates cell growth and division in cancer cells, leading to a significant focus on studies of rapamycin and its analogues as candidate anticancer drugs. However, mTOR inhibitors have failed to produce useful clinical efficacy, potentially because mTOR is also critical in T cells implicated in immunosurveillance. Indeed, recent studies using rapamycin have demonstrated the important role of mTOR in differentiation and induction of the CD8+ memory in T-cell responses associated with antitumor properties. In this study, we demonstrate that rapamycin harms antitumor immune responses mediated by T cells in the setting of cancer vaccine therapy. Specifically, we analyzed how rapamycin affects the antitumor efficacy of a human papilloma virus E7 peptide vaccine (CyaA-E7) capable of eradicating tumors in the TC-1 mouse model of cervical cancer. In animals vaccinated with CyaA-E7, rapamycin administration completely abolished recruitment of CD8+ T cells into TC-1 tumors along with the ability of the vaccine to reduce infiltration of T regulatory cells and myeloid-derived suppressor cells. Moreover, rapamycin completely abolished vaccine-induced cytotoxic T-cell responses and therapeutic activity. Taken together, our results demonstrate the powerful effects of mTOR inhibition in abolishing T-cell-mediated antitumor immune responses essential for the therapeutic efficacy of cancer vaccines.

Topics: Animals; Antibiotics, Antineoplastic; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Disease Models, Animal; Female; Flow Cytometry; Humans; Mice, Inbred C57BL; Myeloid Cells; Neoplasms; Papillomavirus E7 Proteins; Sirolimus; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases; Tumor Burden

Chronic spinal cord injury (SCI) is a formidable hurdle that prevents a large number of injured axons from crossing the lesion, particularly the corticospinal tract (CST). This study shows that Pten deletion in the adult mouse cortex enhances compensatory sprouting of uninjured CST axons. Furthermore, forced upregulation of mammalian target of rapamycin (mTOR) initiated either 1 month or 1 year after injury promoted regeneration of CST axons. Our results indicate that both developmental and injury-induced mTOR downregulation in corticospinal motor neurons can be reversed in adults. Modulating neuronal mTOR activity is a potential strategy for axon regeneration after chronic SCI.. As one of the long descending tracts controlling voluntary movement, the corticospinal tract (CST) plays an important role for functional recovery after spinal cord injury. The regeneration of CST has been a major challenge in the field, especially after chronic injuries. Here we developed a strategy to modulate Pten/mammalian target of rapamycin signaling in adult corticospinal motor neurons in the postinjury paradigm. It not only promoted the sprouting of uninjured CST axons, but also enabled the regeneration of injured axons past the lesion in a mouse model of spinal cord injury, even when treatment was delayed up to 1 year after the original injury. The results considerably extend the window of opportunity for regenerating CST axons severed in spinal cord injuries.

Topics: Animals; Axons; Cerebral Cortex; Dependovirus; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Longitudinal Studies; Mice; Mice, Transgenic; Nerve Regeneration; PTEN Phosphohydrolase; Pyramidal Tracts; Recovery of Function; Sirolimus; Spinal Cord Injuries; Time Factors; Vesicular Glutamate Transport Protein 1

Friedreich's ataxia (FRDA), the most common inherited ataxia in the Caucasian population, is a multisystemic disease caused by a significant decrease in the frataxin level. To identify genes capable of modifying the severity of the symptoms of frataxin depletion, we performed a candidate genetic screen in a Drosophila RNAi-based model of FRDA. We found that genetic reduction in TOR Complex 1 (TORC1) signalling improves the impaired motor performance phenotype of FRDA model flies. Pharmacologic inhibition of TORC1 signalling by rapamycin also restored this phenotype and increased the lifespan and ATP levels. Furthermore, rapamycin reduced the altered levels of malondialdehyde + 4-hydroxyalkenals and total glutathione of the model flies. The rapamycin-mediated protection against oxidative stress is due in part to an increase in the transcription of antioxidant genes mediated by cap-n-collar (Drosophila ortholog of Nrf2). Our results suggest that autophagy is indeed necessary for the protective effect of rapamycin in hyperoxia. Rapamycin increased the survival and aconitase activity of model flies subjected to high oxidative insult, and this improvement was abolished by the autophagy inhibitor 3-methyladenine. These results point to the TORC1 pathway as a new potential therapeutic target for FRDA and as a guide to finding new promising molecules for disease treatment.

Topics: Aconitate Hydratase; Adenosine Triphosphate; Aldehydes; Animals; Animals, Genetically Modified; Antioxidants; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Frataxin; Friedreich Ataxia; Gene Expression; Glutathione; Humans; Immunosuppressive Agents; Iron-Binding Proteins; Longevity; Male; Malondialdehyde; Motor Activity; Oxidative Stress; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sirolimus; Superoxide Dismutase; Transcription Factors

The lysosomal enzyme β-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. In this study, we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and matrix metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release.

Topics: Animals; Cell Membrane; Disease Models, Animal; Exocytosis; Glucuronidase; Immunosuppressive Agents; Lyme Disease; Lysosomal-Associated Membrane Protein 1; Lysosomes; Macrophages; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Models, Biological; Protein Transport; Sirolimus

To evaluate the role of rapamycin (RAPA) in paraquat (PQ)-induced acute lung injury.. Lung tissues were stained with HE and lung histology was observed. Mortality rate, and neutrophil and leukocyte count in blood and bronchoalveolar lavage fluid (BALF) were recorded. Protein content in BALF was determined by Coomassie blue staining. Malondialdehyde (MDA) content, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity in blood were determined by thiobarbituric acid (TBA) assay, pyrogallol autoxidation method, and modified Haefman method, respectively. The NF-κB activity was measured by gel electrophoretic mobility shift assay (EMSA). Carbon dioxide partial pressure (PaCO2), partial pressure of oxygen (PaO2) and pH values were measured by automated blood gas analyzer.. HE staining results demonstrated RAPA alleviated pathological changes of acute alveolitis in SD rats. Trend of protein content in BALF was PQ group>RAPA treatment group>control group (P<0.05). Neutrophil and leukocyte count in RAPA treatment group was significantly lower than PQ group at 3, 5, and 7 days after injection (P<0.05). Trend of MDA content was RAPA treatment group>PQ group>control group (P<0.05). Trend of GSH-Px and SOD activity was control group>RAPA treatment group>PQ group (P<0.05). Compared with PQ group, PaO2 in RAPA treatment group was markedly higher and PaCO2 was lower (P<0.05).. PQ-induced acute lung injury was effectively reversed with RAPA, through inhibition of NF-κB activation.

Topics: Acute Lung Injury; Animals; Biomarkers; Bronchoalveolar Lavage Fluid; Carbon Dioxide; Disease Models, Animal; Glutathione Peroxidase; Hydrogen-Ion Concentration; Lung; Male; Malondialdehyde; Neutrophils; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Paraquat; Phosphorylation; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Superoxide Dismutase; Time Factors

Initial preclinical studies are required during the process of improving polymers, platforms, and drug-eluting systems for new coronary stent designs. Our objective was to analyze the efficacy and safety of new drug-eluting stent models compared with a conventional stent and commercialized drug-eluting stents in an experimental model with healthy porcine coronary arteries.. Sixty stents (conventional stent, new sirolimus-eluting stents: drug-eluting stents 1, 2 and 3; Cypher(®) and Xience(®)) were randomly placed in the coronary arteries of 20 Large White domestic pigs. Angiographic and histomorphometric studies were done 28 days later.. The stents were implanted at a stent/artery ratio of 1.34±0.15, with no significant differences between groups. The new stents showed less late loss and angiographic restenosis than conventional stents (P=.006 and P<.001, respectively). Histologically, restenosis and neointimal area were lower with all the new platforms than with the conventional stents (P<.001 for each variable), and no differences were found vs the drug-eluting stents on the market. Safety data showed that endothelialization was lower with drug-eluting stents than with conventional stents, except for drug-eluting stent 3 (P=.084). Likewise, inflammation was lower with drug-eluting stent 3 than with other stents.. The new drug-eluting stent platforms studied are associated with less restenosis than conventional stents and showed no significant differences in safety or efficacy vs commercialized drug-eluting stents.

Topics: Animals; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Immunosuppressive Agents; Prosthesis Design; Random Allocation; Sirolimus; Sus scrofa; Swine

TDP-43 is a multi-functional RNA/DNA-binding protein, well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-U. Currently, there are no effective drugs for these neurodegenerative diseases. We describe the generation and characterization of a new fly model of ALS-TDP with transgenic expression of the Drosophila ortholog of TDP-43, dTDP, in adult flies under the control of a temperature-sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. Diminished lifespan as well as impaired locomotor activities of the flies following induction of dTDP overexpression have been observed. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of these neurons. To counter the defects in this fly model of ALS-TDP, we have examined the therapeutic effects of the autophagy activator, rapamycin. Although harmful to the control flies, administration of 400 μM rapamycin before the induction of dTDP overexpression can significantly reduce the number of neurons bearing dTDP (+) aggregates, as well as partially rescue the diminished lifespan and locomotive defects of the ALS-TDP flies. Furthermore, we identify S6K, a downstream mediator of the TOR pathway, as one genetic modifier of dTDP. In sum, this Drosophila model of ALS-TDP under temporal and spatial control presents a useful new genetic tool for the screening and validation of therapeutic drugs for ALS. Furthermore, the data support our previous finding that autophagy activators including rapamycin are potential therapeutic drugs for the progression of neurodegenerative diseases with TDP-43 proteinopathies.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Genetically Modified; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Motor Activity; Mutation; Neurons; Sirolimus

Multipotent mesenchymal stromal cells (MSCs) are distinguished by their ability to differentiate into a number of stromal derivatives of interest for regenerative medicine, but they also have immunoregulatory properties that are being tested in a number of clinical settings.. We show that brief incubations with rapamycin, everolimus, FK506 or cyclosporine A increase the immunosuppressive potency of MSCs and other cell types.. The treated MSCs are up to 5-fold more potent at inhibiting the induced proliferation of T lymphocytes in vitro. We show that this effect probably is due to adsorption of the drug by the MSCs during pre-treatment, with subsequent diffusion into co-cultures at concentrations sufficient to inhibit T-cell proliferation. MSCs contain measurable amounts of rapamycin after a 15-min exposure, and the potentiating effect is blocked by a neutralizing antibody to the drug. With the use of a pre-clinical model of acute graft-versus-host disease, we demonstrate that a low dose of rapamycin-treated but not untreated umbilical cord-derived MSCs significantly inhibit the onset of disease.. The use of treated MSCs may achieve clinical end points not reached with untreated MSCs and allow for infusion of fewer cells to reduce costs and minimize potential side effects.

Topics: Animals; Antibodies, Neutralizing; Cell Proliferation; Coculture Techniques; Cyclosporine; Disease Models, Animal; Everolimus; Female; Graft vs Host Disease; Humans; Immune Tolerance; Immunosuppression Therapy; Immunosuppressive Agents; Lymphocyte Activation; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Sirolimus; T-Lymphocytes; Tacrolimus; Umbilical Cord

Cannabidiol (CBD), a major non-psychotomimetic constituent of Cannabis sativa, has therapeutic potential for certain psychiatric and neurological disorders. Studies in laboratory animals and limited human trials indicate that CBD has anticonvulsant and neuroprotective properties. Its effects against cocaine neurotoxicity, however, have remained unclear. Thus, the present study tested the hypothesis that CBD protects against cocaine-induced seizures and investigated the underlying mechanisms. CBD (30 mg/kg) pre-treatment increased the latency and reduced the duration of cocaine (75 mg/kg)-induced seizures in mice. The CB1 receptor antagonist, AM251 (1 and 3mg/kg), and the CB2 receptor antagonist, AM630 (2 and 4 mg/kg), failed to reverse this protective effect, suggesting that alternative mechanisms are involved. Synaptosome studies with the hippocampus of drug-treated animals revealed that cocaine increases glutamate release, whereas CBD induces the opposite effect. Finally, the protective effect of this cannabinoid against cocaine-induced seizure was reversed by rapamycin (1 and 5mg/kg), an inhibitor of the mammalian target of rapamycin (mTOR) intracellular pathway. In conclusion, CBD protects against seizures in a model of cocaine intoxication. These effects possibly occur through activation of mTOR with subsequent reduction in glutamate release. CBD should be further investigated as a strategy for alleviating psychostimulant toxicity.

Topics: Anesthetics, Local; Animals; Antiemetics; Cannabidiol; Cocaine; Disease Models, Animal; Dose-Response Relationship, Drug; Glutamic Acid; Immunosuppressive Agents; Indoles; Male; Mice; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

NOBORI biolimus A9-eluting stent (BES) is the third generation drug eluting stent (DES) with only abluminal biodegradable polymer. Recent clinical trials have indicated that the BES is non-inferior to the XIENCE V everolimus-eluting stent (EES). Meanwhile, potential superiority of biodegradable polymer BES over current generation DES has not been addressed. The aim of this preclinical study was to assess and compare the biocompatibility of both BES and EES in porcine coronary arteries.. BES with length of 24-mm (n=9) and EES with length of 23-mm (n=9) were both implanted in porcine coronary arteries. At 28 days endothelium-dependent vasomotion was assessed by acetylcholine (Ach) and subsequently measurements of endothelial superoxide production, histological evaluations and microarray gene analyses were performed.. Angiographic and histological in-stent stenoses were significantly suppressed in BES compared with EES. Histopathological assessment showed lower inflammatory score as well as fibrin and injury scores in BES as compared with EES. On the contrary, paradoxical vasoconstriction to Ach was frequently observed in EES-treated vessels compared with BES-treated vessels. Additionally, gene expressions of inflammatory cytokines and chemokines were upregulated in vessels treated with EES compared with BES in microarray pathway specific analyses.. Implantation of BES revealed less inflammation and foreign-body immunoreaction than EES, suggesting more enhanced biocompatibility of BES compared with EES at 28 days in porcine coronary arteries.

Topics: Animals; Biocompatible Materials; Coronary Stenosis; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Immunosuppressive Agents; Materials Testing; Sirolimus; Swine; Treatment Outcome

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies.

Topics: Animals; Autophagy; Cells, Cultured; Disease Models, Animal; Dogs; Enzyme Inhibitors; Humans; Induced Pluripotent Stem Cells; Macrolides; Models, Biological; Oxidation-Reduction; Primary Cell Culture; Proteolysis; Retinal Photoreceptor Cell Outer Segment; Retinal Pigment Epithelium; Sirolimus; Valproic Acid; Vitelliform Macular Dystrophy

Obliterative bronchiolitis (OB) ranks as the major obstacle for long-term survival of lung transplantation patients. Rapamycin (Rapa) has recently been confirmed as an immunosuppressant for antirejection due to its suppressive role in T cell activation. Here, we explore the effect of Rapa combined with immature dendritic cells (imDCs) on OB in trachea allograft rats.. The effect of bone marrow-derived imDCs or Rapa-imDCs on lymphocyte cells and CD4+ T cells were evaluated by methyl thiazolyl tetrazolium and flow cytometry. Tracheal transplantation was performed from Lewis rats to Wistar recipients. Recipient rats received Rapa+imDCs for 10 consecutive days after implantation. Allograft rejection was assessed by micro-CT image, hematoxylin/eosinHE staining and flow cytometry. The underlying mechanism was also investigated.. Rapa-imDCs inhibited lymphocyte and CD4+ T cell growth. Furthermore, Rapa-imDC treatment induced T cell hyporesponsiveness by attenuating T cell differentiation into IFN-x03B3;-producing T cells (Th1), but increased CD4+CD25+Foxp3+ T cell (Treg) contents. Importantly, Rapa-imDC administration ameliorated airway obliteration symptoms and CD4+ and CD8+ T cell infiltration. Furthermore, the proinflammatory factor levels of IL-6, TNF-α, IFN-x03B3; and IL-17 were decreased, concomitant with the upregulation of immunosuppressive cytokines IL-10 and TGF-β1. Further analysis confirmed that Rapa-imDC treatment attenuated the amounts of infiltrated IL-17+CD4+ T cells (Th17 cells) and Th1 cells, but increased Treg contents in the spleens of recipients.. This research may corroborate a protective role of Rapa-imDCs in OB by regulating the balance between effector T cells and Tregs, suggesting a potential applicable strategy to treat OB after lung transplantation.

Topics: Allografts; Animals; Antigen Presentation; Bronchiolitis Obliterans; Cytokines; Dendritic Cells; Disease Models, Animal; Graft Rejection; Immunomodulation; Immunophenotyping; Immunosuppressive Agents; Inflammation Mediators; Lymphocyte Activation; Male; Rats; Sirolimus; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Cyclosporine A (CsA) is widely used as an immunosuppressor in transplantation. Previous studies reported that CsA induces autophagy and that chronic treatment with CsA results in accumulation of autophagosomes and reduced autophagic clearance. Autophagy is a prosurvival process that promotes recovery from acute kidney injury by degrading misfolded proteins produced in the kidney. In the present study, we used TMBIM6-expressing HK-2, human kidney tubular cells (TMBIM6 cells) and Tmbim6 knockout (tmbim6(-/-)) mice. When exposed to CsA, the TMBIM6 cells maintained autophagy activity by preventing autophagosome accumulation. With regard to signaling, PRKKA/AMPK phosphorylation and mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) expression and its downstream target TFEB (transcription factor EB), a lysosome biogenesis factor, were regulated in the TMBIM6 cells. Lysosomal activity was highly increased or stably maintained in the presence of TMBIM6. In addition, treatment of tmbim6(-/-) mice with CsA resulted in increased autophagosome formation and decreased lysosome formation and activity. We also found that tmbim6(-/-) mice were susceptible to CsA-induced kidney injury. Taken together, these results indicate that TMBIM6 protects against CsA-induced nephrotoxicity both in vitro and in vivo by inducing autophagy and activating lysosomes.

Topics: Animals; Apoptosis; Autophagy; Cyclosporine; Disease Models, Animal; Humans; Immunosuppressive Agents; Kidney; Lysosomes; Mice, Knockout; Microtubule-Associated Proteins; Phagosomes; Sirolimus

Qiliqiangxin (QL) has been used for the treatment of chronic heart failure in China. Accumulating evidence suggests QL's cardio-protective effects on continuous myocardial ischemia. However, it is unclear whether QL has beneficial effects on cardiac ischemia-reperfusion (I/R) injury.. A mouse model of cardiac I/R was established by ligation of the left anterior descending coronary artery for 45 minutes followed by reperfusion. The mice were treated with QL for three days before surgery and continually after I/R. Triphenyltetrazolium chloride staining, echocardiography and Masson's trichrome staining were used to determine infarct size, cardiac function, and fibrosis, respectively. Expression levels of phospho-mTOR (Ser2448), mTOR, phospho-4EBP (Ser65), 4EBP, phospho-Akt (Ser473) and Akt were detected by Western blotting.. At 1 day after I/R, QL treatment significantly reduced the infarct size of mice exposed to I/R. At 7 days after I/R, mortality was reduced in QL treated animals in comparison with the control group. In addition, QL treated mice showed increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at 1 and 7 days after I/R. In agreement, Masson's trichrome staining demonstrated that interstitial fibrosis was less pronounced in QL treated mice compared with controls, suggesting that adverse left ventricular remodeling is attenuated in QL treated mice. Moreover, western blotting analysis demonstrated that QL activated the mTOR pathway, while mTOR inhibition via Rapamycin abolished the protective effects of QL against I/R injury.. This study suggests that QL attenuates the progression of cardiac remodeling after I/R likely via mTOR activation. This represents a new application for QL in the prevention of I/R injury.

Topics: Animals; Disease Models, Animal; Drugs, Chinese Herbal; Male; Mice; Myocardial Reperfusion Injury; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Remodeling

Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that controls protein synthesis in the nervous system. Here, we characterized the role of protein synthesis regulation due to mTOR signaling in rat dorsal root ganglion (DRG) following plantar incision. The number of phosphorylated mTOR (p-mTOR)-positive neurons was increased 2-4days after the incision. Rapamycin inhibited p-mTOR expression in the DRG and thermal hypersensitivity 3days but not 1day after the incision. Vesicular glutamate transporter 2 (VGLUT2) expression was increased after the plantar incision, which was inhibited by rapamycin. These results demonstrated that tissue injury induces phosphorylation of mTOR and increased protein level of VGLUT2 in the DRG neurons. mTOR phosphorylation involves in maintenance of injury-induced thermal hypersensitivity.

Topics: Analgesics; Animals; Disease Models, Animal; Foot Injuries; Ganglia, Spinal; Hindlimb; Hot Temperature; Hyperalgesia; Male; Neurons; Pain; Pain Threshold; Phosphorylation; Posterior Horn Cells; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; TRPV Cation Channels; Vesicular Glutamate Transport Protein 2

Despite antiviral therapy, the mortality rate of herpes simplex virus encephalitis (HSE) remains high and many surviving patients harbor neurological sequelae. Although viral replication is responsible for substantial neurological damages, an exaggerated inflammatory response could also contribute to this process. Artesunate (ART) and rapamycin (RAPA) have shown some benefits in the treatment of herpes simplex virus infections. Herein, we evaluated the benefit of combining ART or RAPA with valacyclovir (VACV) in a murine model of HSE. Infected mice were treated with VACV (1mg/mL in drinking water) from day 3 post-infection (p.i.) combined or not with daily intraperitoneal administration of ART (30mg/kg) or RAPA (20mg/kg) from days 4 to 13 p.i. Viral load, infectious titers, cytokine and chemokine levels were measured in brain homogenates on days 5, 7 and 9. The survival rates of mice treated with VACV and ART or RAPA were higher than with VACV alone (71.9% versus 43.2% for ART and 66.7% versus 43.2% for RAPA; both P⩽0.05) but no significant difference was seen in the brain viral loads. Levels of IL-1β, IL-2 (both P⩽0.05), IL-6, IFN-γ (both P⩽0.01), CCL2 (P⩽0.01), CCL3 and CCL4 (both P⩽0.05) were reduced in mice treated with VACV combined with ART versus VACV alone. Levels of IL-6, IL-1β and IFN-γ slightly increased on day 7 in mice treated with VACV combined with RAPA compared to VACV alone and then decreased on day 9. Our results suggest that immunomodulatory compounds such as ART or RAPA could benefit antiviral therapy in HSE.

Topics: Acyclovir; Administration, Oral; Animals; Antiviral Agents; Artemisinins; Artesunate; Brain; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Encephalitis, Herpes Simplex; Female; Immunologic Factors; Injections, Intraperitoneal; Mice, Inbred BALB C; Sirolimus; Survival Analysis; Treatment Outcome; Valacyclovir; Valine; Viral Load

Down's syndrome (DS) is the most prevalent genetic intellectual disability. Memory deficits significantly contribute to the cognitive dysfunction in DS. Previously, we discovered that mTOR-dependent local translation, a pivotal process for some forms of synaptic plasticity, is deregulated in a DS mouse model. Here, we report that these mice exhibit deficits in both synaptic plasticity (i.e., BDNF-long term potentiation) and the persistence of spatial long-term memory. Interestingly, these deficits were fully reversible using rapamycin, a Food and Drug Administration-approved specific mTOR inhibitor; therefore, rapamycin may be a novel pharmacotherapy to improve cognition in DS.

Topics: Animals; Brain-Derived Neurotrophic Factor; CA1 Region, Hippocampal; Disease Models, Animal; Down Syndrome; Long-Term Potentiation; Male; Maze Learning; Memory Disorders; Memory, Long-Term; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Nootropic Agents; Sirolimus; Spatial Memory; Synaptic Transmission; Tissue Culture Techniques; TOR Serine-Threonine Kinases

Fetal hemoglobin-inducing therapies are disease-modifying and ameliorate the pain phenotype in sickle cell disease (SCD). Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, increases HbF in erythroid precursor cells in vitro. We hypothesized that rapamycin would increase HbF levels and improve nociception phenotype in SCD mice. We used sine-wave electrical stimulation to examine nocifensive phenotype and evaluate myelinated [2000Hz (Aβ-fiber) and 250Hz (Aδ-fiber)] and unmyelinated (5Hz C-fibers)] sensory fiber function. Rapamycin significantly increased γ-globin mRNA and HbF levels [+2.3% (0.7, 3.9), mean increase (95% confidence interval, CI), p=0.006]. In homozygous (sickling) mice, long- (16 weeks), but not short-term (6 weeks), rapamycin treatment increased 2000Hz and 250Hz current thresholds in a pattern that varied according to sex. In male, but not female mice, rapamycin (compared with vehicle) was associated with increases in 2000Hz [21Units (7, 35), mean difference (95% CI), p=0.009 for sex∗treatment interaction] and 250Hz [9Units (1, 16), p=0.01] current thresholds. In rapamycin-treated homozygotes, HbF levels directly correlated with myelinated [2000Hz(Aβ-fiber, r=0.58, p=0.01) and 250Hz(Aδ-fiber, r=0.6, p=0.01)] but not unmyelinated sensory fiber current thresholds. These findings suggest that in SCD mice, rapamycin increases HbF and modulates current thresholds of myelinated fibers. Therefore, mTOR signaling might be implicated in the pathobiology of SCD.

Topics: Anemia, Sickle Cell; Animals; Bone Marrow; Disease Models, Animal; Female; Fetal Hemoglobin; Gene Expression Regulation; Genotype; Male; Mice; Mice, Transgenic; Nociception; Phenotype; Sensory Thresholds; Sirolimus; Thermosensing

Mesenchymal stem cells (MSCs) can protect bone marrow transplantation (BMT) recipients from the lethal acute graft-versus-host disease (aGVHD) development. However, the mechanisms underlying the anti-inflammatory properties of MSCs in aGVHD remain to be elucidated. The immunoregulatory properties of MSCs are mediated by their production of anti-inflammatory molecules, including IL-10 and TGF-β. On the other hand, MSCs can also produce proinflammatory cytokines during their normal growth, such as IL-1β and IL-6. These opposite actions may limit their therapeutic application in aGVHD. Therefore, optimization of the functional properties of MSCs can increase their benefits.. The expressions of mRNA and protein were analyzed by real-time PCR and western blotting, respectively. Expression of MSC markers was assessed by flow cytometry. An animal model of aGVHD was established by transplanting C57BL/6 donor bone marrow cells and spleen cells into lethally irradiated BALB/c recipient mice. The recipient mice were divided into the control group and the therapy [adipose tissue-derived human MSCs (Ad-hMSCs) or rapamycin-treated Ad-hMSCs] groups. The survival, body weight and clinical score of aGVHD in transplanted mice were monitored.. Rapamycin pre-treatment of Ad-hMSCs increased mRNA synthesis of IL-10, indoleamine 2,3-dioxygenase, and TGF-β compared with untreated Ad-hMSCs. Rapamycin-treated Ad-hMSCs suppressed clonal expansion of interleukin-17-producing CD4(+) T (Th17) cells more effectively than untreated cells. mRNA expression of autophagy markers such as ATG5, LC3A and LC3B was significantly increased in the rapamycin-treated Ad-hMSCs compared with untreated Ad-hMSCs. Transmission electron microscopy revealed that Ad-hMSCs exposure to rapamycin resulted in the appearance of autophagic vacuoles. Interestingly, in vitro migration efficiency of rapamycin-treated Ad-hMSCs toward the CD4(+) T cells was increased significantly compared with the untreated cells. And, these effects were associated with autophagy induction capacity of rapamycin. In vivo, the inhibiting properties of MSCs on the clinical severities of aGVHD were greater in the mice receiving rapamycin-treated Ad-hMSCs compared with untreated Ad-hMSCs. The beneficial effects of rapamycin treatment in Ad-hMSCs shown in vivo were associated with a reduction of Th17 cells and an increase in regulatory T cells.. Rapamycin can optimize the immunomodulatory potential of Ad-hMSCs, suggesting a promising strategy of MSC use in aGVHD treatment.

Topics: Abdominal Fat; Animals; Antigens, CD; Autophagy; Bone Marrow Transplantation; Cells, Cultured; Disease Models, Animal; Female; Graft vs Host Disease; Humans; Immunomodulation; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Inbred BALB C; Mice, Inbred C57BL; Receptors, Chemokine; Sirolimus; T-Lymphocyte Subsets

Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our previous study found that the novel anti-inflammatory cytokine IL-35 could suppress aGVHD in patients after allo-HSCT. In this study, we used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2) F1 (BDF1, H-2b×d) mice as recipients to create a model of aGVHD and explore the relationship between IL-35 and aGVHD. The mice receiving IL-35 survived longer than did the control mice. We observed that treatment with IL-35 and RAPA could reduce the incidence of aGVHD. Additionally, this treatment inhibited intestinal and thymic epithelial cell apoptosis and liver infiltration by the donor T-cells, thereby ameliorating the enteropathy and liver injury caused by aGVHD. We found that IL-35 and RAPA also markedly suppressed TNF-α and IL-17A expression and enhanced IFN-γ expression in the intestine and liver. We measured Tregs in spleen and found that IL-35 and RAPA treatment expanded the number of Tregs in spleen. We found that the phosphorylation of STAT1 and STAT4 were inhibited in mice with aGVHD. In contrast, STAT1 and STAT4 were phosphorylated when the mice were treated with IL-35. IL-35 may have therapeutic potential in the treatment of aGVHD after allo-HSCT.

Topics: Animals; Apoptosis; Cytokines; Disease Models, Animal; Epithelial Cells; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Immunosuppressive Agents; Interleukins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Sirolimus; Survival Analysis; T-Lymphocytes

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-D-aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDA-induced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1-positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells.

Topics: Animals; Disease Models, Animal; Everolimus; Extracellular Signal-Regulated MAP Kinases; In Situ Nick-End Labeling; Inflammation; Intravitreal Injections; Leukocyte Common Antigens; Male; N-Methylaspartate; Neuroprotective Agents; Phosphorylation; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Retina; Retinal Diseases; Retinal Ganglion Cells; Sirolimus; TOR Serine-Threonine Kinases

The perforant pathway projection from layer II of the entorhinal cortex to the hippocampal dentate gyrus is especially important for long-term memory formation, and is preferentially vulnerable to developing a degenerative tauopathy early in Alzheimer's disease (AD) that may spread over time trans-synaptically. Despite the importance of the perforant pathway to the clinical onset and progression of AD, a therapeutic has not been identified yet that protects it from tau-mediated toxicity. Here, we used an adeno-associated viral vector-based mouse model of early-stage AD-type tauopathy to investigate effects of the mTOR inhibitor and autophagy stimulator rapamycin on the tau-driven loss of perforant pathway neurons and synapses. Focal expression of human tau carrying a P301L mutation but not eGFP as a control in layer II of the lateral entorhinal cortex triggered rapid degeneration of these neurons, loss of lateral perforant pathway synapses in the dentate gyrus outer molecular layer, and activation of neuroinflammatory microglia and astroglia in the two locations. Chronic systemic rapamycin treatment partially inhibited phosphorylation of a mechanistic target of rapamycin substrate in brain and stimulated LC3 cleavage, a marker of autophagic flux. Compared with vehicle-treated controls, rapamycin protected against the tau-induced neuronal loss, synaptotoxicity, reactive microgliosis and astrogliosis, and activation of innate neuroimmunity. It did not alter human tau mRNA or total protein levels. Finally, rapamycin inhibited trans-synaptic transfer of human tau expression to the dentate granule neuron targets for the perforant pathway, likely by preventing the synaptic spread of the AAV vector in response to pathway degeneration. These results identify systemic rapamycin as a treatment that protects the entorhinal cortex and perforant pathway projection from tau-mediated neurodegeneration, axonal and synapse loss, and neuroinflammatory reactive gliosis. The findings support the potential for slowing the progression of AD by abrogating tau-mediated neurotoxicity at its earliest neuropathological stages.

Topics: Alzheimer Disease; Animals; Axons; Dentate Gyrus; Disease Models, Animal; Entorhinal Cortex; Hippocampus; Humans; Male; Memory, Long-Term; Mice; Microglia; Neurodegenerative Diseases; Neurons; Perforant Pathway; Phosphorylation; Sirolimus; Synapses; tau Proteins; Tauopathies; TOR Serine-Threonine Kinases

We created implantable intraocular devices capable of constant and continuous rapamycin release on the scale of months to years.. Polycaprolactone (PCL) thin films were used to encapsulate rapamycin to create implantable and biodegradable intraocular devices. Different film devices were studied by modifying the size, thickness, and porosity of the PCL films.. In vitro release of rapamycin was observed to be constant (zero-order) through 14 weeks of study. Release rates were tunable by altering PCL film porosity and thickness. In vivo release of rapamycin was observed out through 16 weeks with concentrations in the retina-choroid in the therapeutic range. Rapamycin concentration in the blood was below the lower limit of quantification. The drug remaining in the device was chemically stable in vitro and in vivo, and was sufficient to last for upwards of 2 years of total release. The mechanism of release is related to the dissolution kinetics of crystalline rapamycin.. Microporous PCL thin film devices demonstrate good ocular compatibility and the ability to release rapamycin locally to the eye over the course of many weeks.

Topics: Absorbable Implants; Animals; Anterior Eye Segment; Delayed-Action Preparations; Disease Models, Animal; Drug Delivery Systems; Follow-Up Studies; Immunosuppressive Agents; Rabbits; Sirolimus; Time Factors; Uveitis

The tumor suppressor gene FBXW7 is deleted and mutated in many different types of human cancers. FBXW7 primarily exerts its tumor suppressor activity by ubiquitinating different oncoproteins including mTOR. Here we used gene transcript profiling to gain a deeper understanding of the role of FBXW7 in tumor development and to determine the influence of mTOR inhibition by rapamycin on tumor transcriptome and biological functions. In comparison to tumors from p53 single heterozygous (p53+/-) mice, we find that radiation-induced thymic lymphomas from Fbxw7/p53 double heterozygous (Fbxw7+/-p53+/-) mice show significant deregulation of cholesterol metabolic processes independent of rapamycin treatment, while cell cycle related genes were upregulated in tumors from placebo treated Fbxw7+/-p53+/- mice, but not in tumors from rapamycin treated Fbxw7+/-p53+/- mice. On the other hand, tumors from rapamycin treated Fbxw7+/-p53+/- mice were enriched for genes involved in the integrated stress response, an adaptive mechanism to survive in stressful environments. Finally, we demonstrated that the Fbxw7 gene signatures identified in mouse tumors significantly overlap with FBXW7 co-expressed genes in human cancers. Importantly these common FBXW7 gene signatures between mouse and human are predictive for disease-free survival in human colon, breast and lung adenocarcinoma cancer patients. These results provide novel insights into the role of FBXW7 in tumor development and have identified a number of potential targets for therapeutic intervention.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cholesterol; Databases, Genetic; Disease Models, Animal; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genotype; Heterozygote; Humans; Lymphoma; Mice, Knockout; Neoplasms, Radiation-Induced; Phenotype; Protein Kinase Inhibitors; Sirolimus; Thymus Neoplasms; TOR Serine-Threonine Kinases; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Two new dimeric epipolythiodiketopiperazines, preussiadins A (1) and B (2), together with two known diastereomers, leptosins C (6) and A (7), were obtained from the mycelia of a Preussia typharum isolate. The structures of the new compounds were established by spectroscopic methods, and the absolute configurations of 1 and 2 were assigned by chemical transformations and comparisons of quantum chemical ECD and VCD calculations to experimental data. Compound 1 exhibited potent cytotoxic activity in the NCI-60 cell line panel with an average LC50 value of 251 nM. Further studies demonstrated that 1 circumvents P-glycoprotein-mediated drug resistance, yet had no significant antitumor activity in a xenograft UACC-62 melanoma model.

Topics: Animals; Antineoplastic Agents; Ascomycota; ATP Binding Cassette Transporter, Subfamily B, Member 1; Disease Models, Animal; Drug Screening Assays, Antitumor; Humans; Melanoma; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Piperazines

Few data exist with regard to the polymer integrity of drug-eluting stents (DES) in vivo. This study aims to investigate the integrity of the polymer layer of 4 polymer-coated DES in vivo. We assessed the morphology of the polymer layer of sirolimus-eluting stent (SES; Cypher Select™), paclitaxel-eluting stent (PES; Taxus Liberté™), zotarolimus-eluting stent (ZES; Endeavour RX™) and everolimus-eluting stent (EES; Xience V™) by scanning electron microscopy after balloon expansion at nominal and high pressures in the coronary arteries of 3 pigs. Effects of kissing balloon procedure were also explored. The polymer layer of SES, PES and EES were damaged in less than 3 % of the surface area with high pressure procedures, whereas the damaged area reached 38.0 ± 2.6 % in ZES (P < 0.01). The polymer integrity differed greatly among DES after balloon inflation in vivo. This should be taken into account when placing DES in tortuous vessels, calcified, as well as bifurcation lesions because the polymer layer may be easily damaged in these lesions.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blood Vessel Prosthesis Implantation; Coated Materials, Biocompatible; Coronary Restenosis; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Immunosuppressive Agents; Paclitaxel; Prosthesis Design; Sirolimus; Swine

Abstract We evaluated the efficacy of the anti-CD20 monoclonal antibody rituximab in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus for treating diffuse large B-cell lymphoma (DLBCL). The combination of rituximab and everolimus was more effective for inhibiting cell growth compared with single-agent therapy. An increase in G0/G1 cell cycle arrest and an increased population of cells in apoptosis were observed in the combination treatment group. The addition of rituximab reduced the overexpression of p-AKT caused by the negative feedback loop of everolimus and had an enhanced effect on inhibition of mTOR signaling, thus providing a rationale for this synergistic effect. Furthermore, combination treatment was also more effective than treatment with either agent alone for inhibiting the growth of DLBCL xenografts. Our study provides preclinical evidence and a theoretical basis for combination therapy with rituximab and everolimus in DLBCL.

Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Synergism; Everolimus; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Nude; Rituximab; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; CD11b Antigen; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Immunosuppressive Agents; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Pain Measurement; Schwann Cells; Sciatic Nerve; Sciatica; Sirolimus; Time Factors

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Endoplasmic Reticulum; Endothelial Cells; Female; Fetal Death; Homozygote; Mice; Mice, Mutant Strains; Mice, Transgenic; Mitochondria; Neovascularization, Pathologic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Yolk Sac

In the present study, the ability of metformin to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was analyzed in mice maintained on either an overweight control diet or an obesity-inducing diet. Rapamycin was included for comparison, and a combination of metformin and rapamycin was also evaluated. Metformin (given in the drinking water) and rapamycin (given topically) inhibited development of both papillomas and squamous cell carcinomas in overweight and obese mice in a dose-dependent manner. A low-dose combination of these two compounds displayed an additive inhibitory effect on tumor development. Metformin treatment also reduced the size of papillomas. Interestingly, all treatments seemed to be at least as effective for inhibiting tumor formation in obese mice, and both metformin and rapamycin were more effective at reducing tumor size in obese mice compared with overweight control mice. The effect of metformin on skin tumor development was associated with a significant reduction in TPA-induced epidermal hyperproliferation. Furthermore, treatment with metformin led to activation of epidermal AMP-activated protein kinase (AMPK) and attenuated signaling through mTOR complex (mTORC)-1 and p70S6K. Combinations of metformin and rapamycin were more effective at blocking epidermal mTORC1 signaling induced by TPA consistent with the greater inhibitory effect on skin tumor promotion. Collectively, the current data demonstrate that metformin given in the drinking water effectively inhibited skin tumor promotion in both overweight and obese mice and that the mechanism involves activation of epidermal AMPK and attenuated signaling downstream of mTORC1.

Topics: Adenylate Kinase; Adiponectin; Animals; Body Weight; Carcinogenesis; Carcinoma, Squamous Cell; Diet; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Insulin; Insulin-Like Growth Factor I; Leptin; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Obese; Multiprotein Complexes; Neoplasms, Experimental; Obesity; Overweight; Papilloma; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Skin Neoplasms; Tetradecanoylphorbol Acetate; TOR Serine-Threonine Kinases

The present study investigated the involvement of the PI3K, GSK-3β, heme oxygenase-1 (HO-1) and mTOR in the antidepressant-like effect of ascorbic acid in the tail suspension test (TST). Male Swiss mice were pretreated with ascorbic acid (1 mg/kg, p.o.) or vehicle and 45 min after, LY294002 (10 μg/site, i.c.v., reversible PI3K inhibitor), rapamycin (0.2 nmol/site, i.c.v., selective mTOR inhibitor), zinc protoporphyrin (ZnPP - 10 ng/site, i.c.v., HO-1 inhibitor) or vehicle was administered. We also investigated the synergistic effect of ascorbic acid (0.1 mg/kg, p.o., sub-effective dose in the TST) with lithium chloride (10 mg/kg, p.o., non-selective GSK-3β inhibitor), AR-A014418 (0.01 μg/site, i.c.v., selective GSK-3β inhibitor) or cobalt protoporphyrin (CoPP - 0.01 μg/site, i.c.v., HO-1 inducer) in the TST. The antidepressant-like effect of ascorbic acid (1 mg/kg, p.o.) was prevented by the treatment of mice with LY294002, rapamycin or ZnPP. In addition, sub-effective doses of lithium chloride, AR-A014418 or CoPP, combined with a sub-effective dose of ascorbic acid produced a synergistic antidepressant-like effect. We also demonstrated that 1 h after its administration, ascorbic acid increased the phosphorylation of p70S6K and the immunocontent of PSD-95 in the hippocampus of mice. These results indicate that the antidepressant-like effect of ascorbic acid in the TST might be dependent on the activation of PI3K and mTOR, inhibition of GSK-3β as well as induction of HO-1, reinforcing the notion that these are important targets for antidepressant activity and contributing to better elucidate the mechanisms underlying the antidepressant-like effect of ascorbic acid.

Topics: Animals; Antidepressive Agents; Ascorbic Acid; Depression; Disease Models, Animal; Disks Large Homolog 4 Protein; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Exploratory Behavior; Gene Expression Regulation; Guanylate Kinases; Hindlimb Suspension; Hippocampus; Immobility Response, Tonic; Male; Membrane Proteins; Mice; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. Apc(Min/+) mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve Apc(Min/+) mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of Apc(Min/+) mice. We show that eRapa improved survival of Apc(Min/+) mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the Apc(Min/+) mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy.

Topics: Adenomatous Polyposis Coli; Animals; Chemistry, Pharmaceutical; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Genes, APC; Intestinal Mucosa; Longevity; Mechanistic Target of Rapamycin Complex 1; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Neoplasm Transplantation; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Topics: Actins; Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Survival; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Everolimus; Hepatic Stellate Cells; Humans; Immunosuppressive Agents; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Muscle, Smooth; Myofibroblasts; Neoplasm Transplantation; Neovascularization, Pathologic; Rats; Rats, Wistar; Sirolimus

Overactivation of the mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of syndromic forms of autism spectrum disorders (ASDs), such as tuberous sclerosis complex, neurofibromatosis 1, and fragile X syndrome. Administration of mTORC1 (mTOR complex 1) inhibitors (e.g. rapamycin) in syndromic mouse models of ASDs improved behavior, cognition, and neuropathology. However, since only a minority of ASDs are due to the effects of single genes (∼10%), there is a need to explore inhibition of mTOR activity in mouse models that may be more relevant to the majority of nonsyndromic presentations, such as the genetically inbred BTBR T(+)Itpr3(tf)/J (BTBR) mouse model of ASDs. BTBR mice have social impairment and exhibit increased stereotypic behavior. In prior work, d-cycloserine, a partial glycineB site agonist that targets the N-methyl-d-aspartate (NMDA) receptor, was shown to improve sociability in both Balb/c and BTBR mouse models of ASDs. Importantly, NMDA receptor activation regulates mTOR signaling activity. The current study investigated the ability of rapamycin (10mg/kg, i.p.×four days), an mTORC1 inhibitor, to improve sociability and stereotypic behavior in BTBR mice. Using a standard paradigm to assess mouse social behavior, rapamycin improved several measures of sociability in the BTBR mouse, suggesting that mTOR overactivation represents a therapeutic target that mediates or contributes to impaired sociability in the BTBR mouse model of ASDs. Interestingly, there was no effect of rapamycin on stereotypic behaviors in this mouse model.

Topics: Animals; Behavior, Animal; Child Development Disorders, Pervasive; Disease Models, Animal; Enzyme Inhibitors; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Mutant Strains; Multiprotein Complexes; Sirolimus; Social Behavior; Stereotyped Behavior; TOR Serine-Threonine Kinases

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.

Topics: Animals; Animals, Genetically Modified; Carcinogenesis; Cell Line, Tumor; Chordoma; Disease Models, Animal; Disease Progression; Humans; Immunohistochemistry; Mutation; Notochord; Organ Specificity; Proto-Oncogene Proteins p21(ras); Sirolimus; Survival Analysis; Zebrafish

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide. It is well known that the activation of PI3K/AKT/mTOR and the Ras/MAPK signaling pathway plays a critical role in cellular metabolism, growth and proliferation, and its inhibitors have been used as therapeutic drugs for hepatocellular carcinoma. Cetuximab, a chimerical monoclonal EGFR IgG1 antibody, can block the binding of EGF or other ligands to EGFR and thus inhibit ligands-induced receptor phosphorylation. In the present study, we found that rapamycin could enhance the antiproliferation effect of cetuximab in both HepG2 cells and Huh-7 cells and arrest the cell cycle. Cetuximab in combination with rapamycin had synergistic effects on inhibiting the phosphrylation of proteins in PI3K/AKT/mTOR and Ras/MAPK signaling pathway. Combination of cetuximab with rapamycin treatment significantly suppressed the HCC development in HepG2 cells-xenografted mice and improved the survival. Cetuximab and rapamycin inhibited the growth of HCC both in vitro and in vivo. These results suggest that the combination therapy using the inhibitors for both EGFR and PI3K/AKT/mTOR signaling pathways may be a novel therapeutic approach for HCC.

Topics: Animals; Antibodies, Monoclonal, Humanized; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cetuximab; Disease Models, Animal; Drug Synergism; Female; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

In advanced breast cancer, bone metastases occur in 70 % of patients. Managing the devastating pain associated with the disease is difficult. Rapamycin is an immunomodulatory drug that targets the mammalian target of rapamycin pathway. Rapamycin has been shown to decrease osteolysis associated with metastatic breast cancer in pre-clinical models and to reduce pain in inflammatory and neuropathic models. The aim of this study was to evaluate the effectiveness of rapamycin in reducing pain associated with experimental osteolytic metastases. Bone cancer was induced by intra-tibial injections of murine mammary carcinoma cells (4T1) in immunocompetent BALB/c mice and treated intraperitoneally for up to 5 weeks with vehicle, rapamycin or pamidronate (a bisphosphonate currently used to reduce bone loss in bone cancer patients). The control group received intra-tibial injection with saline (sham) and was treated with vehicle intraperitoneally. Cancer-induced osteolysis was observed histologically and radiographically 2-3 weeks following cancer inoculation and gradually increased with time. Measures of evoked nociceptive behaviors including sensitivity to mechanical, thermal, and cold stimuli and spontaneous nociceptive behaviors (limping, guarding) were evaluated. Significant hypersensitivity to sensory stimuli developed in cancer-bearing mice compared to sham 3 weeks following inoculation. Rapamycin decreased or delayed the development of cancer-induced mechanical, heat, and cold hypersensitivity, while pamidronate reduced heat and cold hypersensitivity. Both rapamycin and pamidronate had a partial protective effect on the spontaneous nociceptive behaviors, limping and guarding. Our data suggest that rapamycin may have efficacy in the management of pain associated with metastatic breast cancer.

Topics: Animals; Antibiotics, Antineoplastic; Bone Density Conservation Agents; Bone Neoplasms; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Female; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Osteolysis; Pain; Pain Measurement; Pamidronate; Sirolimus; TOR Serine-Threonine Kinases

High sugar consumption and diabetes increase the risk of developing Alzheimer's disease (AD) by unknown mechanisms. Using an animal model of AD, here we show that high sucrose intake induces obesity with changes in central and peripheral insulin signaling. These pre-diabetic changes are associated with an increase in amyloid-β production and deposition. Moreover, high sucrose ingestion exacerbates tau phosphorylation by increasing Cdk5 activity. Mechanistically, the sucrose-mediated increase in AD-like pathology results from hyperactive mammalian target of rapamycin (mTOR), a key nutrient sensor important in regulating energy homeostasis. Specifically, we show that rapamycin, an mTOR inhibitor, prevents the detrimental effects of sucrose in the brain without altering changes in peripheral insulin resistance. Overall, our data suggest that high sucrose intake and dysregulated insulin signaling, which are known to contribute to the occurrence of diabetes, increase the risk of developing AD by upregulating brain mTOR signaling. Therefore, early interventions to modulate mTOR activity in individuals at high risk of developing diabetes may decrease their AD susceptibility.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cyclin-Dependent Kinase 5; Diabetes Mellitus; Dietary Sucrose; Disease Models, Animal; Female; Insulin; Insulin Resistance; Mice; Mice, Transgenic; Molecular Targeted Therapy; Phosphorylation; Signal Transduction; Sirolimus; tau Proteins; TOR Serine-Threonine Kinases

The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, we found that DAF-18/PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR.

Topics: Aging; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Forkhead Transcription Factors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Insulin; Insulin-Like Growth Factor I; Nerve Degeneration; Nerve Regeneration; Phosphotransferases (Alcohol Group Acceptor); PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Time Factors; Transcription Factors

Erlotinib and Rapamycin are both in clinical use and experimental inhibition of their respective molecular targets, EGFR and mTORC1, has improved recovery from spinal cord injury. Our aim was to determine if daily Erlotinib or Rapamycin treatment started directly after spinal contusion injury in rats improves locomotion function or recovery of bladder function.. Stockholm, Sweden.. Rats were subjected to contusion injuries and treated during the acute phase with either Erlotinib or Rapamycin. Recovery of bladder function was monitored by measuring residual urine volume and hindlimb locomotion assessed by open-field observations using the BBB rating scale as well as by automated registration of gait parameters. Body weights were monitored. To determine whether Erlotinib and Rapamycin inhibit the same signaling pathway, a cell culture system and western blots were used.. Erlotinib accelerated locomotor recovery and slightly improved bladder recovery; however, we found no long-term improvements of locomotor function. Rapamycin did neither improved locomotor function nor bladder recovery. In vitro studies confirmed that Erlotinib and Rapamycin both inhibit the EGFR-mTORC1 signaling pathway.. We conclude that none of these two drug regimes improved long-term functional outcome in our current model of spinal cord injury. Nevertheless, oral treatment with Erlotinib may offer modest temporary advantages, whereas treatment with Rapamycin does not.

Topics: Administration, Oral; Animals; Disease Models, Animal; Erlotinib Hydrochloride; Female; Hindlimb; Immunosuppressive Agents; Locomotion; Protein Kinase Inhibitors; Quinazolines; Rats, Sprague-Dawley; Recovery of Function; Sirolimus; Spinal Cord Injuries; Urinary Bladder

The role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) in the maintenance of cardiac homeostasis still remains controversial. This study was designed to evaluate the role of cardiomyocyte-specific PTEN in the maintenance of cardiac homeostasis and the underlying mechanisms involved with a focus on autophagy, an evolutionarily conserved pathway for protein degradation. Cardiomyocyte-specific PTEN((flox/flox))/α-myosin heavy chain Cre mice, henceforth referred to as CM-PTENKO, were generated by crossing the floxed PTEN mice with α-myosin heavy chain Cre mice driven by a Cre recombinase promoter. The adult PTEN(-/-) mice displayed the phenotype of established hypertrophic cardiomyopathy, including unfavorable geometric, functional, and histological changes. Furthermore, cardiomyocyte-specific PTEN knockout mice exhibited increased cardiac mammalian target of rapamycin although suppressed autophagy. Treatment with rapamycin (2 mg/kg per day, IP), an inhibitor of mammalian target of rapamycin, for 1 month effectively reversed the established hypertrophic cardiomyopathy in CM-PTENKO mice. With rapamycin treatment, autophagy activity was significantly restored in the heart of CM-PTENKO mice. Taken together, our results demonstrate an essential role for cardiomyocyte PTEN in maintaining cardiac homeostasis under physiological condition. Cardiomyocyte-specific deletion of PTEN results in the development of hypertrophic cardiomyopathy possibly through a mechanism associated with mammalian target of rapamycin hyperactivation and autophagy suppression.

Topics: Animals; Autophagy; Cardiomyopathy, Hypertrophic; Disease Models, Animal; Homeostasis; Integrases; Male; Mice; Mice, Knockout; Myocardium; Myocytes, Cardiac; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Myosins

AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target.. We sought to identify the pro-growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage.. In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate-stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor-signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth.. Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of γ2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

Topics: AMP-Activated Protein Kinases; Animals; Cardiomyopathy, Hypertrophic, Familial; Cell Division; Cell Enlargement; Disease Models, Animal; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Knock-In Techniques; Genetic Complementation Test; Glucose-6-Phosphate; Glycogen; Glycogen Storage Disease; Glycogen Synthase; Insulin Resistance; Mice; Myocardium; Myocytes, Cardiac; Pre-Excitation Syndromes; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated.. Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors.. The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner.. mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.

Topics: Animals; Blotting, Western; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Humans; Immunosuppressive Agents; Mice; Muscle, Smooth, Vascular; Osteoblasts; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Calcification

Defect of autophagy is common to many neurodegenerative disorders because it serves as a major degradation pathway for the clearance of various aggregate-prone proteins. Mammalian target of rapamycin (mTOR) signaling, which is recognized as the most important negative regulator of autophagy, is also involved in neurodegenerative diseases. However, the role of mTOR and its dependent autophagy in normal brain during aging remains unknown. Furthermore, caloric restriction (CR) is frequently used as a tool to study mechanisms behind aging and age-associated diseases because CR can prevent age-related diseases and prolong lifespan in several model organisms. Inhibiting mTOR and promoting autophagy activity play roles in aging delayed by CR. However, whether CR can ameliorate age-related cognition deficits by inhibiting mTOR and activate autophagy in hippocampus needs to be further investigated. Here we showed a decline of autophagic degradation in mice hippocampus in correlation with age-dependent cognitive dysfunction, whereas the activity of mTOR and its upstream brain-derived neurotrophic factor (BDNF)/phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling was decreased with aging. In addition, facilitating the mTOR pathway successfully declines and sustains autophagic degradation with aging in hippocampus by CR treatment and is involved in CR by ameliorating age-related cognitive deficits.

Topics: Age Factors; Aging; Animals; Autophagy; Brain; Brain-Derived Neurotrophic Factor; Caloric Restriction; Cognition Disorders; Disease Models, Animal; Food Deprivation; Gene Expression Regulation; Male; Maze Learning; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Time Factors

Autophagy is essential to neuronal homeostasis, and its impairment is implicated in the development of neurodegenerative pathology. However, the underlying mechanisms and consequences of this phenomenon remain a matter of conjecture. We show that misexpression of human tau in Drosophila induces accumulation of autophagic intermediates with a preponderance of large vacuoles, which we term giant autophagic bodies (GABs), which are reminiscent of dysfunctional autophagic entities. Lowering basal autophagy reduces GABs, whereas increasing autophagy decreases mature autolysosomes. Induction of autophagy is also associated with rescue of the tauopathy phenotype, suggesting that formation of GABs may be a compensatory mechanism rather than a trigger of neurodegeneration. Last, we show that the peculiar Biondi bodies observed in the choroid epithelium of both elderly and Alzheimer's disease human brains express immunoreactive markers similar to those of GABs. Collectively, these data indicate that autophagic gridlock contributes to the development of pathology in aging and neurodegeneration.

Topics: Aging; Animals; Animals, Genetically Modified; Autophagy; Biomarkers; Disease Models, Animal; Drosophila; Female; Gene Expression Regulation; Genotype; Humans; Neurodegenerative Diseases; Retina; Sirolimus; tau Proteins

Recently, evidence indicated that the rapamycin-eluting stent which was used worldwide may contribute to an increased risk for thrombosis. On the contrary, other researchers found it was safe. Thus, it is necessary to clarify the effect of rapamycin on thrombosis and the corresponding mechanisms.. The effects of rapamycin in vivo were evaluated by modified deep vein thrombosis animal model. The platelets were from healthy volunteers and the platelet-endothelium (purchased from ATCC) adhesion in cultured endothelial cells was assessed. Membrane rufflings in endothelial cells were examined by confocal and electron microscope. Thrombus formation increased in rats that were injected with rapamycin. Electron microscope analysis exhibited microvilli on the rapamycin-treated endothelium in rats. Rapamycin enhanced membrane ruffling in human umbilical vein endothelial cells (HUVECs) and adhesion of platelets to HUVECs. The platelet-HUVECs adhesion was attenuated when cells were treated with cytochalacin B. Inhibition of autophagy by 3-methyladenine led to suppression of membrane ruffles in HUVECs and augmentation of platelet-endothelial adhesion.. In conclusion, we found that endothelial membrane remodeling induced by rapamycin is crucial for the adhesion of platelets to endothelial cells and thereby for thrombosis in vivo, and that the endothelial membrane remodeling is autophagy dependent.

Topics: Adenine; Animals; Autophagy; Blood Platelets; Cell Adhesion; Cell Membrane; Cytochalasin B; Disease Models, Animal; Human Umbilical Vein Endothelial Cells; Humans; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Thrombosis

Hepatocyte transplantation is proposed as a solution for liver failure. The allotransplantation of hepatocytes has been studied extensively, however, graft rejection remains a major problem. The aim of the present study was to evaluate the immunosuppressive activity of mycophenolate mofetil (MMF), sirolimus, and their combination in an experimental model of hepatocyte allotransplantation in rats with acute liver failure.. Five male Wistar rats were used as hepatocyte donors and 60 male Lewis rats as recipients. The recipients were divided into five groups of 12 animals each. Group 1: no treatment. Group 2: cyclosporine. Group 3: sirolimus. Group 4: MMF. Group 5: MMF and sirolimus. All surviving animals were preserved for 15 days. For the induction of acute liver failure the recipients were injected with N-dimethyl-nitrosamine 24 hr before transplantation. The isolated hepatocytes were transplanted intrasplenically.. Analysis of the results showed a statistically significant prolongation of animal survival for groups 3, 4, and 5. More animals in group 5 survived than those in groups 3 and 4, although the difference was not statistically significant. Transplant hepatocyte survival was significantly better in groups 3, 4, and 5. Hepatocytes transplanted in the spleen of animals of group 5 showed better survival compared with those of groups 3 and 4.. Use of MMF and sirolimus, as monotherapy or in combination, is both effective and safe as immunosuppressive treatment in hepatocyte transplantation, as was proven in this experimental protocol.

Topics: Animals; Disease Models, Animal; Graft Rejection; Hepatocytes; Immunosuppressive Agents; Liver Failure, Acute; Male; Mycophenolic Acid; Rats, Inbred Lew; Rats, Wistar; Sirolimus; Spleen

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

Topics: Animals; Biological Transport; Cyclosporine; Disease Models, Animal; Female; Humans; Immunization; Interferon-gamma; Interleukin-7; Macrophages; Macular Degeneration; Male; Mice; Oxidative Stress; Pyrroles; Retinal Pigment Epithelium; Sirolimus; T-Lymphocytes

Mammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates many processes, including cell growth and the immune response. mTOR is also dysregulated in several neurological diseases, such as traumatic brain injury (TBI), stroke, and neurodegenerative disease. However, the role of mTOR in intracerebral hemorrhage (ICH) remains unexplored. The aims of our study were to determine whether inhibiting mTOR signaling could affect the outcome after ICH and to investigate the possible underlying mechanism.. A rat ICH model was induced by intracerebral injection of collagenase IV into the striatum, and mTOR activation was inhibited by administration of rapamycin. mTOR signaling activation was determined by western blotting. Neurobehavioral deficit after ICH was determined by a set of modified Neurological Severity Scores (mNSS). The levels of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and cytokines were examined using flow cytometry and ELISA, respectively.. Our results demonstrated thatmTOR signaling was activated 30 minutes and returned to its basal level 1 day after ICH. Increased p-mTOR, which mean that mTOR signaling was activated, was predominantly located around the hematoma. Rapamycin treatment significantly improved the neurobehavioral deficit after ICH, increased the number of Tregs, increased levels of interleukin-10 and transforming growth factor-β and reduced interferon-γ both in peripheral blood and brain.. Our study suggests that mTOR improves ICH outcome and modulates immune response after ICH.

Topics: Animals; Blood Transfusion, Autologous; Cerebral Cortex; Cerebral Hemorrhage; Collagenases; Corpus Striatum; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Forkhead Transcription Factors; Gene Expression Regulation; Male; Nervous System Diseases; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; Time Factors; TOR Serine-Threonine Kinases

Accumulation of amyloid-β peptides (Aβ) within brain is a major pathogenic hallmark of Alzheimer's disease (AD). Emerging evidence suggests that autophagy, an important intracellular catabolic process, is involved in Aβ clearance. Here, we investigated whether temsirolimus, a newly developed compound approved by Food and Drug Administration and European Medicines Agency for renal cell carcinoma treatment, would promote autophagic clearance of Aβ and thus provide protective effects in cellular and animal models of AD. HEK293 cells expressing the Swedish mutant of APP695 (HEK293-APP695) were treated with vehicle or 100nM temsirolimus for 24h in the presence or absence of 3-methyladenine (5mM) or Atg5-siRNA, and intracellular Aβ levels as well as autophagy biomarkers were measured. Meanwhile, APP/PS1 mice received intraperitoneal injection of temsirolimus (20mg/kg) every 2 days for 60 days, and brain Aβ burden, autophagy biomarkers, cellular apoptosis in hippocampus, and spatial cognitive functions were assessed. Our results showed that temsirolimus enhanced Aβ clearance in HEK293-APP695 cells and in brain of APP/PS1 mice in an autophagy-dependent manner. Meanwhile, temsirolimus attenuated cellular apoptosis in hippocampus of APP/PS1 mice, which was accompanied by an improvement in spatial learning and memory abilities. In conclusion, our study provides the first evidence that temsirolimus promotes autophagic Aβ clearance and exerts protective effects in cellular and animal models of AD, suggesting that temsirolimus administration may represent a new therapeutic strategy for AD treatment. Meanwhile, these findings emphasize the notion that many therapeutic agents possess pleiotropic actions aside from their main applications.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antineoplastic Agents; Autophagy; Brain; Disease Models, Animal; HEK293 Cells; Humans; Male; Maze Learning; Memory; Mice, Transgenic; Neuroprotective Agents; Sirolimus; TOR Serine-Threonine Kinases

To explore the effects of rapamycin-induced autophagy on apoptosis in a rat model of acute spinal cord injury (SCI), and to explore the effect of rapamycin on apoptosis in primary spinal cord cell culture.. SCI was induced at T10 in female adult Sprague-Dawley rats. After injury was induced, the rats were injected with rapamycin and/or methylprednisolone and were sacrificed at various days after injury. Apoptosis and autophagy were examined with TUNEL staining and electron microscopy. Hind limb function was assessed by the Gale scale.. The expression of the apoptosis-related protein caspase-3 did not significantly increase until 21 days following injury, while increases in LC3II and LC3I began 10 days after injury, but then declined. TUNEL staining and electron microscopy confirmed that following injury autophagy occurred before apoptosis, but by 14 days after the injury, the level of autophagy had decreased significantly while the level of apoptosis showed a continued increase. Following treatment with rapamycin, apoptosis was significantly higher than in the vehicle control group, but significantly lower than in the sham-operated group, showing a protective effect of rapamycin. Gale scale grades in rats treated with rapamycin were significantly higher compared with the vehicle control group, suggesting a functional effect of rapamycin-induced inhibition of apoptosis.. The results indicate that rapamycin significantly improved the prognosis of acute SCI in rats by inhibiting cell apoptosis. Rapamycin might be useful as a therapeutic agent for acute SCI.

Topics: Animals; Apoptosis; Autophagy; Blotting, Western; Disease Models, Animal; Female; Flow Cytometry; In Situ Nick-End Labeling; Microscopy, Electron, Transmission; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Recovery of Function; Sirolimus; Spinal Cord Injuries

IgA nephropathy (IgAN) is the most frequent glomerulonephritis worldwide. Different therapeutic approaches have been tested against IgAN. The present study was designed to explore the renoprotective potential of low-dose mammalian target of rapamycin (mTOR) inhibitor rapamycin in an IgAN rat model and the possible mechanism of action.. After establishing an IgAN model, the rats were randomly divided into four groups: control, control with rapamycin treatment, IgAN model, and IgAN model with rapamycin treatment. Coomassie Brilliant Blue was utilized to measure 24-hour urinary protein levels. Hepatic and renal function was determined with an autoanalyzer. Proliferation was assayed via 5-bromo-2'-deoxyuridine incorporation. Real-time PCR and immunohistochemistry were utilized to detect the expression of α-SMA, collagen I, collagen III, TGF-β1 and platelet-derived growth factor. Western blotting and immunohistochemistry were performed to determine p-S6 protein levels.. Low-dose mTOR inhibitor rapamycin prevented an additional increase in proteinuria and protected kidney function in a model of IgAN. Rapamycin directly or indirectly interfered with multiple key pathways in the progression of IgAN to end-stage renal disease: (1) reduced the deposition of IgA and inhibited cell proliferation; (2) decreased the expression of fibrosis markers α-SMA and type III collagen, and (3) downregulated the expression of the profibrotic growth factors platelet-derived growth factor and TGF-β1. The expression of p-S6 was significantly elevated in IgAN rats.. The mTOR pathway was activated in IgAN rats and the early application of low-dose mTOR inhibitor rapamycin may slow the renal injury of IgAN in rats.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Disease Progression; Fibrosis; Glomerulonephritis, IGA; Immunosuppressive Agents; Kidney; Kidney Glomerulus; Liver; Male; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

The aim of this study was to determine the effect of everolimus and tacrolimus pretreatments on renal morphology and function in a rat ischemia reperfusion (I/R) model. Twenty-eight male Sprague-Dawley rats were randomly assigned to saline + sham operation, saline + I/R (IR), tacrolimus + I/R (TRL + I/R) and everolimus + I/R (ERL + I/R) groups. Saline and active treatments were administered intraperitoneally for seven consecutive days before the surgery. The suprarenal aorta was clamped to achieve warm ischemia, except in the sham group. Right nephrectomy was performed in all animals and histology was examined. Renal function was assessed on post-operative Day 7 by Tc-99m dimercaptosuccinic acid (DMSA) scintigraphy, glomerular filtration rate (GFR) and serum biochemistry. Both everolimus and tacrolimus preserved serum creatinine and blood urea nitrogen levels, but only everolimus preserved GFR (0.74 ± 0.36, 1.20 ± 0.37 and 2.24 ± 0.32 mL/min for I/R, TRL + I/R and ERL + I/R, respectively, P < 0.001). %ID values for sham, I/R, TRL + I/R and ERL + I/R were 55 ± 3, 47 ± 4, 45 ± 6 and 62 ± 7 (P < 0.001). On histologic evaluation, ERL + I/R showed less tubular damage and necrosis than I/R, as well as TRL + I/R. Within the confines of this rat warm ischemia model, everolimus pre-treatment was useful in preserving renal function following I/R injury. The possibility of using everolimus as a pre-conditioning agent for I/R injury in kidney transplantation should be further explored.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Everolimus; Glomerular Filtration Rate; Immunosuppressive Agents; Ischemic Preconditioning; Kidney; Kidney Transplantation; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Tacrolimus; Warm Ischemia

Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease affecting 4-8% of men older than 60 years. No pharmacologic strategies limit disease progression, aneurysm rupture, or aneurysm-related death. We examined the ability of rapamycin to limit the progression of established experimental AAAs.. AAAs were created in 10-12-week-old male C57BL/6J mice via the porcine pancreatic elastase (PPE) infusion method. Beginning 4 days after PPE infusion, mice were treated with rapamycin (5 mg/kg/day) or an equal volume of vehicle for 10 days. AAA progression was monitored by serial ultrasound examination. Aortae were harvested for histological analyses at sacrifice.. Three days after PPE infusion, prior to vehicle or rapamycin treatment, aneurysms were enlarging at an equal rate between groups. In the rapamycin group, treatment reduced aortic enlargement by 38%, and 53% at 3 and 10 days, respectively. On histological analysis, medial elastin and smooth muscle cell populations were relatively preserved in the rapamycin group. Rapamycin treatment also reduced mural macrophage density and neoangiogenesis.. Rapamycin limits the progression of established experimental aneurysms, increasing the translational potential of mechanistic target of rapamycin-related AAA inhibition strategies.

Topics: Animals; Aortic Aneurysm, Abdominal; Disease Models, Animal; Disease Progression; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Sirolimus; Treatment Outcome

Acute lung injury (ALI) is characterized by pulmonary inflammation and edema. Innate immune cells (e.g., neutrophils and macrophages) are major contributors to inflammation in ALI. Less is known regarding the role of T cells. We examined the effects of rapamycin on inflammation in a LPS-induced murine model of ALI. Rapamycin was administered before and after initiation of injury. Inflammatory parameters, including bronchoalveolar lavage cell counts, T cell surface markers (i.e., cytotoxic T lymphocyte antigen 4 [CTLA4] and fork head-winged helix transcription factor [Foxp3]), T cell activation (CD69), IL-6, and IL-10 were analyzed. Rapamycin significantly decreased inflammatory parameters and decreased Foxp3, CTLA4, and CD69 in CD4(+) T cells. Rapamycin administration before or after the onset of lung injury, as well as systemically or by pulmonary routes, ameliorates inflammation in ALI.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Biomarkers; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cells, Cultured; CTLA-4 Antigen; Cytokines; Disease Models, Animal; Female; Immunity, Innate; Inflammation Mediators; Lipopolysaccharides; Lung; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Pneumonia; Sirolimus; T-Lymphocytes; Time Factors

The utility of animal models for the prediction of drug-eluting stent (DES) efficacy in human clinical trials is still unclear. The familial hypercholesterolemic swine (FHS) model has been shown to induce a human-like neointimal response to bare metal stent (BMS) implantation. However, its utility to discriminate efficacy signals following DES implantation is unknown. In this study, we aimed to test the efficacy and healing response of several everolimus-eluting stent (EES) platforms in the coronary territory of the FHS.. A total of 19 EES platforms (SYNERGY=6, SYNERGY½-dose=7, and PROMUS Element=6) and an identical BMS control (Element=6) were implanted into the coronary arteries of nine FHS. All implants were performed under intravascular ultrasound guidance using a 1.2 : 1 overstretch ratio. At 30 days, the vascular response to the implant was evaluated by quantitative coronary angiography, optical coherence tomography, and histology.. At 28 days, all EES platforms showed a significant decrease in angiographic late lumen loss (between 27 and 37%) compared with the BMS control group. This finding was confirmed both by optical coherence tomography (mean neointimal thickness=28-42% reduction) and by histology (mean neointimal thickness=44-55% reduction). All EES platforms showed similar degrees of neointimal inhibition. The presence of moderate to severe para-strut inflammation was observed in 83% of the stent sections in the BMS group compared with 28.6% in the SYNERGY½-dose group and 0% in the SYNERGY and PROMUS groups (P=0.0002). There was a 68-95% reduction in MMP9 expression in the media in all EES platforms compared with the BMS controls. The presence of mild to moderate para-strut fibrin deposits ranged from 66.7 to 83.4% in all EES platforms compared with 16.7% in the EBMS group.. The FHS coronary injury model showed the efficacy of several EES platforms compared with an identical BMS control. Everolimus eluted from different polymeric platforms showed lower levels of inflammation and slightly higher fibrin deposits compared with BMS controls.

Topics: Animals; Blood Vessel Prosthesis Implantation; Coronary Angiography; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Humans; Hyperlipoproteinemia Type II; Immunosuppressive Agents; Male; Models, Cardiovascular; Neointima; Polymers; Sirolimus; Swine; Tomography, Optical Coherence; Treatment Outcome; Ultrasonography, Interventional

Both experimental and clinical studies have suggested that any potential treatment of polycystic kidney disease (PKD) should start early and last for a long time to be effective, with unavoidable side reactions and considerable costs. The aim of the present study was to test how low doses of rapamycin (RAPA; 0.15 mg/kg ip for 4 days/wk), tolvaptan (TOLV; 0.005% in diet), or AEZ-131 (AEZ; a novel ERK inhibitor, 30 mg/kg for 3 days/wk by gavage), alone and in association, affect the progression of polycystic renal disease in PCK rats. Rats were treated for 8 wk starting at 4-6 wk of age. The efficacy of low doses of such drugs in inhibiting their respective targets was confirmed by immunoblot experiments. Compared with rats in the control (CON) group, RAPA treatment caused a significant reduction in cyst volume density (CVD; -19% vs. the CON group) and was numerically similar to that in TOLV-treated rats (-18%, not significiant), whereas AEZ treatment was not effective. RAPA + TOLV treatment resulted in a significantly lower CVD (-49% vs. the CON group) and was associated with a striking decrease in cAMP response element-binding protein phosphorylation, and similar data were detected in RAPA + AEZ-treated rats (-42%), whereas TOLV + AEZ treatment had virtually no effect. RAPA administration significantly lessened body weight gain, whereas TOLV administration resulted a mild increase in diuresis and a significant increase in cAMP urinary excretion. Histological data of tubular proliferation were in full agreement with CVD data. In conclusion, this study demonstrates that the association of low doses of RAPA, TOLV, and AEZ slows the progression of PKD with limited side effects, suggesting the use of combined therapies also in clinical trials.

Topics: Animals; Benzazepines; Cyclic AMP; Disease Models, Animal; Disease Progression; Drug Therapy, Combination; Enzyme Inhibitors; Kidney; Male; MAP Kinase Signaling System; Polycystic Kidney Diseases; Rats; Rats, Inbred Strains; Rats, Mutant Strains; Rats, Sprague-Dawley; Sirolimus; Tolvaptan; TOR Serine-Threonine Kinases; Treatment Outcome

The role played by autophagy after ischemia/reperfusion (I/R) in the retina remains unknown. Our study investigated whether ischemic injury in the retina, which causes an energy crisis, would induce autophagy. Retinal ischemia was induced by elevation of the intraocular pressure and modulation of autophagic markers was analyzed at the protein levels in an early and late phase of recovery. Following retinal ischemia an increase in LC3BII was first observed in the early phase of recovery but did not stay until the late phase of recovery. Post-ischemic induction of autophagy by intravitreal rapamycin administration did not provide protection against the lesion induced by the ischemic stress. On the contrary, an increase in the number of apoptotic cells was observed following I/R in the rapamycin treated retinas.

Topics: Animals; Apoptosis; Autophagy; Disease Models, Animal; Immunosuppressive Agents; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retina; Retinal Diseases; Sirolimus

Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

Topics: Angelman Syndrome; Animals; Carrier Proteins; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hemizygote; Hippocampus; Homer Scaffolding Proteins; Immunosuppressive Agents; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Pyridines; Receptor, Metabotropic Glutamate 5; Signal Transduction; Sirolimus; Ubiquitin-Protein Ligases

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that occur spontaneously, or from benign plexiform neurofibromas, in the context of the genetic disorder Neurofibromatosis Type 1 (NF1). The current standard treatment includes surgical resection, high-dose chemotherapy, and/or radiation. To date, most targeted therapies have failed to demonstrate effectiveness against plexiform neurofibromas and MPNSTs. Recently, several studies suggested that the mTOR and MAPK pathways are involved in the formation and progression of MPNSTs. Everolimus (RAD001) inhibits the mTOR and is currently FDA approved for several types of solid tumors. PD-0325901 (PD-901) inhibits MEK, a component of the MAPK pathway, and is currently in clinical trials. Here, we show in vitro than MPNST cell lines are more sensitive to inhibition of cellular growth by Everolimus and PD-901 than immortalized human Schwann cells. In combination, these drugs synergistically inhibit cell growth and induce apoptosis. In two genetically engineered mouse models of MPNST formation, modeling both sporadic and NF1-associated MPNSTs, Everolimus, or PD-901 treatment alone each transiently reduced tumor burden and size, and extended lifespan. However, prolonged treatment of each single agent resulted in the development of resistance and reactivation of target pathways. Combination therapy using Everolimus and PD-901 had synergistic effects on reducing tumor burden and size, and increased lifespan. Combination therapy allowed persistent and prolonged reduction in signaling through both pathways. These data suggest that co-targeting mTOR and MEK may be effective in patients with sporadic or NF1-associated MPNSTs.

Topics: Animals; Apoptosis; Benzamides; Blotting, Western; Cell Proliferation; Cells, Cultured; Diphenylamine; Disease Models, Animal; Drug Synergism; Everolimus; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; Neoplasm Grading; Neurilemmoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Schwann Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Abnormal levels of mammalian target of rapamycin (mTOR) signaling have been recently implicated in the pathophysiology of neurodegenerative diseases, such as Alzheimer's disease (AD). However, the implication of mTOR in diabetes mellitus (DM)-related cognitive dysfunction still remains unknown. In the present study, we found that phosphorylated mTOR at Ser2448, phosphorylated p70S6K at Thr421/Ser424 and phosphorylated tau at Ser396 were significantly increased in the hippocampus of streptozotocin (STZ)-induced diabetic mice when compared with control mice. A low dose of rapamycin was used to elucidate the role of mTOR signaling in DM-related cognitive deficit. Rapamycin restored abnormal mTOR/p70S6K signaling and attenuated the phosphorylation of tau protein in the hippocampus of diabetic mice. Furthermore, the spatial learning and memory function of diabetic mice significantly impaired compared with control mice, was also reversed by rapamycin. These findings indicate that mTOR/p70S6K signaling pathway is hyperactive in the hippocampus of STZ-induced diabetic mice and inhibiting mTOR signaling with rapamycin prevents the DM-related cognitive deficits partly through attenuating the hyperphosphorylation of tau protein.

Topics: Animals; Cognition Disorders; Diabetes Mellitus, Experimental; Disease Models, Animal; Hippocampus; Maze Learning; Mice; Phosphorylation; Signal Transduction; Sirolimus; tau Proteins; TOR Serine-Threonine Kinases

The "mechanistic target of rapamycin" (mTOR) is a central controller of growth, proliferation and/or motility of various cell-types ranging from adipocytes to immune cells, thereby linking metabolism and immunity. mTOR signaling is overactivated in obesity, promoting inflammation and insulin resistance. Therefore, great interest exists in the development of mTOR inhibitors as therapeutic drugs for obesity or diabetes. However, despite a plethora of studies characterizing the metabolic consequences of mTOR inhibition in rodent models, its impact on immune changes associated with the obese condition has never been questioned so far. To address this, we used a mouse model of high-fat diet (HFD)-fed mice with and without pharmacologic mTOR inhibition by rapamycin. Rapamycin was weekly administrated to HFD-fed C57BL/6 mice for 22 weeks. Metabolic effects were determined by glucose and insulin tolerance tests and by indirect calorimetry measures of energy expenditure. Inflammatory response and immune cell populations were characterized in blood, adipose tissue and liver. In parallel, the activities of both mTOR complexes (e. g. mTORC1 and mTORC2) were determined in adipose tissue, muscle and liver. We show that rapamycin-treated mice are leaner, have enhanced energy expenditure and are protected against insulin resistance. These beneficial metabolic effects of rapamycin were associated to significant changes of the inflammatory profiles of both adipose tissue and liver. Importantly, immune cells with regulatory functions such as regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were increased in adipose tissue. These rapamycin-triggered metabolic and immune effects resulted from mTORC1 inhibition whilst mTORC2 activity was intact. Taken together, our results reinforce the notion that controlling immune regulatory cells in metabolic tissues is crucial to maintain a proper metabolic status and, more generally, comfort the need to search for novel pharmacological inhibitors of the mTOR signaling pathway to prevent and/or treat metabolic diseases.

Topics: Adipose Tissue; Animals; Cell Proliferation; Dietary Fats; Disease Models, Animal; Female; Immunosuppressive Agents; Insulin Resistance; Liver; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Myeloid Cells; Obesity; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Topics: Animals; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Everolimus; Humans; Hyperlipoproteinemia Type II; Male; Neointima; Sirolimus

Our previous study demonstrated that diabetes increases in-stent restenosis following rapamycin-eluting stent placement, which was defined as rapamycin resistance. However, the underlying mechanisms of rapamycin resistance remain to be determined. In the present study, male apolipoprotein E-deficient (ApoE-/-) mice were randomly divided into control and diabetic groups. Diabetes was induced by injecting streptozocin (STZ). The hyperglycemic state, defined as a fasting plasma glucose level >13 mmol/l, was maintained for 8 weeks. At the end of the administration, the plasma levels of triglycerides (TG) and total cholesterol (TC) were significantly elevated in the diabetic group compared with the control mice (all P<0.01). The present study revealed that diabetes increased the atherosclerotic plaque size of the aortic root (P<0.01) and the content of vascular smooth muscle cells (VSMCs) in the atherosclerotic lesion (P<0.01). Furthermore, the protein expression and phosphorylation of mammalian target of rapamycin (mTOR), 4E-binding protein 1 and ribosomal S6 kinase 1 (P<0.01) were significantly decreased in the diabetic mice compared with the control group. The decrease in the expression and phosphorylation of mTOR and its downstream kinases may be one of the molecular mechanisms underlying rapamycin resistance.

Topics: Animals; Apolipoproteins E; Diabetes Mellitus, Experimental; Disease Models, Animal; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Plaque, Atherosclerotic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/- 2 vs 56.5+/- 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.

Topics: Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Animals, Genetically Modified; Antibiotics, Antineoplastic; beta Catenin; Cell Differentiation; Cell Line; Colon; Colonic Polyps; Disease Models, Animal; Disease Progression; Epithelial Cells; Humans; Intestinal Mucosa; Mice; Sirolimus; SOX9 Transcription Factor; Wnt Signaling Pathway

Interstitial lung disease (ILD) is a well-known adverse effect of mammalian target of rapamycin (mTOR) inhibitors. However, it remains unknown how lung toxicities are induced by mTOR inhibitors. Here, we constructed a mouse model of mTOR inhibitor-induced ILD using temsirolimus and examined the pathogenesis of the disease. Male ICR mice were treated with an intraperitoneal injection of different doses of temsirolimus (3 or 30 mg·kg(-1)·wk(-1)) or vehicle. Temsirolimus treatment increased capillary-alveolar permeability and induced neutrophil infiltration and fibrinous exudate into the alveolar space, indicating alveolar epithelial and/or endothelial injury. It also induced macrophage depletion and the accumulation of excessive surfactant phospholipids and cholesterols. Alveolar macrophage depletion is thought to cause surfactant lipid accumulation. To further examine whether temsirolimus has cytotoxic and/or cytostatic effects on alveolar macrophages and alveolar epithelial cells, we performed in vitro experiments. Temsirolimus inhibited cell proliferation and viability in both alveolar macrophage and alveolar epithelial cells. Temsirolimus treatment caused some signs of pulmonary inflammation, including upregulated expression of several proinflammatory cytokines in both bronchoalveolar lavage cells and lung homogenates, and an increase in lymphocytes in the bronchoalveolar lavage fluid. These findings indicate that temsirolimus has the potential to induce alveolar epithelial injury and to deplete alveolar macrophages followed by surfactant lipid accumulation, resulting in pulmonary inflammation. This is the first study to focus on the pathogenesis of mTOR inhibitor-induced ILD using an animal model.

Topics: Animals; Cytokines; Disease Models, Animal; Lipid Metabolism; Macrophages, Alveolar; Male; Mice; Mice, Inbred ICR; Phospholipids; Pneumonia; Pulmonary Alveoli; Pulmonary Surfactants; Sirolimus

Cisplatin-induced ototoxicity affects a high percentage of new cancer patients worldwide. The detailed mechanism of cisplatin-induced ototoxicity is not completely understood. We investigated whether rapamycin could protect rats from cisplatin-induced ototoxicity.. Forty-eight male Wistar rats were randomly divided into six groups. Three groups were intraperitoneally (IP) infused with cisplatin at a dose of 16 mg/kg and immediately injected with either dimethylsulfoxide (DMSO), rapamycin, or chloroquine (CQ). The remaining three groups were treated with rapamycin, CQ, or saline alone. The auditory brainstem response (ABR) test was performed to detect the rats' hearing status. Serum was isolated to measure the level of the oxidative marker malondialdehyde (MDA), the basilar membrane was prepared to count the outer hair cell loss, and soft tissue samples extracted from the cochleae were lysed to analyze the microtubule-associated protein light chain 3 (LC3) and Beclin-1.. The rapamycin treatment significantly attenuated cisplatin-induced hearing loss, decreased oxidative stress, and alleviated the hair cell damage that was associated with the upregulation of the LC3-II/GAPDH ratio and increased Beclin-1 expression.. Our results demonstrated that rapamycin has an otoprotective effect; it attenuates cisplatin-induced ototoxicity, probably by attenuating oxidative damage and inducing autophagy.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cisplatin; Cochlea; Disease Models, Animal; Ear, Inner; Evoked Potentials, Auditory, Brain Stem; Hair Cells, Auditory, Outer; Hearing Loss, Sensorineural; Humans; Male; Malondialdehyde; Microtubule-Associated Proteins; Oxidative Stress; Rats; Rats, Wistar; Sirolimus

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1β compared with D-CSC. Using blocking antibodies, we verified that IL1β hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1β acts intracranially inducing IKKβ signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1β itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKβ signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1β secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

Topics: Animals; Cellular Senescence; Disease Models, Animal; Female; Humans; Mice; Mice, SCID; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Resveratrol; Signal Transduction; Sirolimus; Stem Cell Transplantation; Stilbenes

To assess the late postinterventional response to iliac stenting in atheromatous rabbits using the Xience V everolimus-eluting stent (Xience V EES; Abbott Vascular) and the Resolute zotarolimus-eluting stent (Resolute ZES; Medtronic Vascular) with the MultiLink Vision bare metal stent (BMS; Abbott Vascular) as a reference.. Xience V EES and Resolute ZES were developed to overcome shortcomings of first-generation DES.. Functional and microscopic changes were assessed by organ bath experiments and histopathologic examination. Gene expression was investigated using RT-PCR.. After 91 days, re-endothelialization was nearly complete (BMS: 93 ± 3%; Resolute ZES: 92 ± 2%; Xience V EES: 94 ± 3%; P = 0.10). Neointima thickness was similar in Resolute ZES (0.17 ± 0.08 mm) and BMS (0.17 ± 0.09 mm), and reduced in Xience V EES (0.03 ± 0.01 mm; P < 0.0001). Xience V EES had less peri-strut inflammation compared with BMS (P = 0.001) and Resolute ZES (P = 0.0001), while arterial segments distal to Xience V EES were more sensitive to acetylcholine than those distal to BMS and Resolute ZES (P = 0.02). Lectin-like oxidized receptor-1 was overexpressed in stented arteries (P < 0.001), whereas thrombomodulin was downregulated in Resolute ZES (P = 0.01) and BMS (P = 0.02) compared to unstented arteries of rabbits on regular chow. No significant changes were seen for vascular cell adhesion molecule-1, nitric oxide synthase 3, or endothelin-1.. At 3-month follow-up, nearly complete re-endothelialization was achieved for all stent groups. Xience V EES induced greater suppression of neointimal growth and peri-strut inflammation, higher vasorelaxation to acetylcholine, and expression of thrombomodulin at the level of unstented controls.

Topics: Acetylcholine; Angioplasty, Balloon; Animals; Atherosclerosis; Disease Models, Animal; Down-Regulation; Drug-Eluting Stents; Endothelium, Vascular; Everolimus; Iliac Artery; Inflammation; Neointima; Rabbits; Scavenger Receptors, Class E; Sirolimus; Thrombomodulin; Vasodilator Agents

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disease in the USA. In human ADPKD studies, sirolimus, a mammalian target of rapamycin complex 1 (mTORC1) inhibitor, had little therapeutic effect. While sirolimus robustly inhibits mTORC1, it has a minimal effect on mTOR complex 2 (mTORC2). Polycystic kidneys of Pkd2WS25/- mice, an orthologous model of human ADPKD caused by a mutation in the Pkd2 gene, had an early increase in pS6 (marker of mTORC1) and pAktSer(473) (marker of mTORC2). To investigate the effect of combined mTORC1 and 2 inhibition, Pkd2WS25/- mice were treated with an mTOR anti-sense oligonucleotide (ASO) that blocks mTOR expression thus inhibiting both mTORC1 and 2. The mTOR ASO resulted in a significant decrease in mTOR protein, pS6 and pAktSer(473). Pkd2WS25/- mice treated with the ASO had a normalization of kidney weights and kidney function and a marked decrease in cyst volume. The mTOR ASO resulted in a significant decrease in proliferation and apoptosis of tubular epithelial cells. To demonstrate the role of mTORC2 on cyst growth, Rictor, the functional component of mTORC2, was silenced in Madin-Darby canine kidney cell cysts grown in 3D cultures. Silencing Rictor significantly decreased cyst volume and expression of pAktSer(473). The decreased cyst size in the Rictor silenced cells was reversed by introduction of a constitutively active Akt1. In vitro, combined mTORC1 and 2 inhibition reduced cyst growth more than inhibition of mTORC1 or 2 alone. In conclusion, combined mTORC1 and 2 inhibition has therapeutic potential in ADPKD.

Topics: Animals; Carrier Proteins; Cell Proliferation; Disease Models, Animal; Dogs; Gene Expression Regulation; Genetic Therapy; Humans; Kidney; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; Oligonucleotides, Antisense; Polycystic Kidney Diseases; Rapamycin-Insensitive Companion of mTOR Protein; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Rapamycin has been reported to inhibit mesenchymal cell proliferation in a murine model of pulmonary fibrosis. In the present study, we examined the effects of rapamycin on vascular remodeling including intraluminal myofibroblast proliferation in a murine model of allergic vasculitis with eosinophil infiltration.. C57BL/6 mice were sensitized with ovalbumin (OVA) and alum. The positive controls were exposed to aerosolized OVA daily for 7 days. The other group of mice was administered with rapamycin (1mg/kg) intraperitoneally, in parallel with daily exposure to aerosolized OVA for 7 days. On the 3rd and 7th day, bronchoalveolar lavage (BAL) was performed and the lungs were excised for pathological analysis. Cell differentials were determined and concentrations of IL-4, IL-5, IL-13 and TGF-β in the BAL fluid (BALF) were measured. Semi-quantitative analysis of pathological changes in the pulmonary arteries was evaluated according to the severity of vasculitis.. The number of eosinophils in BALF was reduced significantly in the mice treated with rapamycin compared to the positive control. There was a significant decrease in the TGF-β concentration of the BALF in the rapamycin-treated group compared to that of the positive control. The pathological scores were reduced significantly in the rapamycin-treated group compared to the positive control group. Intraluminal myofibroblasts in pulmonary arteries were reduced dramatically in the rapamycin-treated group compared to the positive control group.. Rapamycin suppressed pulmonary vascular remodeling in a murine model of allergic vasculitis with eosinophil infiltration through reducing eosinophil infiltration and TGF-β production in the lung and inhibition against biological action of TGF-β.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Disease Progression; Eosinophils; Female; Humans; Hypersensitivity; Immunosuppressive Agents; Lung Diseases; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Artery; Sirolimus; Transforming Growth Factor beta; Vascular Remodeling; Vasculitis

Intimal hyperplasia produces restenosis (re-narrowing) of the vessel lumen following vascular intervention. Drugs that inhibit intimal hyperplasia have been developed, however there is currently no clinical method of perivascular drug-delivery to prevent restenosis following open surgical procedures. Here we report a poly(ε-caprolactone) (PCL) sheath that is highly effective in preventing intimal hyperplasia through perivascular delivery of rapamycin. We first screened a series of bioresorbable polymers, i.e., poly(lactide-co-glycolide) (PLGA), poly(lactic acid) (PLLA), PCL, and their blends, to identify desired release kinetics and sheath physical properties. Both PLGA and PLLA sheaths produced minimal (<30%) rapamycin release within 50days in PBS buffer. In contrast, PCL sheaths exhibited more rapid and near-linear release kinetics, as well as durable integrity (>90days) as evidenced in both scanning electron microscopy and subcutaneous embedding experiments. Moreover, a PCL sheath deployed around balloon-injured rat carotid arteries was associated with a minimum rate of thrombosis compared to PLGA and PLLA. Morphometric analysis and immunohistochemistry revealed that rapamycin-loaded perivascular PCL sheaths produced pronounced (85%) inhibition of intimal hyperplasia (0.15±0.05 vs 1.01±0.16), without impairment of the luminal endothelium, the vessel's anti-thrombotic layer. Our data collectively show that a rapamycin-loaded PCL delivery system produces substantial mitigation of neointima, likely due to its favorable physical properties leading to a stable yet flexible perivascular sheath and steady and prolonged release kinetics. Thus, a PCL sheath may provide useful scaffolding for devising effective perivascular drug delivery particularly suited for preventing restenosis following open vascular surgery.

Topics: Animals; Cardiovascular Agents; Carotid Artery Injuries; Carotid Stenosis; Cell Proliferation; Chemistry, Pharmaceutical; Delayed-Action Preparations; Disease Models, Animal; Drug Carriers; Hyperplasia; Kinetics; Linear Models; Male; Neointima; Polyesters; Rats; Rats, Sprague-Dawley; Sirolimus; Solubility; Technology, Pharmaceutical

Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.

Topics: Adrenal Cortex Diseases; Adrenocorticotropic Hormone; Animals; Apoptosis; bcl-Associated Death Protein; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Disease Models, Animal; Gene Knockout Techniques; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In a variety of neurodegenerative tauopathies including Alzheimer's disease, frontotemporal dementia and some types of Parkinson's disease, tau protein is abnormally hyperphosphorylated by several kinases and eventually aggregates to form neurofibrillary tangles, a neurotoxic pathological characteristic that closely correlates with cognitive impairments. Hence, targeting hyperphosphorylated tau protein has now been considered as a valid therapeutic approach for these neurodegenerative tauopathies. As a newly developed analog of rapamycin, temsirolimus was approved by the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of renal cell carcinoma. Recent findings suggested that temsirolimus also provided beneficial effects in animal models of Huntington's disease and spinocerebellar ataxia type 3, two neurodegenerative diseases caused by accumulation of aberrant proteins within brain. To date, the therapeutic potentials of temsirolimus in neurodegenerative tauopathies have not been determined. Herein, we demonstrated for the first time that temsirolimus treatment effectively enhanced autophagic clearance of hyperphosphorylated tau in okadaic acid-incubated SH-SY5Y cells and in brain of P301S transgenic mice. Meanwhile, we showed that inactivation of glycogen synthase kinase-3β, the most important tau kinase, might contribute to the temsirolimus-induced reduction of tau hyperphosphorylation in these two tauopathy models. More importantly, temsirolimus administration rescued spatial learning and memory impairments in P301S transgenic mice. These findings highlight temsirolimus administration as a potential therapeutic strategy for neurodegenerative tauopathies.

Topics: Animals; Autophagy; Brain; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Memory Disorders; Mice, Inbred C57BL; Mice, Transgenic; Neuroprotective Agents; Okadaic Acid; Phosphorylation; Sirolimus; Spatial Learning; Spatial Memory; tau Proteins; Tauopathies

Atopic dermatitis (AD), a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. Recently, rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, has been reported to play a critical role in immune responses and has emerged as an effective immunosuppressive drug. In this study, we assessed whether inhibition of mTOR signalling could suppress dermatitis in mice. Rapamycin was topically applied to inflamed skin in a murine AD model that was developed by repeated topical application of Dermatophagoides farina body (Dfb) extract antigen twice weekly for 7 weeks in NC/Nga mice. The efficacy of topical rapamycin treatment was evaluated immunologically and serologically. Topical application of rapamycin reduced inflammatory cell infiltration in the dermis, alleviated the increase of serum IgE levels and resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. In addition, increased mTOR phosphorylation in the lesional skin was observed in our murine AD model. Topical application of rapamycin ointment inhibited Dfb antigen-induced dermatitis in NC/Nga mice, promising a new therapy for atopic dermatitis.

Topics: Administration, Topical; Animals; Cytokines; Dermatitis, Atopic; Dermatophagoides farinae; Disease Models, Animal; Female; Immunosuppressive Agents; Mice; Mice, Mutant Strains; Ointments; Phosphorylation; Signal Transduction; Sirolimus; Skin; Tissue Extracts; TOR Serine-Threonine Kinases

Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.

Topics: Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Cardiomegaly; Cell Line; Disease Models, Animal; Enzyme Activation; Estrone; Gene Silencing; Heart Failure; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Mitochondrial Turnover; Multiprotein Complexes; Organ Size; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Ventricular Function

Preclinical evaluation of the vascular response of drug-eluting stents is limited especially in the setting of diabetes mellitus preventing the evaluation of changes in drug-eluting stent design and eluted drugs after clinical use.. Cultured human aortic endothelial cells were used to assess the differences between sirolimus and its analog, everolimus, in the setting of hyperglycemia on various cellular functions necessary for endothelial recovery. A diabetic rabbit model of iliac artery stenting was used to compare histological and morphometric characteristics of the vascular response to everolimus-eluting, sirolimus-eluting, and bare metal stent placement. Under hyperglycemic conditions, sirolimus impaired human aortic endothelial cell barrier function, migration, and proliferation to a greater degree compared with everolimus. In our in vivo model of diabetes mellitus, endothelialization at 28 days was significantly lower and endothelial integrity was impaired in sirolimus-eluting stent compared with both everolimus-eluting and bare metal stents. Neointimal area, uncovered struts, and fibrin deposition were significantly higher in sirolimus-eluting compared with everolimus-eluting and bare metal stents.. Use of everolimus-eluting stent results in improved vascular response in our preclinical models of diabetes mellitus.

Topics: Animals; Aorta; Cell Movement; Cells, Cultured; Diabetes Mellitus; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Everolimus; Fibrin; Humans; Hyperglycemia; Iliac Artery; Male; Neointima; Rabbits; Sirolimus

Glaucoma is a leading cause of irreversible blindness. Injury of retinal ganglion cells (RGCs) accounts for visual impairment of glaucoma. Here, we report rapamycin protects RGCs from death in experimental glaucoma model and the underlying mechanisms. Our results showed that treatment with rapamycin dramatically promote RGCs survival in a rat chronic ocular hypertension model. This protective action appears to be attributable to inhibition of neurotoxic mediators release and/or direct suppression of RGC apoptosis. In support of this mechanism, in vitro, rapamycin significantly inhibits the production of NO, TNF-α in BV2 microglials by modulating NF-κB signaling. In experimental animals, treatment with rapamycin also dramatically inhibited the activation of microglials. In primary RGCs, rapamycin was capable of direct suppression the apoptosis of primary RGCs induced by glutamate. Mechanistically, rapamycin-mediated suppression of RGCs apoptosis is by sparing phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity in cell and animal model. These results demonstrate that rapamycin is neuroprotective in experimental glaucoma, possibly via decreasing neurotoxic releasing and suppressing directly apoptosis of RGCs.

Topics: Animals; Cell Survival; Cells, Cultured; Chronic Disease; Disease Models, Animal; Female; Glaucoma; Neuroprotective Agents; Ocular Hypertension; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Sirolimus

Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor-positive (ER+) breast cancer, but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon.. We analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ breast cancer patients. We generated transgenic (MMTV-TGFα;A (y) /a) and orthotopic/syngeneic (A (y) /a) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole-genome transcriptome microarrays and protein analyses. We used a coculture system to examine the impact of adipocytes/adipokines on breast cancer cell proliferation. All statistical tests were two-sided.. Functional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes (P < .05) linked to cancer hallmarks. Gene enrichment analysis revealed enrichment of AKT-target genes (P = .04) and epithelial-mesenchymal transition genes (P = .03) in patients. Our obese mouse models demonstrated activation of the AKT/mTOR pathway in obesity-accelerated mammary tumor growth (3.7- to 7.0-fold; P < .001; n = 6-7 mice per group). Metformin or everolimus can suppress obesity-induced secretion of adipokines and breast tumor formation and growth (0.5-fold, P = .04; 0.3-fold, P < .001, respectively; n = 6-8 mice per group). The coculture model revealed that adipocyte-secreted adipokines (eg, TIMP-1) regulate adipocyte-induced breast cancer cell proliferation and invasion. Metformin suppress adipocyte-induced cell proliferation and adipocyte-secreted adipokines in vitro.. Adipokine secretion and AKT/mTOR activation play important roles in obesity-accelerated breast cancer aggressiveness in addition to hyperinsulinemia, estrogen signaling, and inflammation. Metformin and everolimus have potential for therapeutic interventions of ER+ breast cancer patients with obesity.

Topics: Adipocytes; Adipokines; Aged; Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Disease Models, Animal; Everolimus; Female; Humans; Kaplan-Meier Estimate; Metformin; Mice; Mice, Transgenic; Middle Aged; Obesity; Postmenopause; Prospective Studies; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome

Castration-resistant prostate cancer is an incurable heterogeneous disease that is characterized by a complex multistep process involving different cellular and biochemical changes brought on by genetic and epigenetic alterations. These changes lead to the activation or overexpression of key survival pathways that also serve as potential therapeutic targets. Despite promising preclinical results, molecular targeted therapies aimed at such signaling pathways have so far been dismal. In the present study, we used a PTEN-deficient mouse model of prostate cancer to show that plasticity in castration-resistant tumors promotes therapeutic escape. Unlike castration-naïve tumors which depend on androgen receptor and PI3K/AKT signal activation for growth and survival, castration-resistant tumors undergo phenotypic plasticity leading to increased intratumoral heterogeneity. These tumors attain highly heterogeneous phenotypes that are characterized by cancer cells relying on alternate signal transduction pathways for growth and survival, such as mitogen-activated protein kinase and janus kinase/signal transducer and activator of transcription, and losing their dependence on PI3K signaling. These features thus enabled castration-resistant tumors to become insensitive to the therapeutic effects of PI3K/AKT targeted therapy. Overall, our findings provide evidence that androgen deprivation drives phenotypic plasticity in prostate cancer cells and implicate it as a crucial contributor to therapeutic resistance in castration-resistant prostate cancer. Therefore, incorporating intratumoral heterogeneity in a dynamic tumor model as a part of preclinical efficacy determination could improve prediction for response and provide better rationale for the development of more effective therapies.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Butadienes; Carcinogenesis; Cell Proliferation; Combined Modality Therapy; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Everolimus; Humans; Male; Mice; Mice, Transgenic; Molecular Targeted Therapy; Nitriles; Orchiectomy; Phenotype; Prostate; Prostatic Neoplasms, Castration-Resistant; PTEN Phosphohydrolase; Receptors, Androgen; Sirolimus

Nonalcoholic fatty liver disease (NAFLD), the accumulation of lipid within hepatocytes, is increasing in prevalence. Increasing fructose consumption correlates with this increased prevalence, and rodent studies directly support fructose leading to NAFLD. The mechanisms of NAFLD and in particular fructose-induced lipid accumulation remain unclear, although there is evidence for a role for endoplasmic reticulum (ER) stress and oxidative stress. We have evidence that NAFLD models demonstrate activation of the target of rapamycin complex 1 (Torc1) pathway. We set out to assess the contribution of ER stress, oxidative stress, and Torc1 up-regulation in the development of steatohepatitis in fructose-treated larval zebrafish. Zebrafish were treated with fructose or glucose as a calorie-matched control. We also treated larvae with rapamycin, tunicamycin (ER stress), or valinomycin (oxidative stress). Fish were stained with oil red O to assess hepatic lipid accumulation, and we also performed quantitative polymerase chain reaction (qPCR)and western blot analysis. We performed immunostaining on samples from patients with NAFLD and nonalcoholic steatohepatitis (NASH). Treatment with fructose induced hepatic lipid accumulation, mitochondrial abnormalities, and ER defects. In addition, fructose-treated fish showed activation of inflammatory and lipogenic genes. Treatment with tunicamycin or valinomycin also induced hepatic lipid accumulation. Expression microarray studies of zebrafish NAFLD models showed an elevation of genes downstream of Torc1 signaling. Rapamycin treatment of fructose-treated fish prevented development of hepatic steatosis, as did treatment of tunicamycin- or valinomycin-treated fish. Examination of liver samples from patients with hepatic steatosis demonstrated activation of Torc1 signaling.. Fructose treatment of larval zebrafish induces hepatic lipid accumulation, inflammation, and oxidative stress. Our results indicate that Torc1 activation is required for hepatic lipid accumulation across models of NAFLD, and in patients.

Topics: Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Endoplasmic Reticulum Stress; Fatty Liver; Fructose; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation; Zebrafish

The underlying causes of endometrial cancer (EMC) are poorly understood, and treatment options for patients with advanced stages of the disease are limited. Mutations in the phosphatase and tensin homologue gene are frequently detected in EMC. Cyclooxygenase 2 (Cox2) and mammalian target of rapamycin complex 1 (mTORC1) are known downstream targets of the phosphatase and tensin homologue protein, and their activities are up-regulated in EMC. However, it is not clear whether Cox2 and mTORC1 are crucial players in cancer progression or whether they work in parallel or cooperatively. In this study, we used a Cox2 inhibitor, celecoxib, and an mTORC1 inhibitor, rapamycin, in mouse models of EMC and in human EMC cell lines to explore the interactive roles of Cox2 and mTORC1 signaling. We found that a combined treatment with celecoxib and rapamycin markedly reduces EMC progression. We also observed that rapamycin reduces Cox2 expression, whereas celecoxib reduces mTORC1 activity. These results suggest that Cox2 and mTORC1 signaling is cross-regulated and cooperatively exacerbate EMC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Carcinoma; Celecoxib; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Cyclosporine, tacrolimus and sirolimus are commonly used in renal transplant recipients to prevent rejection. Various adverse effects of these agents on the multiple organ system have been reported clinically. However, animal studies are necessary to determine and compare these effects on individual organ given the presence of multiple confounding factors and multi-pharmacy in clinical settings. In a physiologically and clinically relevant rat model of unilateral nephrectomy, the long-term impacts of commonly used immunosuppressants at doses equivalent to the therapeutic levels used for post-renal transplant patients on hepatic function and histological changes of the liver were examined. Cyclosporine induced significant hepatocellular injury, impairment of synthetic function of the liver, hyperbilirubinemia and cholestasis, and dyslipidemia accompanied by profound histological changes of hepatic structures on both light and electron microscopic examinations. On the other hand, neither tacrolimus nor sirolimus developed any hepatotoxic effects except for more remarkable dyslipidemia was observed in animals treated with sirolimus. Our study indicates that long-term administration of commonly used immunosuppressants has various impacts on biochemical parameters as well as histological alterations of the liver even at therapeutic levels. These data may therefore provide useful information for judicious selection of immunosuppressive agents based on different clinical settings.

Topics: Animals; Cyclosporine; Disease Models, Animal; Graft Rejection; Immunosuppressive Agents; Kidney Transplantation; Liver; Male; Nephrectomy; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus

Autophagy is significant in myocardial ischemia-reperfusion (IR) injury. Ulinastatin has been demonstrated to protect cardiomyocytes against IR through inducing anti-inflammatory effects. However, whether ulinastatin has an anti‑autophagic effect is yet to be elucidated. The present study aimed to investigate the effect of ulinastatin on the regulation of autophagy during IR injury. Cardiomyocytes of neonatal rats were randomly divided into control, hypoxia-reoxygenation (HR) and ulinastatin groups. In order to investigate whether mammalian target of rapamycin (mTOR) is involved in mediating the protective effect of ulinastatin, cells were treated with the mTOR inhibitor, rapamycin 30 min prior to ulinastatin treatment. To demonstrate the anti-autophagic effect of ulinastatin in vivo, a rat IR model was established. Ulinastatin (1x104 U/kg body weight) was administered 30 min prior to the induction of IR via peritoneal injection. Light chain 3 (LC3), phosphorylated (p)‑mTOR, p‑protein kinase B (Akt) and p‑P70S6 kinase (p‑P70S6K) protein expression were assessed using western blot analysis. In addition, cell vitality, myocardial infarct size and lactate dehydrogenase (LDH) levels were measured. LC3‑Ⅱ protein expression was found to be downregulated, while p‑Akt, p‑mTOR and p‑P70S6K protein expression were observed to be upregulated by ulinastatin. In addition, cell vitality was found to increase and LDH was observed to decrease in the ulinastatin group compared with the HR group in vitro. Furthermore, rapamycin was found to attenuate the myocardial protective effect that is induced by ulinastatin. In vivo, ulinastatin was found to downregulate LC3‑Ⅱ protein expression, and reduce myocardium infarct size and LDH serum levels. These findings indicate that ulinastatin exhibits a myocardial protective effect against IR injury by regulating autophagy through mTOR activation.

Topics: Animals; Autophagy; Cells, Cultured; Disease Models, Animal; Down-Regulation; Glycoproteins; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Phosphorylation; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

Topics: Adolescent; Age Factors; Animals; Autistic Disorder; Autophagy; Child; Child, Preschool; Dendritic Spines; Disease Models, Animal; Exploratory Behavior; Female; Humans; Immunosuppressive Agents; Male; Mice; Mice, Transgenic; Neurons; Sirolimus; Synapses; Temporal Lobe; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Young Adult

Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were first treated with a single intraperitoneal injection of rapamycin or vehicle and then submitted to a second intraperitoneal administration of glucose 4 h later. Our results revealed that intraperitoneal administration of glucose induced hyperglycemia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish at 8 and 17 h, whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle-treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt on Thr-308 or Ser-473. Despite the lack of significant variation in phosphoenolpyruvate carboxykinase mRNA abundance, mRNA abundance for glucokinase (GK), glucose 6-phosphatase (G6Pase) I and II, and fructose 1,6-bisphosphatase (FBPase) was reduced by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppression of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1-dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout.

Topics: Animals; Blood Glucose; Disease Models, Animal; Fish Proteins; Fructose-Bisphosphatase; Glucokinase; Gluconeogenesis; Hyperglycemia; Liver; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Oncorhynchus mykiss; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Chronic inflammation is an underlying risk factor for colorectal cancer. No direct evidence has proven that inflammation in the colon promotes carcinogenesis. STAT3 plays an important role in the development of colitis-associated colorectal cancer (CAC). There is crosstalk between the mammalian target of rapamycin complex 1 (mTORC1) and the STAT3 pathways. The aim of the present study was to confirm that colitis promotes CAC and if so, to explore the function of the STAT3 and mTORC1 pathways in CAC. C57BL/6 mice were treated with axozymethane (AOM) and dextran sulfate sodium (DSS) to induce CAC. By varying the concentration of DSS (0, 1 and 2% respectively), we mimicked the CAC model with different degrees of inflammation and determined the risk of carcinogenesis. Expression of the STAT3 and mTORC1 pathways was detected. Finally, rapamycin, an mTORC1 inhibitor, was used to treat the CAC model. Tumor load, protein and gene expression of chemokines were determined. The multiplicity and tumor load of the high inflammation group were higher than those of the low inflammation group. Immunohistochemical staining and western blot analysis revealed that activation of the STAT3 and mTORC1 pathways increased gradually in the inflammation tissues and tumors. When we treated the mice with rapamycin, the tumor incidence, multiplicity and tumor load decreased. In addition, rapamycin widely suppressed the expression of pro‑inflammatory and anti-inflammatory chemokines in the tissues, including tumor necrosis factor-α, interferon-γ, IL-6, IL-10 and IL-12α. In conclusion, inflammation promotes the development of CAC via the STAT3 and mTORC1 pathways, which may be a viable treatment strategy for the chemoprevention of CAC.

Topics: Animals; Antineoplastic Agents; Azoxymethane; Cell Transformation, Neoplastic; Colitis; Colorectal Neoplasms; Cytokines; Dextran Sulfate; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

The success of immunotherapeutic approaches targeting glioblastoma multiforme (GBM) demands a robust antiglioma T-cell cytotoxic and memory response. Recent evidence suggests that rapamycin regulates T-cell differentiation. Herein, we tested whether administration of rapamycin could enhance the efficacy of immunotherapy utilizing Fms-like tyrosine kinase 3 ligand (Ad-Flt3L) and thymidine kinase/ganciclovir (Ad-TK/GCV). Using the refractory rat RG2 glioma model, we demonstrate that administration of rapamycin with Ad-Flt3L + Ad-TK/GCV immunotherapy enhanced the cytotoxic activity of antitumor CD8(+) T cells. Rats treated with rapamycin + Ad-Flt3L + Ad-TK/GCV exhibited massive reduction in the tumor volume and extended survival. Rapamycin administration also prolonged the survival of Ad-Flt3L + Ad-TK/GCV-treated GL26 tumor-bearing mice, associated with an increase in the frequency of tumor-specific and IFNγ(+) CD8(+) T cells. More importantly, rapamycin administration, even for a short interval, elicited a potent long-lasting central memory CD8(+) T-cell response. The enhanced memory response translated to an increased frequency of tumor-specific CD8(+) T cells within the tumor and IFNγ release, providing the mice with long-term survival advantage in response to tumor rechallenge. Our data, therefore, point to rapamycin as an attractive adjuvant to be used in combination with immunotherapy in a phase I clinical trial for GBM.

Topics: Adenoviridae; Animals; Antigens; Antigens, Surface; Cell Line, Tumor; Cytokines; Dendritic Cells; Disease Models, Animal; Ganciclovir; Genetic Therapy; Genetic Vectors; Glioblastoma; Glioma; Immunologic Memory; Immunophenotyping; Immunotherapy; Membrane Proteins; Mice; Rats; Signal Transduction; Sirolimus; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Thymidine Kinase; TOR Serine-Threonine Kinases

In the central nervous system, demyelinating diseases, such as multiple sclerosis, result in devastating long-term neurologic damage, in part because of the lack of effective remyelination in the adult human brain. One model used to understand the mechanisms regulating remyelination is cuprizone-induced demyelination, which allows investigation of remyelination mechanisms in adult animals following toxin-induced demyelination. Unfortunately, the degree of demyelination in the cuprizone model can vary, which complicates understanding the process of remyelination. Previous work in our laboratory demonstrated that the Akt/mTOR pathway regulates active myelination. When given to young postnatal mice, the mTOR inhibitor, rapamycin, inhibits active myelination. In the current study, the cuprizone model was modified by the addition of rapamycin during cuprizone exposure. When administered together, cuprizone and rapamycin produced more complete demyelination and provided a longer time frame over which to investigate remyelination than treatment with cuprizone alone. The consistency in demyelination will allow a better understanding of the mechanisms initiating remyelination. Furthermore, the slower rate of remyelination provides a longer window of time in which to investigate the diverse contributing factors that regulate remyelination. This new model of cuprizone-induced demyelination could potentially aid in identification of new therapeutic targets to enhance remyelination in demyelinating diseases.

Topics: Analysis of Variance; Animals; Basic Helix-Loop-Helix Transcription Factors; Body Weight; Brain; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Immunosuppressive Agents; Ki-67 Antigen; Male; Mice; Mice, Inbred C57BL; Monoamine Oxidase Inhibitors; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Receptor, Platelet-Derived Growth Factor alpha; Sirolimus

Previous studies have demonstrated that autophagy is involved in the pathogenesis of human cytomegalovirus (HCMV) infection. However, whether autophagy is regulated by murine cytomegalovirus (MCMV) infection has not yet been investigated. The purpose of these studies was to determine how autophagy is affected by MCMV infection of the retinal pigment epithelial (RPE) cells and whether there is a functional relationship between autophagy and apoptosis; and if so, how regulation of autophagy impacts apoptosis.. RPE cells were isolated from C57BL/6 mice and infected with MCMV K181. The cells were cultured in medium containing rapamycin, chloroquine, or ammonium chloride. Green fluorescent protein-light chain 3 (GFP-LC3) plasmid was transfected to RPE cells, and the GFP-LC3 positive puncta were counted. Electron microscopic (EM) images were taken to visualize the structure of the autophagic vacuoles. Western blot was performed to detect the expression of related proteins. Trypan blue exclusion assay was used to measure the percentage of viable cells.. Although the LC3B-II levels consistently increased during MCMV infection of RPE cells, administration of chloroquine or ammonium chloride increased LC3B-II expression only at the early stage of infection (6 h post-inoculation [p.i.] and 12 h p.i.), not at or after 24 h p.i. The punctate autophagic vacuoles in the GFP-LC3 transfected RPE cells were counted using light microscopy or by EM examination, the number of autophagic vacuoles was significantly increased in the MCMV-infected RPE cells compared to the uninfected controls. Compared to untreated MCMV-infected control cells, rapamycin treatment resulted in a significant decrease in the cleaved caspase 3 levels as well as a significant decrease in the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR and in the ratio of phosphorylated P70S6K to total P70S6K. In contrast, chloroquine treatment resulted in a significant increase in the cleaved caspase 3 levels in the MCMV-infected RPE cells.. Autophagic vacuole accumulation was detected during MCMV infection of RPE cells. In contrast, autophagic flux was greatly decreased at or after 24 h p.i. The results suggest that MCMV might have a strategy for inhibiting or blocking autophagy activity by targeting a later autophagy process, such as the formation of autolysosomes or degradation of their content. Our data also suggest that there is a functional relationship between autophagy and apoptosis, which plays an important role during MCMV infection of the RPE.

Topics: Animals; Apoptosis; Autophagy; Caspase 3; Chloroquine; Cytomegalovirus Infections; Disease Models, Animal; Green Fluorescent Proteins; Herpesviridae Infections; Humans; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Muromegalovirus; Recombinant Fusion Proteins; Retinal Pigment Epithelium; Sirolimus; Transfection; Vacuoles

The immunosuppressant drug rapamycin was reported to have an antiaging activity, which was attributed to the TORC1 inhibition that inhibits cell proliferation and increases autophagy. However, rapamycin also exhibits a number of harmful adverse effects. Whether rapamycin can be developed into an antiaging agent remains unclear.. We demonstrated that rapamycin at micro-doses (below the TORC1 inhibiting concentration) exhibits a cell-protective activity: (1) It protects cultured neurons against neurotoxin MPP(+) and H2O2. (2) It increases survival time of neuron in culture. (3) It maintains the nonproliferative state of cultured senescent human fibroblasts and prevents cell death induced by telomere dysfunction. (4) In animal models, it decreased the cerebral infarct sizes induced by acute ischemia and dramatically extended the life span of stroke prone spontaneously hypertensive rats (SHR-SPs).. We propose that rapamycin at micro-dose can be developed into an antiaging agent with a novel mechanism.

Topics: Aging; Animals; beta-Galactosidase; Brain Infarction; Brain Ischemia; Cell Death; Cell Line, Transformed; Cerebral Cortex; Disease Models, Animal; Fibroblasts; Humans; Hydrogen Peroxide; Immunosuppressive Agents; Male; Mice, Inbred C57BL; Phosphorylation; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Tacrolimus

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/- mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2-/-MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived "mesenchymal" cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.

Topics: Adipocytes; Angiomyolipoma; Animals; Cell Line; Cluster Analysis; Disease Models, Animal; Enzyme Activation; Female; Gene Expression Profiling; Gene Expression Regulation; Gene Knockout Techniques; Humans; Lung; Lymphangioleiomyomatosis; Mice; Phospholipases A2; Sirolimus; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Up-Regulation

Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.. Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).. Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.. These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Topics: Animals; Cartilage, Articular; Disease Models, Animal; Immunosuppressive Agents; Injections, Intra-Articular; Male; Mice; Osteoarthritis; Sirolimus

Low-dose IL-2 administration can control autoimmunity by specifically activating CD4(+) Foxp3(+) regulatory T cells (Tregs). Here, we studied IL-2-based immunotherapy in experimental graft-versus-host disease (GVHD). IL-2 administration to donor mice induced a dose-dependent expansion of Tregs in the graft but was insufficient to control GVHD. IL-2 administration to allogeneic-grafted recipient mice activated T-conventional cells (Tcons) and did not prevent GVHD. This loss of IL-2 selectivity toward Tregs was explained by an IL-2-induced increase in the IL-2 receptor α-chain expression on Tcons. Finally, in xeno-GVHD generated by human PBMCs transplanted into immunodeficient mice, low-dose IL-2 increased Treg frequencies but did neither control pro-inflammatory cytokine production by pathogenic Tcons, nor prevented GVHD. Furthermore, combination of low-dose IL-2 with rapamycin was ineffective in this model. Our results indicate that limitations on the use of IL-2 during acute GVHD are likely due to the massive activation of the allogeneic T cells unique to this setting.

Topics: Acute Disease; Allografts; Animals; Antineoplastic Agents; Disease Models, Animal; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Heterografts; Humans; Immunosuppressive Agents; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Transgenic; Sirolimus; T-Lymphocytes, Regulatory

While rapamycin treatment has been reported to have a putatively negative effect on glucose homeostasis in mammals, it has not been tested in polygenic models of type 2 diabetes. One such mouse model, NONcNZO10/LtJ, was treated chronically with rapamycin (14 ppm encapsulated in diet) and monitored for the development of diabetes. As expected, rapamycin treatment accelerated the onset and severity of hyperglycemia. However, development of nephropathy was ameliorated, as both glomerulonephritis and IgG deposition in the subendothelial tuft were markedly reduced. Insulin production and secretion appeared to be inhibited, suppressing the developing hyperinsulinemia present in untreated controls. Rapamycin treatment also reduced body weight gain. Thus, rapamycin reduced some of the complications of diabetes despite elevating hyperglycemia. These results suggest that multiple factors must be evaluated when assessing the benefit vs. hazard of rapamycin treatment in patients that have overt, or are at risk for, type 2 diabetes. Testing of rapamycin in combination with insulin sensitizers is warranted, as such compounds may ameliorate the putative negative effects of rapamycin in the type 2 diabetes environment.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Female; Humans; Hyperglycemia; Hyperinsulinism; Insulin; Insulin Secretion; Male; Mice; Sirolimus

Topics: Animals; Caloric Restriction; Disease Models, Animal; Dose-Response Relationship, Drug; Electron Transport Complex I; Humans; Leigh Disease; Mice; Molecular Targeted Therapy; Saccharomyces cerevisiae; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Coronary Restenosis; Cytokines; Disease Models, Animal; DNA; Drug-Eluting Stents; Gene Expression Regulation; Immunosuppressive Agents; Postoperative Period; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus

Drug-eluting stents (DES) in percutaneous coronary intervention are more effective in preventing in-stent restenosis compared with bare metal stents (BMS); however, DES may cause late stent thrombosis, which has limited its use. In this study, the functional properties of a newly developed DES (RAP/CS/HEP), in which rapamycin was abluminally-loaded onto a chitosan/heparin coating stent (CS/HEP), were investigated in large animal artery injury models.. The effectiveness of BMS, RAP (the traditional version of rapamycin DES), CS/HEP and RAP/CS/HEP stents in preventing coagulation and promoting re-endothelialisation was examined and compared in the porcine coronary artery models with arteriovenous shunt, high load thrombus and coronary balloon injury at day 7 and 28, respectively, after stent implantation. The re-endothelialisation on these stents was further evaluated in terms of endothelial gene expression using quantitative RT-PCR.. In the porcine coronary artery injury models, both RAP and RAP/CS/HEP stents were potent in reducing neointimal thickness, thus enlarging lumen area efficiently in the stented artery region compared with BMS and CS/HEP. RAP/CS/HEP stents facilitated re-endothelialisation and inhibited thrombosis more efficiently than BMS and RAP. Consistent with this, the expression of endothelial genes, such as CD31, CD34, eNOS and VEGF, was significantly elevated with RAP/CS/HEP stents compared with RAP and BMS stents.. Abluminal coating of rapamycin onto the endothelialisation-accelerated CS/HEP stent and may prove to be an efficient treatment for tackling the late stent thrombosis associated with the traditionally circumferential RAP stent. This new RAP/CS/HEP stent system exhibits considerably improved therapeutic activity.

Topics: Animals; Anticoagulants; Biocompatible Materials; Chitosan; Coronary Artery Disease; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Heparin; Immunosuppressive Agents; Sirolimus; Swine

Sepsis-induced cardiac dysfunction is a severe clinical problem. It is evident that rapamycin can protect heart from pathological injuries. However, there are no data demonstrating rapamycin reverse cardiac dysfunction induced by sepsis. In this study, Lipopolysaccharide (LPS) was administrated to mice and H9c2 cells. After treatment, we further determined cardiac function by echocardiography, ANP, BNP and inflammatory markers by qPCR and apoptosis by TUNEL staining. Moreover, mTORC1 signaling pathway and Akt activity were measured by Western blots. We found that rapamycin attenuated cardiac dysfunction, increase in ANP and BNP as well as apoptosis induced by LPS both in mice and in H9c2 cells. Unexpectedly, LPS did not significantly affect the mRNA levels of TNF-α and IL-6. Furthermore, rapamycin further reduced the decrease in mTORC1 signaling and Akt activity induced by LPS. In conclusion, rapamycin can protect heart from LPS induced damages by inhibition mTORC1 signaling and elevation of Akt activity.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Disease Models, Animal; Echocardiography; Heart; Immunosuppressive Agents; In Situ Nick-End Labeling; Lipopolysaccharides; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Rats; Real-Time Polymerase Chain Reaction; Sepsis; Sirolimus; TOR Serine-Threonine Kinases

To investigate whether temporary placement of a paclitaxel or rapamycin eluting stent is more effective to reduce stenting induced inflammatory reaction and scaring than a bared stent in benign cardia stricture models.. Eighty dog models of stricture were randomly divided into a control group (CG, n=20, no stent insertion), a bare stent group (BSG, n=20), a paclitaxel eluting (Pacl-ESG, n=20) and a rapamycin eluting stent group (Rapa-ESG, n=20), with one-week stent retention. Lower-oesophageal-sphincter pressure (LOSP), 5-minute barium height (5-mBH) and cardia diameter were assessed before, immediately after the procedure, and regularly for 6 months. Five dogs in each group were euthanized for histological examination at each follow-up assessment.. Stent insertion was well tolerated, with similar migration rates in three groups. At 6 months, LOSP and 5-mBH improved in Pacl-ESG and Rapa-ESG compared to BSG (p<0.05), with no difference between Pacl-ESG and Rapa-ESG (p>0.05). Cardia kept more patency in the Pacl-ESG and Rapa-ESG than in BSG (p<0.05). Reduced peak inflammatory reactions and scarring occurred in the Pacl-ESG and Rapa-ESG compared to BSG (p<0.05), with a similar outcome in the Pacl-ESG and Rapa-ESG (p>0.05).. Paclitaxel or rapamycin-eluting stents insertion led to better outcomes than bare stents in benign cardia stricture models.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Cardia; Cicatrix; Constriction, Pathologic; Disease Models, Animal; Dogs; Drug-Eluting Stents; Esophageal Sphincter, Lower; Esophageal Stenosis; Female; Humans; Inflammation; Male; Manometry; Paclitaxel; Random Allocation; Sirolimus; Time Factors

To evaluate the mTORC1 (mammalian target of rapamycin complex 1) pathway in meningiomas and to explore mTORC1 as a therapeutic target in meningioma cell lines and mouse models.. Tissue microarrays (53 meningiomas of all WHO grades) were stained for phosphorylated polypeptides of mTOR, Akt, and the mTORC1 targets 4EBP1 and p70S6K, the latter being the consensus marker for mTORC1 activity. Expression of proteins and mRNAs was assessed by Western blotting and real-time PCR in 25 tumors. Cell lines Ben-Men-1 (benign), IOMM-Lee and KT21 (malignant), and pairs of merlin-positive or -negative meningioma cells were used to assess sensitivity toward mTORC1 inhibitors in methyl-tetrazolium and bromodeoxyuridine (BrdUrd) assays. The effect of temsirolimus (20 mg/kg daily) on tumor weight or MRI-estimated tumor volume was tested by treatment of eight nude mice (vs. 7 controls) carrying subcutaneous IOMM-Lee xenografts, or of eight (5) mice xenotransplanted intracranially with IOMM-Lee (KT21) cells in comparison to eight (5) untreated controls.. All components of the mTORC1 pathway were expressed and activated in meningiomas, independent of their WHO grade. A significant dosage-dependent growth inhibition by temsirolimus and everolimus was observed in all cell lines. It was slightly diminished by merlin loss. In the orthotopic and subcutaneous xenograft models, temsirolimus treatment resulted in about 70% growth reduction of tumors (P < 0.01), which was paralleled by reduction of Ki67 mitotic index (P < 0.05) and reduction of mTORC1 activity (p70S6K phosphorylation) within the tumors.. mTORC1 inhibitors suppress meningioma growth in mouse models, although the present study did not measure survival.

Topics: Animals; Apoptosis; Blotting, Western; Cell Adhesion; Cell Proliferation; Disease Models, Animal; Genes, Neurofibromatosis 2; Humans; Immunoenzyme Techniques; Male; Mechanistic Target of Rapamycin Complex 1; Meningeal Neoplasms; Meningioma; Mice; Mice, Nude; Multiprotein Complexes; Neoplasm Grading; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autophagy; Disease Models, Animal; Frontotemporal Dementia; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunosuppressive Agents; Mice; Mice, Transgenic; Microtubules; Neurofibrillary Tangles; Phosphorylation; Sirolimus; tau Proteins; Tauopathies; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Up-Regulation

Parkinson's disease (PD) is a neurodegenerative disease, in which oxidative stress and mitochondrial dysfunction are responsible for neuronal apoptosis. Rapamycin plays a crucial role in reducing oxidative stress and protecting the mitochondria. However, its protective role in PD has not yet been fully elucidated. In this study, we report that pre-treatment with rapamycin provides behavioral improvements, protects against the loss of dopaminergic neurons, and alleviates mitochondrial ultrastructural injuries in a rat model of PD. Peroxide levels were lower and antioxidant activities were higher in PD rats pre-treated with rapamycin compared to the PD rats pre-treated with the vehicle. Furthermore, pre-treatment with rapamycin significantly elevated the expression of anti-apoptotic markers and reduced the levels of pro-apoptotic markers compared to pre-treatment with the vehicle. In conclusion, our results demonstrated that rapamycin reduced oxidative stress and alleviated mitochondrial injuries in the 6-hydroxydopamine (6-OHDA)-induced rat model of PD, which may subsequently contribute to its anti-apoptotic effects. The ability of rapamycin to exhibit neuroprotection in a rat model of PD may be related to its antioxidant capabilities.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Disease Models, Animal; Dopaminergic Neurons; Female; Mitochondria; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sirolimus

Pathological phenotypes in inclusion body myopathy (IBM) associated with Paget disease of the bone (PDB), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (IBMPFD/ALS) include defective autophagosome and endosome maturation that result in vacuolation, weakness and muscle atrophy. The link between autophagy and IBMPFD/ALS pathobiology has been poorly understood. We examined the AKT-FOXO3 and MTOR pathways to characterize the regulation of autophagy in IBMPFD/ALS mouse muscle. We identified a defect in MTOR signaling that results in enhanced autophagosome biogenesis. Modulating MTOR signaling may therefore be a viable therapeutic target in IBMPFD/ALS.

Topics: Adenosine Triphosphatases; Animals; Autophagy; Cell Cycle Proteins; Disease Models, Animal; Frontotemporal Dementia; Humans; Mice; Muscle Weakness; Mutant Proteins; Myositis, Inclusion Body; Osteitis Deformans; Phagosomes; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Valosin Containing Protein

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ER(T) under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

Topics: Adenocarcinoma; Alleles; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Endometrial Neoplasms; Endometrium; Epithelial Cells; Everolimus; Female; Gene Deletion; Humans; Hyperplasia; Integrases; Male; Mice; Mice, Knockout; Precancerous Conditions; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; PTEN Phosphohydrolase; Recombination, Genetic; Sirolimus; Stromal Cells; Tamoxifen; Thyroid Gland; Thyroid Neoplasms

Inflammatory bowel disease is characterized by dysregulated immune responses in inflamed intestine, with dominance of interleukin-17 (IL-17)--producing cells and deficiency of regulatory T (Treg) cells. The aim of this study was to investigate the effect and mechanisms of sirolimus, an inhibitor of the mammalian target of rapamycin, on immune responses in a murine model of Crohn's disease. Murine colitis was induced by intrarectal administration of 2,4,6-trinitrobenzene sulphonic acid at day 0. Mice were then treated intraperitoneally with sirolimus daily for 3 days. The gross and histological appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. Sirolimus treatment significantly decreased all macroscopic, microscopic and histopathological parameters of colitis that were analysed. The therapeutic effects of sirolimus were associated with a down-regulation of pro-inflammatory cytokines tumour necrosis factor-α, IL-6 and IL-17A. Intriguingly, sirolimus administration resulted in a prominent up-regulation of the regulatory cytokine transforming growth factor-β. Supporting the hypothesis that sirolimus directly affects the functional activity of CD4+ CD25+ Treg cells, we observed a remarkable enhancement of FoxP3 expression in colon tissues and isolated CD4+ T cells of sirolimus-treated mice. Simultaneously, sirolimus treatment led to a significant reduction in the number of CD4+ IL-17A+ T cells in the mesenteric lymph node cells as well as IL-17A production in mesenteric lymph node cells. Therefore, sirolimus may offer a promising new therapeutic strategy for the treatment of inflammatory bowel disease.

Topics: Animals; Anti-Inflammatory Agents; CD4 Lymphocyte Count; Cells, Cultured; Colitis; Colon; Disease Models, Animal; Forkhead Transcription Factors; Immunosuppressive Agents; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-17; Interleukin-6; Lymph Nodes; Male; Mice; Mice, Inbred BALB C; Phenotype; Sirolimus; Th17 Cells; Time Factors; Transforming Growth Factor beta; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.

Topics: Animals; Autophagy; Disease Models, Animal; Female; Fibroblast Growth Factor 2; Heat-Shock Proteins; Humans; Motor Neurons; Neuroprotective Agents; PC12 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recovery of Function; Sequestosome-1 Protein; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases; Ubiquitinated Proteins

Cognitive impairments are prominent sequelae of prolonged continuous seizures (status epilepticus; SE) in humans and animal models. While often associated with dendritic injury, the underlying mechanisms remain elusive. The mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated following SE. This pathway modulates learning and memory and is associated with regulation of neuronal, dendritic, and glial properties. Thus, in the present study we tested the hypothesis that SE-induced mTORC1 hyperactivation is a candidate mechanism underlying cognitive deficits and dendritic pathology seen following SE. We examined the effects of rapamycin, an mTORC1 inhibitor, on the early hippocampal-dependent spatial learning and memory deficits associated with an episode of pilocarpine-induced SE. Rapamycin-treated SE rats performed significantly better than the vehicle-treated rats in two spatial memory tasks, the Morris water maze and the novel object recognition test. At the molecular level, we found that the SE-induced increase in mTORC1 signaling was localized in neurons and microglia. Rapamycin decreased the SE-induced mTOR activation and attenuated microgliosis which was mostly localized within the CA1 area. These findings paralleled a reversal of the SE-induced decreases in dendritic Map2 and ion channels levels as well as improved dendritic branching and spine density in area CA1 following rapamycin treatment. Taken together, these findings suggest that mTORC1 hyperactivity contributes to early hippocampal-dependent spatial learning and memory deficits and dendritic dysregulation associated with SE.

Topics: Animals; Dendrites; Dendritic Spines; Disease Models, Animal; Electroencephalography; Gliosis; Hippocampus; Ion Channels; Male; Maze Learning; Mechanistic Target of Rapamycin Complex 1; Memory Disorders; Microglia; Multiprotein Complexes; Neurons; Phosphorylation; Pilocarpine; Rats; Ribosomal Protein S6 Kinases; Sirolimus; Status Epilepticus; TOR Serine-Threonine Kinases

Autophagy is a biological process during which cells digest organelles in their cytoplasm and recycle the constituents. The impact of autophagy in the heart, however, remains unclear in part because of the inability to noninvasively image this process in living animals.. Here, we report the use of fluorescence molecular tomography and a cathepsin-activatable fluorochrome to image autophagy in the heart in vivo after ischemia/reperfusion and rapamycin (RAP) therapy. We show that cathepsin-B activity in the lysosome is upregulated by RAP and that this allows the expanded lysosomal compartment in autophagy to be imaged in vivo with fluorescence molecular tomography. We further demonstrate that the delivery of diagnostic nanoparticles to the lysosome by endocytosis is enhanced during autophagy. The upregulation of autophagy by RAP was associated with a 23% reduction (P<0.05) of apoptosis in the area at risk and a 45% reduction in final infarct size (19.6±5.6% of area at risk with RAP versus 35.9±9.1% of area at risk without RAP; P<0.05).. The ability to perform noninvasive tomographic imaging of autophagy in the heart has the potential to provide valuable insights into the pathophysiology of autophagy, particularly its role in cardiomyocyte salvage. Although additional data are needed, our study supports the investigation of RAP therapy in patients with acute coronary syndromes.

Topics: Animals; Apoptosis; Autophagy; Cardiovascular Agents; Cathepsin B; Disease Models, Animal; Enzyme Activation; Female; Fluorescent Dyes; Ischemic Postconditioning; Lysosomes; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microspheres; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Optical Imaging; Radionuclide Imaging; Sirolimus; Time Factors; Tomography; X-Ray Microtomography

To validate optical coherence tomography (OCT) imaging for assessment of vascular healing in a preclinical animal model and human autopsy cases and to translate the findings to the assessment of vascular healing after drug-eluting stent implantation in clinical practice.. Drug-eluting and bare metal stents were imaged 28 and 42 days after implantation in atherosclerotic rabbits using OCT and simultaneously evaluated by histology. After coregistration with histology, gray-scale signal intensity (GSI) was measured for identified mature or immature neointimal tissue. Autopsy specimens were imaged with OCT and GSI values correlated with histology. Finally, prospective OCT imaging and GSI measurements were acquired in 10 patients undergoing follow-up 6 months after stenting with drug-eluting stents. Histopathologic and OCT morphometric analysis of implanted stents showed excellent correlation. Neointimal growth and vessel healing at 28 days in the animal model best correlated with human stented arteries at 6 months. In animal and human autopsy specimens, mature neointimal tissue consistently showed higher GSI values. Receiver operating characteristic curve analysis displayed high sensitivity and specificity for detection of mature neointima in animal (96% and 79%, respectively) and human autopsy (89% and 71%, respectively) data. In patients undergoing OCT follow-up 6 months after drug-eluting stent implantation, prospective GSI analysis revealed that a minimum of 27.7% of areas above stent struts represented mature neointima.. Novel GSI analysis of OCT imaging data allows distinction between mature and immature neointimal tissue in animal models, autopsy specimens, and patients undergoing invasive surveillance in simple atherosclerotic lesions.

Topics: Aged; Angioplasty, Balloon, Coronary; Animals; Autopsy; Coronary Artery Disease; Disease Models, Animal; Drug-Eluting Stents; Female; Humans; Male; Metals; Middle Aged; Neointima; Neovascularization, Physiologic; Prospective Studies; Prosthesis Failure; Rabbits; Reproducibility of Results; ROC Curve; Sirolimus; Species Specificity; Tomography, Optical Coherence

Fibrocytes are integral in the development of fibroproliferative disease. The CXCL12/CXCR4 chemokine axis has been shown to play a central role in fibrocyte migration and the development of bronchiolitis obliterans (BO) after lung transplantation. Inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin has been shown to decrease expression of both CXCR4 and its receptor agonist CXCL12. Thus, we hypothesized that rapamycin treatment would decrease fibrocyte trafficking into tracheal allografts and prevent BO.. A total alloantigenic mismatch murine heterotopic tracheal transplant (HTT) model of BO was used. Animals were either treated with rapamycin or dimethyl sulfoxide (DMSO) for 14 days after tracheal transplantation. Fibrocyte levels were assessed by flow cytometry, and allograft neutrophil, CD3(+) T-cell, macrophage, and smooth muscle actin (SMA) levels were assessed by immunohistochemistry. Tracheal luminal obliteration was assessed on hematoxylin and eosin (H&E) stains.. Compared with DMSO-treated controls, rapamycin-treated mice showed a significant decrease in fibrocyte levels in tracheal allografts. Fibrocyte levels in recipient blood showed a similar pattern, although it was not statistically significant. Furthermore, animals treated with rapamycin showed a significant decrease in tracheal allograft luminal obliteration compared with controls. Based on immunohistochemical analyses, populations of α-SMA-positive (α-SMA(+)) cells, neutrophils, CD3(+) T cells, and macrophages were all decreased in rapamycin-treated allografts versus DMSO controls.. Rapamycin effectively reduces recruitment of fibrocytes into tracheal allografts and mitigates development of tracheal luminal fibrosis. Further studies are needed to determine the cellular and molecular mechanisms that mediate the protective effect of rapamycin against BO.

Topics: Animals; Bronchiolitis Obliterans; Cell Movement; Chemokine CXCL12; Disease Models, Animal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Receptors, CXCR4; Sirolimus; TOR Serine-Threonine Kinases; Trachea; Transplantation, Homologous

The aim of the current study was to assess whether placement of the biodegradable rapamycin-eluting nano-fiber membrane-covered metal stent is followed by less fibroblast proliferation and tissue hyperplasia compared with bare stents in experimental stricture in a dog model.. A total of 80 dog models of stricture were randomly divided into a control group (n = 20, no stent insertion), a bare stent group (BSG, n = 20, 1-week retention), and two drug-eluting stent sub-groups (DESG-1w, n = 20, 1-week retention; DESG-4w, n = 20, 4-week retention). Lower esophageal sphincter (LES) pressure, 5-minute barium height (5-mBH), and cardia diameter were assessed before, immediately after the procedure, and regularly thereafter for 6 months. Five dogs in each group were euthanized for histological examination at each follow-up assessment.. Stent insertion was well tolerated, with similar migration rates (0 % in BSG vs. 7.5 % in DESGs; P = 0.5441). At 6 months, LES pressure and 5-mBH improved in DESG-1w (26.70 ± 5.02 mmHg and 6.50 ± 2.98 cm) and DESG-4w (20.16 ± 5.50 mmHg and 1.54 ± 0.98 cm) compared with BSG (39.94 ± 5.22 mmHg and 11.1 ± 5.46 cm) (P < 0.05), with DESG-4w being more stable than DESG-1w (P < 0.05). The cardia maintained greater patency in the DESGs (7.10 ± 3.09 mm in DESG-1w; 9.16 ± 3.77 mm in DESG-4w) than in the BSG (1.86 ± 2.45 mm; P < 0.05). Reduced peak inflammatory reactions and scarring occurred in DESGs compared with the BSG (P < 0.05), with a better outcome in DESG-4w than in DESG-1w (P < 0.05).. In this experimental stricture model, rapamycin-eluting stents were more effective than bare stents for the reduction of fibroblast proliferation and tissue hyperplasia after stent placement. Furthermore, 4-week retention of the drug-eluting stent led to a better outcome than 1-week retention.

Topics: Absorbable Implants; Animals; Cardia; Cell Proliferation; Constriction, Pathologic; Disease Models, Animal; Dogs; Drug-Eluting Stents; Esophageal Sphincter, Lower; Esophageal Stenosis; Female; Fibroblasts; Immunosuppressive Agents; Male; Manometry; Nanofibers; Pressure; Random Allocation; Sirolimus

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain.

Topics: Animals; Disease Models, Animal; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Immunosuppressive Agents; Male; Neuralgia; Neurogenic Inflammation; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Spinal Cord; Spinal Nerves; Statistics, Nonparametric; Time Factors; TOR Serine-Threonine Kinases

Hippocampal demyelination, a common feature of postmortem multiple sclerosis (MS) brains, reduces neuronal gene expression and is a likely contributor to the memory impairment that is found in >40% of individuals with MS. How demyelination alters neuronal gene expression is unknown.. To explore whether loss of hippocampal myelin alters expression of neuronal microRNAs (miRNAs), we compared miRNA profiles from myelinated and demyelinated hippocampi from postmortem MS brains and performed validation studies.. A network-based interaction analysis depicts a correlation between increased neuronal miRNAs and decreased neuronal genes identified in our previous study. The neuronal miRNA miR-124 was increased in demyelinated MS hippocampi and targets mRNAs encoding 26 neuronal proteins that were decreased in demyelinated hippocampus, including the ionotrophic glutamate receptors AMPA2 and AMPA3. Hippocampal demyelination in mice also increased miR-124, reduced expression of AMPA receptors, and decreased memory performance in water maze tests. Remyelination of the mouse hippocampus reversed these changes.. We establish here that myelin alters neuronal gene expression and function by modulating the levels of the neuronal miRNA miR-124. Inhibition of miR-124 in hippocampal neurons may provide a therapeutic approach to improve memory performance in MS patients.

Topics: Animals; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Humans; Immunosuppressive Agents; Memory Disorders; Mice; MicroRNAs; Monoamine Oxidase Inhibitors; Multiple Sclerosis; Neurons; Postmortem Changes; Receptors, AMPA; RNA, Messenger; Sirolimus

Because of the toxicity associated with myeloablative conditioning, nonmyeloablative regimens are increasingly being used in vulnerable patient populations. For patients with sickle cell disease, stable mixed chimerism has proven sufficient to reverse the phenotype. Because the vast majority of patients do not have an HLA-matched sibling, a safe nonmyeloablative regimen that could be applied to the haploidentical setting would be ideal. We employed a mismatched mouse model using BALB/c donors and C57BL/6 recipients. Recipient mice were conditioned with 200 cGy TBI and sirolimus or CSA with or without post transplant Cy (PT-Cy). Our data show that when sirolimus or PT-Cy alone is given to C57BL/6 recipients, donor cells are not detected. However, when sirolimus is administered for 15 or 31 days starting 1 day before or up to 6 days after transplant with PT-Cy, all mice maintain stable mixed chimerism. In contrast, conventional therapy employing CSA with or without PT-Cy does not result in stable mixed chimerism. Lastly, mice with stable mixed chimerism after sirolimus display decreased reactivity to donor Ag both in vitro and in vivo. These data identify a novel strategy for inducing mixed chimerism for the treatment of nonmalignant hematologic diseases.

Topics: Animals; Cyclophosphamide; Disease Models, Animal; Drug Synergism; Female; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Sirolimus; Skin Transplantation; Transplantation Chimera

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late onset neurodegenerative disorder caused by aberrant expansion of CGG repeats in 5' UTR of FMR1 gene. The elevated mRNA confers a toxic gain-of-function thought to be the critical event of pathogenesis. Expressing rCGG90 repeats of the human FMR1 5'UTR in Drosophila is sufficient to induce neurodegeneration. Rapamycin has been demonstrated to attenuate neurotoxicity by inducing autophagy in various animal models of neurodegenerative diseases. Surprisingly, we observed rapamycin exacerbated rCGG90-induced neurodegenerative phenotypes through an autophagy-independent mechanism. CGG90 expression levels of FXTAS flies exposed to rapamycin presented no significant differences. We further demonstrated that activation of the mammalian target of rapamycin (mTOR) signaling could suppress neurodegeneration of FXTAS. These findings indicate that rapamycin will exacerbate neurodegeneration, and that enhancing autophagy is insufficient to alleviate neurotoxicity in FXTAS. Moreover, these results suggest mTOR and its downstream molecules as new therapeutic targets for FXTAS by showing significant protection against neurodegeneration.

Topics: Animals; Autophagy; Disease Models, Animal; Drosophila; Enzyme Activation; Fragile X Mental Retardation Protein; Fragile X Syndrome; Humans; Neurodegenerative Diseases; Phenotype; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trinucleotide Repeat Expansion

Previous studies reported that autophagy is activated in human dilated cardiomyopathy (DCM). It is still unknown whether modulating autophagy can improve cardiac function of the failing heart.. We immunized rats with porcine cardiac myosin to set up a model of DCM. Rapamycin, a kind of mTOR inhibitor upregulating autophagy, was given to rats weeks after the immunization at low (1 mg/kg · day i.p.), intermediate (2 mg/kg · day i.p.) and high dose (4 mg/kg · day i.p.) for 2 weeks.. Compared to the control group (ejection fraction, EF = 81.3 ± 3.8%), the average EF decreased in both the DCM group (EF = 56.1 ± 3.3%) and the high-dose rapamycin group (EF = 55.9 ± 3.6%), but recovered in the low-/intermediate-dose rapamycin groups (EF = 64.9 ± 4.6/69.4 ± 4.4%). Phosphorylation of p70s6k and 4E-BP1 decreased and the expression of LC3BI/II increased in all rapamycin groups. Autophagic vacuoles were easily found in these groups. However, body weight was significantly reduced in the rapamycin groups. Furthermore, mortality was increased in the high-dose rapamycin group.. Rapamycin could improve cardiac function of early-stage DCM, but the effect of rapamycin turned out to be biphasic and the effective range appeared narrow.

Topics: Analysis of Variance; Animals; Autoimmune Diseases; Autophagy; Cardiomyopathy, Dilated; Cardiotonic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Rats; Rats, Inbred Lew; Sirolimus; Stroke Volume; TOR Serine-Threonine Kinases; Weight Loss

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFβ1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFβ1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease.

Topics: Animals; Cell Line; Disease Models, Animal; Fibroblasts; Fibrosis; Immunohistochemistry; Kidney; Mechanistic Target of Rapamycin Complex 1; Mice; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Neuropeptides; Proteins; Ras Homolog Enriched in Brain Protein; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Chronic pain is extremely difficult to manage, in part due to lack of progress in reversing the underlying pathophysiology. Since translation of messenger ribonucleic acids (mRNAs) in the peripheral terminal of the nociceptor plays a role in the transition from acute to chronic pain, we tested the hypothesis that transient inhibition of translation in the peripheral terminal of the nociceptor could reverse hyperalgesic priming, a model of transition from acute to chronic pain. We report that injection of translation inhibitors rapamycin and cordycepin, which inhibit translation by different mechanisms, at the peripheral terminal of the primed nociceptor produces reversal of priming in the rat that outlasted the duration of action of these drugs to prevent the development of priming. These data support the suggestion that interruption of translation in the nociceptor can reverse a preclinical model of at least 1 form of chronic pain.. This study provides evidence that ongoing protein translation in the sensory neuron terminals is involved in pain chronification, and local treatment that transiently interrupts this translation may be a useful therapy to chronic pain.

Topics: Animals; Chronic Pain; Deoxyadenosines; Disease Models, Animal; Hyperalgesia; Male; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sirolimus

Drug-eluting stents have proven to be effective in reducing the risk of late restenosis. In order to achieve a controlled and prolonged release of the antiproliferative agent, current drug-eluting stents utilise various biodegradable as well as non-erodible polymeric blends to coat the stent surface and to serve as drug carriers. The utilisation of polymeric compounds in current drug-eluting stents may eventually limit their performance as well as their clinical applicability due to the potential induction of undesirable local reactions. The development of alternative, polymer-free drug carriers has the potential to overcome some of the limitations of current drug-eluting stent formulations. Moreover, improvements in drug carriers may also result in an expansion of the technological possibilities for other intravascular drug delivery systems, such as metal-free or even implant-free solutions. This article describes the structure and the preclinical validation profile of a novel phospholipid encapsulated sirolimus nanocarrier, used as a coating in two formulations: a coronary stent-plus-balloon system and a stand-alone balloon catheter. The nanoparticles provided a stable, even and homogenous coating to the devices in both formulations. Dose-finding studies allowed the most appropriate identification of the best nanoparticle structure associated with an extremely efficient transfer of drug to all layers of the vessel wall, achieving high tissue concentrations that persisted days after the application, with low systemic drug leaks.

Topics: Angioplasty, Balloon; Animals; Cardiovascular Agents; Catheters; Coated Materials, Biocompatible; Delayed-Action Preparations; Disease Models, Animal; Drug Carriers; Drug-Eluting Stents; Femoral Artery; Iliac Artery; Kinetics; Male; Nanoparticles; Phospholipids; Rabbits; Sirolimus; Swine; Tissue Distribution; Vascular System Injuries

Basal-like breast cancer is an aggressive disease for which targeted therapies are lacking. Recent studies showed that basal-like breast cancer is frequently associated with an increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway, which is critical for cell growth, survival, and angiogenesis. To investigate the therapeutic potential of PI3K pathway inhibition in the treatment of basal-like breast cancer, we evaluated the antitumor effect of the mTOR inhibitor MK-8669 and AKT inhibitor MK-2206 in WU-BC4 and WU-BC5, two patient-derived xenograft models of basal-like breast cancer. Both models showed high levels of AKT phosphorylation and loss of PTEN expression. We observed a synergistic effect of MK-8669 and MK-2206 on tumor growth and cell proliferation in vivo. In addition, MK-8669 and MK-2206 inhibited angiogenesis as determined by CD31 immunohistochemistry. Biomarker studies indicated that treatment with MK-2206 inhibited AKT activation induced by MK-8669. To evaluate the effect of loss of PTEN on tumor cell sensitivity to PI3K pathway inhibition, we knocked down PTEN in WU-BC3, a basal-like breast cancer cell line with intact PTEN. Compared with control (GFP) knockdown, PTEN knockdown led to a more dramatic reduction in cell proliferation and tumor growth inhibition in response to MK-8669 and MK-2206 both in vitro and in vivo. Furthermore, a synergistic effect of these two agents on tumor volume was observed in WU-BC3 with PTEN knockdown. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer with loss of PTEN.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Female; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Neoplasms, Basal Cell; Neovascularization, Pathologic; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Posttraumatic epilepsy is a major source of disability following traumatic brain injury (TBI) and a common cause of medically-intractable epilepsy. Previous attempts to prevent the development of posttraumatic epilepsy with treatments administered immediately following TBI have failed. Recently, the mammalian target of rapamycin complex 1 (mTORC1) pathway has been implicated in mechanisms of epileptogenesis and the mTORC1 inhibitor, rapamycin, has been proposed to have antiepileptogenic effects in preventing some types of epilepsy. In this study, we have tested the hypothesis that rapamycin has antiepileptogenic actions in preventing the development of posttraumatic epilepsy in an animal model of TBI. A detailed characterization of posttraumatic epilepsy in the mouse controlled cortical impact model was first performed using continuous video-EEG monitoring for 16 weeks following TBI. Controlled cortical impact injury caused immediate hyperactivation of the mTORC1 pathway lasting at least one week, which was reversed by rapamycin treatment. Rapamycin decreased neuronal degeneration and mossy fiber sprouting, although the effect on mossy fiber sprouting was reversible after stopping rapamycin and did not directly correlate with inhibition of epileptogenesis. Most posttraumatic seizures occurred greater than 10 weeks after TBI, and rapamycin treatment for one month after TBI decreased the seizure frequency and rate of developing posttraumatic epilepsy during the entire 16 week monitoring session. These results suggest that rapamycin may represent a rational treatment for preventing posttraumatic epilepsy in patients with TBI.

Topics: Animals; Disease Models, Animal; Electroencephalography; Epilepsy, Post-Traumatic; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mossy Fibers, Hippocampal; Multiprotein Complexes; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Seizure development in tuberous sclerosis complex (TSC) correlates with the presence of specific lesions called cortical tubers. Moreover, heterozygous TSC animal models do not show gross brain pathology and are seizure-free, suggesting that such pathology is a prerequisite for the development of epilepsy. However, cells within TSC lesions show increased activity of the target of rapamycin complex 1 (TORC1) pathway, and recent studies have implicated this pathway in non-TSC-related animal models of epilepsy and neuronal excitability. These findings imply a direct role for TORC1 in epilepsy. Here, we investigate the effect of increased TORC1 signaling induced by acute biallelic deletion of Tsc1 in healthy adult mice.. Biallelic Tsc1 gene deletion was induced in adult Tsc1 heterozygous and wild-type mice. Seizures were monitored by electroencephalographic and video recordings. Molecular and cellular changes were investigated by Western blot analysis, immunohistochemistry, and electrophysiology.. Mice developed epilepsy a few days after biallelic Tsc1 deletion. Acute gene deletion was not accompanied by any obvious histological changes, but resulted in activation of the TORC1 pathway, enhanced neuronal excitability, and a decreased threshold for protein-synthesis-dependent long-term potentiation preceding the onset of seizures. Rapamycin treatment after seizure onset reduced TORC1 activity and fully abolished the seizures.. Our data indicate a direct role for TORC1 signaling in epilepsy development, even in the absence of major brain pathology. This suggests that TORC1 is a promising target for treating seizures not only in TSC but also in other forms of epilepsy that result from increased TORC1 activation.

Topics: Animals; Carrier Proteins; Disease Models, Animal; Dose-Response Relationship, Drug; Electroencephalography; Epilepsy; Gene Expression Regulation; Hippocampus; Immunosuppressive Agents; Long-Term Potentiation; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Multiprotein Complexes; Neurons; ras Proteins; Receptors, Estrogen; Sequence Deletion; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Tuberous sclerosis complex (TSC) is a dominantly inherited disease with high penetrance and morbidity, and is caused by mutations in either of two genes, TSC1 or TSC2. Most affected individuals display severe neurological manifestations - such as intractable epilepsy, mental retardation and autism - that are intimately associated with peculiar CNS lesions known as cortical tubers (CTs). The existence of a significant genotype-phenotype correlation in individuals bearing mutations in either TSC1 or TSC2 is highly controversial. Similar to observations in humans, mouse modeling has suggested that a more severe phenotype is associated with mutation in Tsc2 rather than in Tsc1. However, in these mutant mice, deletion of either gene was achieved in differentiated astrocytes. Here, we report that loss of Tsc1 expression in undifferentiated radial glia cells (RGCs) early during development yields the same phenotype detected upon deletion of Tsc2 in the same cells. Indeed, the same aberrations in cortical cytoarchitecture, hippocampal disturbances and spontaneous epilepsy that have been detected in RGC-targeted Tsc2 mutants were observed in RGC-targeted Tsc1 mutant mice. Remarkably, thorough characterization of RGC-targeted Tsc1 mutants also highlighted subventricular zone (SVZ) disturbances as well as STAT3-dependent and -independent developmental-stage-specific defects in the differentiation potential of ex-vivo-derived embryonic and postnatal neural stem cells (NSCs). As such, deletion of either Tsc1 or Tsc2 induces mostly overlapping phenotypic neuropathological features when performed early during neurogenesis, thus suggesting that the timing of mTOR activation is a key event in proper neural development.

Topics: Animals; Animals, Newborn; Cell Differentiation; Cell Proliferation; Cell Size; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Embryonic Development; Enzyme Activation; Epilepsy; Gene Silencing; Longevity; Megalencephaly; Mice; Mutagenesis; Myelin Sheath; Neuroglia; Neurons; Sirolimus; STAT3 Transcription Factor; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

While pre-conditioning is induced before stroke onset, ischemic post-conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild-type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild-type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri-infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1-30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase. Post-conditioning did not attenuate infarction in nude rats measured 2 days post-stroke, but improved neurological function in nude rats and reduced brain damage 30 days after stroke. It resulted in increased-activities of Akt and mTOR, S6K and p-4EBP1. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC.

Topics: Animals; Disease Models, Animal; Immunosuppressive Agents; Ischemic Postconditioning; Male; Proto-Oncogene Proteins c-akt; Rats; Rats, Nude; Rats, Sprague-Dawley; Recovery of Function; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Stroke; T-Lymphocytes; TOR Serine-Threonine Kinases

Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anti-Bacterial Agents; Brain; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Humans; Memory; Mice; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase Type III; Nitroarginine; Phosphorylation; Sirolimus; Vasodilation

Tumor dissemination to cervical lymph nodes via lymphatics represents the first step in the metastasis of head and neck squamous cell carcinoma (HNSCC) and is the most significant predictor of tumor recurrence decreasing survival by 50%. The lymphatic suppressing properties of mTOR inhibitors are not yet well understood.. Lymphatic inhibiting effects of rapamycin were evaluated in vitro using two lymphatic endothelial cell (LEC) lines. An orthotopic mouse model of HNSCC (OSC-19 cells) was used to evaluate anti-lymphangiogenic effects of rapamycin in vivo. The incidence of cervical lymph node metastases, numbers of tumor-free lymphatic vessels and those invaded by tumor cells in mouse lingual tissue, and expression of pro-lymphangiogenic markers were assessed.. Rapamycin significantly decreased lymphatic vascular density (p = 0.027), reduced the fraction of lymphatic vessels invaded by tumor cells in tongue tissue (p = 0.013) and decreased metastasis-positive lymph nodes (p = 0.04). Rapamycin also significantly attenuated the extent of metastatic tumor cell spread within lymph nodes (p < 0.0001). We found that rapamycin significantly reduced LEC proliferation and was correlated with decreased VEGFR-3 expression in both LEC, and in some HNSCC cell lines.. The results of this study demonstrate anti-lymphangiogenic properties of mTOR inhibitors in HNSCC. mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing VEGF-C/VEGFR-3 axis and release of soluble VEGFR-2. In a murine HNSCC orthotopic model rapamycin significantly suppressed lymphovascular invasion, decreased cervical lymph node metastasis and delayed the spread of metastatic tumor cells within the lymph nodes.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Disease Models, Animal; Head and Neck Neoplasms; Humans; Lymphangiogenesis; Lymphatic Metastasis; Male; Mice; Mice, SCID; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Hepatic arterial infusion (HAI) of specific anti-tumor drugs can be more effective compared with systemic drug application. Herein, we studied whether HAI of temsirolimus is effective to inhibit tumor growth of colorectal liver metastases after liver resection.. Twenty-four Wistar Albino Glaxo from Rijswijk (WAG/Rij) rats were randomized to four groups and underwent subcapsular implantation of CC531 colorectal cancer cells in the left liver lobe. In two groups, a 70% liver resection (Phx) was performed simultaneously. After 10 d, animals received either a HAI of temsirolimus (CCI-779) or saline solution (controls). Tumor growth was determined on d 10 and 13 using three-dimensional ultrasound. On d 13, tumor tissue was removed for histologic and immunohistochemical analysis.. Sham controls revealed a tumor growth of ∼40% from d 10 to d 13. HAI of temsirolimus completely inhibited this tumor growth. Controls with Phx showed a tumor growth of >60%. In contrast, HAI of temsirolimus in Phx animals did not only inhibit tumor growth but was even capable of decreasing the tumor size by ∼8%. Immunohistochemical analysis of the tumors showed a decreased proliferation rate and an increased cleaved caspase-3 activity, which was associated with a significant reduction of platelet endothelial cell adhesion molecule (PECAM)-1-positive cells after HAI of temsirolimus.. HAI of temsirolimus inhibits tumor growth of CC531 colorectal liver metastases even if a growth-stimulating procedure like Phx is performed. Inhibition of tumor growth is provided by a decrease of tumor vascularization associated with an inhibition of tumor cell proliferation and an induction of tumor cell apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Hepatic Artery; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Male; Neovascularization, Pathologic; Random Allocation; Rats; Rats, Wistar; Sirolimus

The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and to measure the effect on seizure frequency.. Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/day) was administered. Starting 1 month after status epilepticus, mice were monitored by video 9 h per day, every day, for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomic analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections.. Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naive controls. Seizure frequency was similar in vehicle-treated and rapamycin-treated mice.. These findings suggest that mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have antiseizure or antiepileptogenic effects in this model.

Topics: Animals; Axons; Disease Models, Animal; Epilepsy, Temporal Lobe; Female; Male; Mice; Mossy Fibers, Hippocampal; Neurons; Pilocarpine; Sirolimus

Metformin impairs endothelialization of drug eluting stents (DES) due to convergent signaling at the mammalian target of rapamycin (mTOR) pathway. We assessed whether metformin will continue to adversely affect stent endothelialization despite design improvements in newer generation DES.. Rabbit iliac artery stenting with newer generation DES was performed followed by 14 days of either normal chow diet or one with metformin (100 mg/kg/day) added. Scanning electron microscopy was used to assess stent endothelialization after sacrifice.. In the metformin-treated group there was significantly less endothelialization compared to the placebo-treated group. Paclitaxel-eluting stents in placebo-treated group had the greatest degree of endothelialization with significantly less in its metformin-treated counterpart and all-limus eluting stent groups.. Metformin inhibited stent endothelialization in newer generation DES despite improvements in stent design. By impairing stent endothelialization, metformin may increase the risk for thrombotic complications after newer generation DES placement.

Topics: Angioplasty, Balloon; Animals; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Everolimus; Hypoglycemic Agents; Iliac Artery; Immunosuppressive Agents; Male; Metformin; Paclitaxel; Rabbits; Sirolimus; Thrombosis; Tubulin Modulators

This study was designed to evaluate the pharmacokinetic and vascular healing of a second-generation everolimus-eluting stent (EES) and slow-release zotarolimus-eluting stent (R-ZES).. Second-generation DESs have alleviated the safety concerns of late stent thrombosis by addressing issues of polymer biocompatibility and stent design, and optimizing drug loads and release kinetics. No preclinical comparison study exists between these stents.. Rabbit iliac artery stent implantation was performed using Xience Prime EES and Resolute R-ZES. Histomorphometric evaluation was performed at 28 and 60 days after implantation in an induced atheroma model. Endothelial coverage and maturation were assessed by scanning electron microscopy and immuno-labeling at 14 and 28 days following deployment. For pharmacokinetic studies, arterial tissue and stents were retrieved at 3, 14, 28, and 90 days, and blood samples were obtained during the first 24 hours.. Vascular remodeling (percent stenosis, neointimal thickness) was similar in arteries implanted with either stent group. At 28 days, inflammation was significantly less in the EES group as compared to the R-ZES group (inflammation score: 1.59 ± 0.52 vs 2.22 ± 0.69, respectively; P=.044), with no differences observed at 60 days. Endothelial coverage was similar between both groups; however, endothelial maturation above stent struts was significantly higher in the EES group vs R-ZES group at 28 days (33 ± 20% vs 22 ± 21%, respectively; P=.040). Arterial drug level concentrations were also shown to be significantly less in the EES group vs the R-ZES group (P<.0001).. Overall, EES and R-ZES displayed similar remodeling properties with lower arterial drug levels observed in the EES group vs the R-ZES group, which may have led to more rapid endothelial maturation.

Topics: Animals; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Everolimus; Femoral Artery; Iliac Artery; Male; Microscopy, Confocal; Microscopy, Electron, Scanning; Plaque, Atherosclerotic; Rabbits; Sirolimus; Time Factors; Treatment Outcome

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated.. Autophagy is increased in the spinal cord of ALS mice. Dietary supplementation of rapamycin increases autophagy, but does not increase the survival of mutant SOD1 mice. To measure the effect of rapamycin in ALS independent of immunosuppression, we tested the effect of rapamycin in ALS mice deficient of mature lymphocytes. Our results show that rapamycin moderately increases the survival of these ALS mice deficient of mature lymphocytes.. Rapamycin could suppress protective immune responses while enhancing protective autophagy reactions during the ALS disease process. While these opposing effects can cancel each other out, the use of immunodeficient mice allows segregation of effects. Our results indicate that maximal therapeutic benefit may be achieved through the use of compounds that enhance autophagy without causing immune suppression.

Topics: Amyotrophic Lateral Sclerosis; Animals; Autophagy; Blotting, Western; Disease Models, Animal; Immunosuppressive Agents; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Sirolimus; Spinal Cord

The mammalian target of rapamycin (mTOR), a regulator of cellular protein synthesis and cell growth, plays an important role in the progression of renal hypertrophy and renal dysfunction in experimental chronic kidney disease models. Because the mTOR activity is regulated by nutrients including amino acids, we tested the hypothesis that the renoprotective effect of a low-protein diet (LPD) might be associated with the attenuation of the renal mTOR pathway. In this study, 5/6 nephrectomized rats were fed an LPD or a normal protein diet (NPD), and a number of rats that were fed an NPD received rapamycin (1.0 mg kg⁻¹ d⁻¹), a specific inhibitor of mTOR. After 6 weeks, renal tissue was collected to evaluate the activity of the mTOR pathway and histologic changes. The phosphorylation of p70S6k, a kinase in the downstream of mTOR, was significantly higher in the NPD-fed rats that showed progressive renal dysfunction than in the sham-operated rats (NPD). The LPD attenuated the excessive phosphorylation of p70S6k concomitant with reduced proteinuria and improved renal histologic changes in the 5/6 nephrectomized rats. The effects of the LPD were similar to the effects of rapamycin. The expression of phosphorylated p70S6k was significantly correlated with proteinuria (r² = 0.63, P < .001), the glomerular area (r² = 0.60, P < .001), and the number of phosphorylated Smad2-positive cells in the glomerulus (r² = 0.26, P < .05) of these rats. These results suggest that the preventive effect of an LPD on the progression of renal failure is associated with attenuation of the activated mTOR/p70S6k pathway in the rat remnant kidney model.

Topics: Animals; Cell Proliferation; Cells, Cultured; Diet, Protein-Restricted; Dietary Proteins; Disease Models, Animal; Hypertrophy; Kidney; Male; Phosphorylation; Proteinuria; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Rats, Wistar; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Smad2 Protein; TOR Serine-Threonine Kinases

Novel methods to control and treat metastatic breast cancer are needed. Interleukin (IL)-15 is a promising cytokine for cancer immunotherapy, and everolimus is an orally administered mammalian target of rapamycin (mTOR) inhibitor, which is already approved for cancer treatment. In the present study, we investigated the efficacy of IL-15 gene therapy and explored the possibility of combining IL-15 therapy with everolimus to treat metastatic breast cancer.. A plasmid encoding IL-15 and everolimus were given to mice inoculated with 4 T1 mouse breast cancer cells. Tumor size and metastasis were monitored to assess the effect of different treatment regimens. Immunohistochemistry was used to detect CD4⁺, CD8⁺ and NKG2D⁺ cells and also the expression of Ki-67 in tumor tissue; these analyses helped establish the immunization status and tumor proliferation rate of different treatment groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to assess cellular apoptosis in tumor tissues.. Both IL-15 and everolimus significantly decreased tumor size. IL-15 gene therapy increased the proportion of CD4⁺ T and natural killer (NK) cells but had no effect on CD8⁺ T cells. By contrast, everolimus decreased the number of CD8⁺ T cells but had no effect on CD4⁺ T and NK cells compared to the control group. Both IL-15 and everolimus decreased expression of Ki-67 and increased rates of apoptosis. Although effective on their own, no synergistic effect was observed with a combined treatment of everolimus and IL-15 gene therapy.. IL-15 gene therapy was potentially useful for the treatment of metastatic breast cancer. The possibility of combining immunotherapy with everolimus requires further study.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line; Cell Proliferation; Combined Modality Therapy; Disease Models, Animal; Dose-Response Relationship, Drug; Everolimus; Female; Gene Order; Genetic Therapy; Genetic Vectors; Immunotherapy; Interleukin-15; Mice; Neoplasm Metastasis; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN). However, the mechanisms underlying this beneficial effect have not been elucidated.. Passive Heymann Nephritis (PHN) was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa). The remaining six rats were used as the proteinuric control group (PHN) while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC). All rats were sacrificed at the end of the 7th week.. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression.. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Glomerulonephritis, Membranous; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteinuria; Rats; Sirolimus

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by severe striatal atrophy with extensive neuronal loss and gliosis. Although the molecular mechanism is not well understood, experimental studies use the irreversible mitochondrial inhibitor 3-nitropropionic acid (3-NP) to mimic the neuropathological features of HD. In this study, the role of autophagy as a neuroprotective mechanism against 3-NP-induced astrocyte cytotoxicity was evaluated. Autophagy is a catabolic process that is essential for the turnover of cytosolic proteins and organelles and is involved in the modulation of cell death and survival. We showed that 3-NP-induced apoptosis, which was accompanied by Bax and Beclin-1 upregulation, was dependent on acidic vesicular organelle (AVO) formation after a continuous exposure to 3-NP for 12 h. The upregulation of Bax and Beclin-1 as well as AVO formation were normalized 24 h after 3-NP exposure.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Astrocytes; Autophagy; bcl-2-Associated X Protein; Beclin-1; Disease Models, Animal; Huntington Disease; Mice; Nitro Compounds; Propionates; Sirolimus

The Notch1 signaling pathway plays an essential role in cell growth and differentiation. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-cell acute lymphoblastic leukemia (T-ALL) in mice. Here we explored the drug sensitivity of T-ALL cells in two subpopulations of CD8(+)CD4(+) and CD8(+)CD4(-) cells in Notch1-induced T-ALL mice. We found that Notch1 induced T-ALL cells could be decreased by chemotherapeutic drug cyclophosphamide (CTX). CD8(+)CD4(-) T-ALL cells were more sensitive to CTX treatment than CD8(+)CD4(+) T-ALL cells. The percentage of apoptotic cells induced by CTX treatment was higher in CD8(+)CD4(-) T-ALL cells. T-ALL cells were also inhibited by inhibitor of mTORC1 rapamycin. CD8(+)CD4(+) T-ALL cells were more susceptible to rapamycin treatment than CD8(+)CD4(-) T-ALL cells. Rapamycin treatment selectively arrested more CD8(+)CD4(+) T-ALL cells at G0 phase of cell cycle. A combination of the two drugs significantly improved overall survival of T-ALL bearing mice when compared with CTX or rapamycin alone. These results indicated that CD8(+)CD4(+) and CD8(+)CD4(-) leukemia cell populations had distinct drug sensitivity.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; CD8-Positive T-Lymphocytes; Cell Proliferation; Cyclophosphamide; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Mice; Mice, Inbred C57BL; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Real-Time Polymerase Chain Reaction; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Tumor Cells, Cultured

Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo.. Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-β (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 μm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting.. The TGF-β activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7).. Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cicatrix; Corneal Opacity; Corneal Stroma; Disease Models, Animal; Female; Humans; Immunosuppressive Agents; Myofibroblasts; Photorefractive Keratectomy; Rabbits; Sirolimus; Transforming Growth Factor beta

Endovascular interventions on peripheral arteries are limited by high rates of restenosis. Our hypothesis was that adventitial injection of rapamycin nanoparticles would be safe and reduce luminal stenosis in a porcine femoral artery balloon angioplasty model.. Eighteen juvenile male crossbred swine were included. Single-injury (40%-60% femoral artery balloon overstretch injury; n=2) and double-injury models (endothelial denudation injury 2 weeks before a 20%-30% overstretch injury; n=2) were compared. The double-injury model produced significantly more luminal stenosis at 28 days, P=0.002, and no difference in medial fibrosis or inflammation. Four pigs were randomized to the double-injury model and adventitial injection of saline (n=2) or 500 μg of nanoparticle albumin-bound rapamycin (nab-rapamycin; n=2) with an endovascular microinfusion catheter. There was 100% procedural success and no difference in endothelial regeneration. At 28 days, nab-rapamycin led to significant reductions in luminal stenosis, 17% (interquartile range, 12%-35%) versus 10% (interquartile range, 8.3%-14%), P=0.001, medial cell proliferation, P<0.001, and fibrosis, P<0.001. There were significantly fewer adventitial leukocytes at 3 days, P<0.001, but no difference at 28 days. Pharmacokinetic analysis (single-injury model) found rapamycin concentrations 1500× higher in perivascular tissues than in blood at 1 hour. Perivascular rapamycin persisted ≥8 days and was not detectable at 28 days.. Adventitial nab-rapamycin injection was safe and significantly reduced luminal stenosis in a porcine femoral artery balloon angioplasty model. Observed reductions in early adventitial leukocyte infiltration and late medial cell proliferation and fibrosis suggest an immunosuppressive and antiproliferative mechanism. An intraluminal microinfusion catheter for adventitial injection represents an alternative to stent- or balloon-based local drug delivery.

Topics: Adventitia; Angioplasty, Balloon; Animals; Arteritis; Cell Movement; Cell Proliferation; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Femoral Artery; Fibrosis; Injections, Intra-Arterial; Leukocytes; Male; Nanoparticles; Sirolimus; Swine; Tunica Media

Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Topics: Animals; Brain; Disease Models, Animal; Electron Transport Complex I; Glycolysis; Leigh Disease; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Mice, Mutant Strains; Mitochondria; Mitochondrial Diseases; Molecular Targeted Therapy; Multiprotein Complexes; Neuroprotective Agents; Sirolimus; TOR Serine-Threonine Kinases

We developed an experimental autoimmune myositis (EAM) mouse model of polymyositis where we outlined the role of regulatory T (Treg) cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.. EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment), or during 10 days following the last myosin immunization (curative treatment).. Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2) = -0.645, p<0.0001). Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001), which were mostly activated regulatory T cells (CD62L(low)CD44(high): 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001). In rapamycin treated mice, inhibition of proliferation (Ki-67(+)) is more important in effector T cells compared to Tregs cells (p<0.05). Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low) CD62L(high) naive T cell and in CD62L(low) CD44(high) activated T cell.. Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.

Topics: Animals; B-Lymphocytes; CD4-Positive T-Lymphocytes; Disease Models, Animal; Female; Immunosuppressive Agents; Inflammation; Kruppel-Like Transcription Factors; Lymphocyte Count; Lymphopenia; Mice; Muscle Strength; Muscle, Skeletal; Nervous System Autoimmune Disease, Experimental; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory

While neurohumoral antagonists improve outcomes in heart failure (HF), cardiac remodeling and dysfunction progress and outcomes remain poor. Therapies superior or additive to standard HF therapy are needed. Pharmacologic mTOR inhibition by rapamycin attenuated adverse cardiac remodeling and dysfunction in experimental heart failure (HF). However, these studies used rapamycin doses that produced blood drug levels targeted for primary immunosuppression in human transplantation and therefore the immunosuppressive effects may limit clinical translation. Further, the relative or incremental effect of rapamycin combined with standard HF therapies targeting upstream regulators of cardiac remodeling (neurohumoral antagonists) has not been defined. Our objectives were to determine if anti-remodeling effects of rapamycin were preserved at lower doses and whether rapamycin effects were similar or additive to a standard HF therapy (angiotensin receptor blocker (losartan)). Experimental murine HF was produced by transverse aortic constriction (TAC). At three weeks post-TAC, male mice with established HF were treated with placebo, rapamycin at a dose producing immunosuppressive drug levels (target dose), low dose (50% target dose) rapamycin, losartan or rapamycin + losartan for six weeks. Cardiac structure and function (echocardiography, catheterization, pathology, hypertrophic and fibrotic gene expression profiles) were assessed. Downstream mTOR signaling pathways regulating protein synthesis (S6K1 and S6) and autophagy (LC3B-II) were characterized. TAC-HF mice displayed eccentric hypertrophy, systolic dysfunction and pulmonary congestion. These perturbations were attenuated to a similar degree by oral rapamycin doses achieving target (13.3±2.1 ng/dL) or low (6.7±2.5 ng/dL) blood levels. Rapamycin treatment decreased mTOR mediated regulators of protein synthesis and increased mTOR mediated regulators of autophagy. Losartan monotherapy did not attenuate remodeling, whereas Losartan added to rapamycin provided no incremental benefit over rapamycin alone. These data lend support to investigation of low dose rapamycin as a novel therapy in human HF.

Topics: Angiotensin Receptor Antagonists; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Heart; Heart Failure; Humans; Male; Mice; Receptors, Angiotensin; Sirolimus

Recent evidence has depicted a role of macrophage migration inhibitory factor (MIF) in cardiac homeostasis under pathological conditions. This study was designed to evaluate the role of MIF in doxorubicin-induced cardiomyopathy and the underlying mechanism involved with a focus on autophagy.. Wild-type (WT) and MIF knockout (MIF(-/-)) mice were given saline or doxorubicin (20 mg/kg cumulative, i.p.). A cohort of WT and MIF(-/-) mice was given rapamycin (6 mg/kg, i.p.) with or without bafilomycin A1 (BafA1, 3 μmol/kg per day, i.p.) for 1 week prior to doxorubicin challenge. To consolidate a role for MIF in the maintenance of cardiac homeostasis following doxorubicin challenge, recombinant mouse MIF (rmMIF) was given to MIF(-/-) mice challenged with or without doxorubicin. Echocardiographic, cardiomyocyte function, and intracellular Ca(2+) handling were evaluated. Autophagy and apoptosis were examined. Mitochondrial morphology and function were examined using transmission electron microscopy, JC-1 staining, MitoSOX Red fluorescence, and mitochondrial respiration complex assay. DHE staining was used to evaluate reactive oxygen species (ROS) generation. MIF knockout exacerbated doxorubicin-induced mortality and cardiomyopathy (compromised fractional shortening, cardiomyocyte and mitochondrial function, apoptosis, and ROS generation). These detrimental effects of doxorubicin were accompanied by defective autophagolysosome formation, the effect of which was exacerbated by MIF knockout. Rapamycin pretreatment rescued doxorubicin-induced cardiomyopathy in WT and MIF(-/-) mice. Blocking autophagolysosome formation using BafA1 negated the cardioprotective effect of rapamycin and rmMIF.. Our data suggest that MIF serves as an indispensable cardioprotective factor against doxorubicin-induced cardiomyopathy with an underlying mechanism through facilitating autophagolysosome formation.

Topics: Animals; Apoptosis; Autophagy; Calcium Signaling; Cardiomyopathies; Cytoprotection; Disease Models, Animal; Doxorubicin; Electron Transport Chain Complex Proteins; Intramolecular Oxidoreductases; Lysosomes; Macrolides; Macrophage Migration-Inhibitory Factors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Myocardial Contraction; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species; Sirolimus; Time Factors; Ventricular Function, Left; Ventricular Remodeling

To investigate the effect of rapamycin, an mTOR inhibitor, on learning and memory ability of mice with pilocarpine (PILO)-induced seizure.. One hundred and sixty male adult ICR mice were randomly grouped as vehicle control (n=20), rapamycin control (n=20), PILO model (n=40), rapamycin pre-treatment (n=40) and rapamycin post-treatment (n=40). PILO model and rapamycin treatment groups were injected with PILO to induce temporal lobe seizure. Rapamycin was administrated for 3 days before or after seizure. Morris water maze, Y maze and open field were used for the assessment of learning and memory, and FJB and Timm staining were conducted to detect the neuronal cell death and mossy fiber sprouting, respectively.. No significant cell death was observed in the mice with PILO-induced seizure. The learning and memory were impaired in mice 7 to 10 days after PILO-induced seizure, which was evident by prolongation of avoiding latency (P<0.05), decrease in number of correct reaction (P<0.01) and number of crossing (P<0.05). Treatment with rapamycin both pre-and post- PILO injection reversed seizure-induced cognitive impairment. In addition, rapamycin inhibited the mossy fiber sprouting after seizure (P<0.001).. Rapamycin improves learning and memory ability in ICR mice after PILO-induced seizure, and its mechanism needs to be further studied.

Topics: Animals; Cell Death; Disease Models, Animal; Epilepsy; Learning; Memory; Mice; Mice, Inbred ICR; Neurons; Pilocarpine; Sirolimus

To test the local delivery of sirolimus nanoparticles following percutaneous transluminal coronary angioplasty (PTCA) to treat in-stent restenosis (ISR) in a swine model.. Coronary bare-metal stent (BMS) implantation reduces major adverse cardiac events when compared with PTCA; however, ISR rates remain high.. Eighteen swine underwent BMS deployment guided by intravascular ultrasound (IVUS). Of these, 16 developed ISR (1 stent/swine) and underwent angioplasty with a noncompliant balloon (PTCA-NC). The animals were then randomized into four groups for local infusion of sirolimus nanoparticles through a porous balloon catheter, as follows: (1) PTCA-NC alone (control); (2) PTCA-NC + (polylactic acid)-based nanoparticle formulation (anionic 1); (3) PTCA-NC + (polylactic-co-glycolic acid)-based nanoparticle formulation (anionic 2); and (4) PTCA-NC + Eudragit RS nanoparticle formulation (cationic). Coronary angiography and IVUS follow-up were performed 28 days after ISR treatment.. There was one episode of acute coronary occlusion with the cationic formulation. Late area loss was similar in all groups at 28 days according to IVUS. However, luminal volume loss (control = 20.7%, anionic 1 = 4.0%, anionic 2 = 6.7%, cationic = 9.6%; P = 0.01) and neointimal volume gain (control = 68.7%, anionic 1 = 17.4%, anionic 2 = 29.5%, cationic = 31.2%; P = 0.019) were significantly reduced in all treatment groups, especially in anionic 1.. PTCA-NC followed by local infusion of sirolimus nanoparticles was safe and efficacious to reduce neointima in this model, and this strategy may be a promising treatment for BMS ISR. Further studies are required to validate this method in humans.

Topics: Acrylic Resins; Animals; Cardiac Catheters; Cardiovascular Agents; Chemistry, Pharmaceutical; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Equipment Design; Infusions, Parenteral; Lactic Acid; Nanoparticles; Neointima; Percutaneous Coronary Intervention; Polyesters; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Porosity; Sirolimus; Swine; Time Factors; Ultrasonography, Interventional

To evaluate differences in strut coverage, inflammation and endothelialization between two second-generation polymer-based drug-eluting stents (DES) in an atherosclerotic rabbit double-injury iliac artery model at 28 days follow-up.. Rabbits with induced atheroma received bilateral iliac artery stents: everolimus-eluting stent (Xience V EES; Abbott Vascular), zotarolimus-eluting stent (Resolute ZES; Medtronic CardioVascular), or bare-metal stent (BMS; MultiLink Vision; Abbott Vascular). After 28 days, total neointimal coverage examined by scanning electron microscopy was >98% for all three stent types. Neointimal thickness above stent struts was decreased by 50% in Xience V EES (0.06 ± 0.01 mm; P = 0.00001) compared with BMS (0.15 ± 0.03 mm) and Resolute ZES (0.12 ± 0.04 mm). Luminal area was largest for Xience V EES (3.79 ± 0.33 mm(2) ; P = 0.0003 for Xience V EES vs. BMS), followed by Resolute ZES (3.46 ± 0.45 mm(2) ; P = 0.083 for Resolute ZES vs. BMS) and BMS (3.07 ± 0.53 mm(2) ). Percentage area stenosis was smallest for Xience V EES (17.23 ± 3.64%; P = 0.00001), while BMS (30.25 ± 7.48%) and Resolute ZES (30.79 ± 7.15%) did not differ. Endothelial monolayer regrowth was significantly lower in Resolute ZES (65 ± 13%) versus BMS (79 ± 11%; P = 0.004). There was no difference between Xience V EES (74 ± 10%) and BMS. Xience V EES was further associated with a lower number of inflammatory cells surrounding the stent struts (7 ± 2 per strut) in comparison to Resolute ZES (15 ± 6; P = 0.0001) and BMS (17 ± 9; P = 0.0005).. In this atherosclerotic rabbit model, Xience V EES suppressed neointimal thickening better, with normal endothelial regrowth as compared with BMS, and less strut-induced inflammation.

Topics: Angioplasty, Balloon; Animals; Atherosclerosis; Cardiovascular Agents; Cell Proliferation; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Everolimus; Iliac Artery; Inflammation; Male; Neointima; Prosthesis Design; Rabbits; Radiography; Sirolimus; Time Factors; Vascular System Injuries

HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg, every other day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.

Topics: AIDS-Associated Nephropathy; Animals; Cell Proliferation; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Disease Progression; HIV-1; Humans; Kidney; Kidney Glomerulus; Kidney Tubules; Mice; NF-kappa B; Podocytes; Sclerosis; Sirolimus; Transcription Factor AP-1; Transcription, Genetic

A-type lamins, generated from the LMNA gene by differential splicing, are type V intermediate filament proteins that polymerize to form part of the nuclear lamina, and are of considerable medical interest because missense mutations in LMNA give rise to a wide range of dystrophic and progeroid syndromes. Among these are dilated cardiomyopathy and two forms of muscular dystrophy (limb-girdle and Emery-Dreifuss), which are modeled in lmna (-/-) mice and mice engineered to express human disease mutations. Our recent study demonstrates that cardiac and skeletal muscle pathology in lmna (-/-) mice can be attributed to elevated MTORC1 signaling leading to impairment of autophagic flux. An accompanying paper from another laboratory shows similar impairments in mice engineered to express the LMNA H222P associated with dilated cardiomyopathy in humans and also in left ventricular tissue from human subjects. MTORC1 inhibition with rapalogs restores autophagic flux and improves cardiac function in both mouse models, and extends survival in the lmna (-/-) mice. These findings elaborate a potential treatment option for dilated cardiomyopathy and muscular dystrophy associated with LMNA mutation and supplement growing evidence linking impaired autophagy to human disease.

Topics: Animals; Autophagy; Cardiomyopathy, Dilated; Disease Models, Animal; Humans; Lamin Type A; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Muscular Dystrophy, Animal; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Poor cell survival severely limits the beneficial effects of stem cell therapy for peripheral arterial disease (PAD). This study was designed to investigate the role of mammalian target of rapamycin (mTOR) in the survival and therapeutic function of transplanted murine adipose-derived stromal cells (mADSCs) in a murine PAD model. mADSCs (1.0 × 10(7)) were isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein-positive transgenic mice, intramuscularly implanted into the hind limb of C57BL/6 mice after femoral artery ligation/excision, and monitored using noninvasive bioluminescence imaging (BLI). Although engrafted mADSCs produced antiapoptotic/proangiogenic effects in vivo by modulating the inflammatory and angiogenic cytokine response involving the mTOR pathway, longitudinal BLI revealed progressive death of post-transplant mADSCs within ~4 weeks in the ischemic hind limb. Selectively targeting mTOR complex-1 (mTORC1) using low-dose rapamycin treatment with mADSCs attenuated proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor-alpha [TNF-α]) expression and neutrophil/macrophage infiltration, which overtly promoted mADSCs viability and antiapoptotic/proangiogenic efficacy in vivo. However, targeting dual mTORC1/mTORC2 using PP242 or high-dose rapamycin caused IL-1β/TNF-α upregulation and anti-inflammatory IL-10, IL-6, and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 downregulation, undermining the survival and antiapoptotic/proangiogenic action of mADSCs in vivo. Furthermore, low-dose rapamycin abrogated TNF-α secretion by mADSCs and rescued the cells from hypoxia/reoxygenation-induced death in vitro, while PP242 or high-dose rapamycin exerted proinflammatory effects and promoted cell death. In conclusion, mTORC1 and mTORC2 may differentially regulate inflammation and affect transplanted mADSCs' functional survival in ischemic hind limb. These findings uncover that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the translation of cell-based PAD therapy.

Topics: Adipocytes; Animals; Apoptosis; Cell Proliferation; Cell Survival; Disease Models, Animal; Down-Regulation; Femoral Artery; Green Fluorescent Proteins; Hindlimb; Inflammation; Interleukin-10; Interleukin-1beta; Interleukin-6; Ischemia; Luciferases, Firefly; Luminescent Measurements; Macrophages; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiprotein Complexes; Neovascularization, Pathologic; Neutrophils; Peripheral Arterial Disease; Proteins; Sirolimus; Stromal Cells; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors

Rapamycin, an inhibitor of target-of-rapamycin, extends lifespan in mice, possibly by delaying aging. We recently showed that rapamycin halts the progression of Alzheimer's (AD)-like deficits, reduces amyloid-beta (Aβ) and induces autophagy in the human amyloid precursor protein (PDAPP) mouse model. To delineate the mechanisms by which chronic rapamycin delays AD we determined proteomic signatures in brains of control- and rapamycin-treated PDAPP mice. Proteins with reported chaperone-like activity were overrepresented among proteins up-regulated in rapamycin-fed PDAPP mice and the master regulator of the heat-shock response, heat-shock factor 1, was activated. This was accompanied by the up-regulation of classical chaperones/heat shock proteins (HSPs) in brains of rapamycin-fed PDAPP mice. The abundance of most HSP mRNAs except for alpha B-crystallin, however, was unchanged, and the cap-dependent translation inhibitor 4E-BP was active, suggesting that increased expression of HSPs and proteins with chaperone activity may result from preferential translation of pre-existing mRNAs as a consequence of inhibition of cap-dependent translation. The effects of rapamycin on the reduction of Aβ, up-regulation of chaperones, and amelioration of AD-like cognitive deficits were recapitulated by transgenic over-expression of heat-shock factor 1 in PDAPP mice. These results suggest that, in addition to inducing autophagy, rapamycin preserves proteostasis by increasing chaperones. We propose that the failure of proteostasis associated with aging may be a key event enabling AD, and that chronic inhibition of target-of-rapamycin may delay AD by maintaining proteostasis in brain. Read the Editorial Highlight for this article on doi: 10.1111/jnc.12098.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; DNA-Binding Proteins; Heat Shock Transcription Factors; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Up-Regulation

Inhibition of mTOR is commonly considered a valid target in cancer treatment, but this assertion does not address effects on the immune microenvironment that may be detrimental to cancer treatment. Here we show how administration of the mTOR inhibitor RAD001 (everolimus) results in the occurrence of distant metastasis in a rat model of pancreatic cancer. RAD001 was administered twice weekly for 4.5 weeks as a single treatment or combined with [(177)Lu-DOTA,Tyr3]octreotate ((177)Lu-DOTATATE), where the latter targets the somatostatin receptor-2. The hypothesized synergistic therapeutic effect of RAD001 combined with (177)Lu-DOTATATE was, however, not observed in our experiments. The combination was shown to be less effective than (177)Lu-DOTATATE alone. Unexpectedly, tumor metastasis was observed in 77% of the subjects treated with RAD001, either alone or as part of the combination treatment. This was a striking effect, because metastasis did not occur in control or (177)Lu-DOTATATE-treated animals, including those where the primary tumor was surgically removed. These findings may be important clinically among noncompliant patients or patients that discontinue RAD001 therapy because of adverse effects.

Topics: Animals; Antineoplastic Agents; Carcinoma, Neuroendocrine; Cell Line, Tumor; Disease Models, Animal; Everolimus; Humans; Male; Mice; Mice, Nude; Neoplasm Metastasis; Pancreatic Neoplasms; Rats; Rats, Inbred Lew; Sirolimus; TOR Serine-Threonine Kinases

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder caused by mutation in either Tsc1 or Tsc2 genes that leads to the hyper activation of the mTOR pathway, a key signalling pathway for synaptic plasticity. TSC is characterized by benign tumors arising in different organs and severe neuropsychiatric symptoms, such as epilepsy, intellectual disability, autism, anxiety and depressive behaviour. Rapamycin is a potent inhibitor of mTOR and its efficacy in treating epilepsy and neurological symptoms remains elusive. In a mouse model in which Tsc1 has been deleted in embryonic telencephalic neural stem cells, we analyzed anxiety- and depression-like behaviour by elevated-plus maze (EPM), open-field test (OFT), forced-swim test (FST) and tail-suspension test (TST), after chronic administration of rapamycin. In addition, spectral analysis of background EEG was performed. Rapamycin-treated mutant mice displayed a reduction in anxiety- and depression-like phenotype, as shown by the EPM/OFT and FST, respectively. These results were inline with EEG power spectra outcomes. The same effects of rapamycin were observed in wild-type mice. Notably, in heterozygous animals we did not observe any EEG and/or behavioural variation after rapamycin treatment. Together these results suggest that both TSC1 deletion and chronic rapamycin treatment might have a role in modulating behaviour and brain activity, and point out to the potential usefulness of background EEG analysis in tracking brain dysfunction in parallel with behavioural testing.

Topics: Animals; Behavior, Animal; Disease Models, Animal; Electroencephalography; Maze Learning; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Sirolimus; Treatment Outcome; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Use of the mTOR inhibitor (mTORi) sirolimus to replace calcineurin inhibitors in kidney transplantation has been associated with improved renal function but, in a proportion of cases, also with de novo or exacerbated proteinuria. Experimental deficiency of vascular endothelial growth factor (VEGF) induces proteinuria and mTOR is required for VEGF production and signalling. We therefore explored the impact of sirolimus on the development of chronic allograft dysfunction (CAD) in the rat, with a focus on VEGF biology.. Lewis rats received F344 kidney allografts and were treated with 24 weeks of cyclosporine or sirolimus. Controls included allografts treated with cyclosporine for 10 days only and isografts treated with cyclosporine or sirolimus for 24 weeks. Kidney injury (proteinuria and histology) and expression of VEGF and VEGF-receptor (VEGFR; immunohistochemistry, laser capture micro-dissection and quantitative RT-PCR) were assessed.. Allograft controls developed proteinuria, tubulointerstitial fibrosis and atrophy, glomerulosclerosis, vasculopathy and leucocyte accumulation. Proteinuria was significantly reduced in both treatment groups but significantly more in cyclosporine treated animals. Tubulointerstitial damage, glomerulosclerosis and leucocyte accumulation were significantly attenuated in both treatment groups; however, vasculopathy was reduced only by sirolimus. Significantly diminished expression of VEGF and VEGFR mRNA and protein was evident in the sirolimus group. In vitro, sirolimus reduced VEGF production by podocytes (P < 0.05) and inhibited VEGF-induced proliferation of podocytes, endothelial and mesangial cells.. Cyclosporine and sirolimus retard development of CAD in this rat model. Sirolimus exhibits greater protection against vasculopathy but induces proteinuria; effects are likely to be related to inhibition of VEGF signalling.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cyclosporine; Disease Models, Animal; Immunosuppressive Agents; In Vitro Techniques; Kidney Transplantation; Male; Podocytes; Proteinuria; Rats; Rats, Inbred F344; Rats, Inbred Lew; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sirolimus; Transplantation, Homologous; Vascular Diseases; Vascular Endothelial Growth Factor A

Immunosuppressants are an important cause of posttransplantation diabetes mellitus. We have shown that tacrolimus and sirolimus induce hyperglycemia and hyperinsulinemia in normal rats. We hypothesized that metformin, given concurrently with tacrolimus and/or sirolimus, prevents disturbances in glucose and insulin metabolism.. Eight groups (n=6) of normal Sprague-Dawley rats were studied: four groups received tacrolimus, sirolimus, tacrolimus/sirolimus, or control for 14 days, and four more groups received similar treatments along with metformin. Daily glucoses were measured. All rats were administered an oral glucose challenge before sacrifice. Pancreata were analyzed by terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling staining and immunohistochemistry.. Tacrolimus, sirolimus, and tacrolimus/sirolimus impaired glucose tolerance compared to control. Sirolimus and tacrolimus/sirolimus also increased random blood glucose levels. Sirolimus alone resulted in hyperinsulinemia after oral glucose challenge compared to control. In the sirolimus/metformin and tacrolimus/sirolimus/metformin groups, mean daily random glucose was no longer increased, although the response to glucose challenge was still impaired. Metformin decreased pancreatic exocrine and trended to decrease endocrine apoptosis in tacrolimus/sirolimus group and reduced islet insulin content in sirolimus group.. This is the first study to show that metformin can improve immunosuppressant-induced hyperglycemia, when administered concurrently, and reduces exocrine apoptosis (reducing the impact on potential islet progenitor cells).

Topics: Animals; Apoptosis; Biomarkers; Blood Glucose; Disease Models, Animal; Glucose Tolerance Test; Hyperglycemia; Hyperinsulinism; Hypoglycemic Agents; Immunohistochemistry; Immunosuppressive Agents; In Situ Nick-End Labeling; Insulin; Male; Metformin; Pancreas, Exocrine; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus; Time Factors

Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression.. In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed.. Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression.. In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Topics: Animals; Aorta; Apoptosis; Blotting, Western; Cell Line; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Glucose; Glucose Transporter Type 1; Hyperplasia; Hypertrophy; Immunohistochemistry; Indican; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nephrectomy; Phloretin; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Renal Insufficiency; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases; RNA, Messenger; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation; Uremia

Esophageal squamous cell carcinoma (ESCC) remains one of the most aggressive cancers with poor prognosis regardless of a several reports that indicate a better therapeutic efficacy using some new chemotherapeutic agents. Recent drug development has contributed to an improved specificity to suppress mTOR activity by which many types of malignancies can be explosively progressed. Temsirolimus (CCI-779, TricelTM) is one of recently synthesized analogs of rapamycin and has provided better outcomes for patients with renal cell carcinoma. In this study, we experimentally evaluated an efficacy of targeting mTOR by temsirolimus for ESCC treatment, with an assessment of its survival advantage using an advanced ESCC animal model. First, we confirmed that the expression of phosphorylated mTOR was increased in 46 of 58 clinical ESCC tumor tissues (79.3%) and appeared to get strengthened with tumor progression. All of ESCC cell lines used in this study revealed an increase of mTOR phosphorylation, accompanied with the upregulation of hypoxia inducible factor-I α (HIF-1α), one of the critical effectors regulated by mTOR. Temsirolimus treatment apparently suppressed the activation of mTOR and its downstream effectors, resulting in the reduced ability of ESCC cell proliferation. Finally, the weekly administration of temsirolimus significantly diminished the size of subcutaneous tumors (vehicle, 3261.6 ± 722.0; temsirolimus, 599.2 ± 122.9; p = 0.007) in nude mice and effectively prolonged orthotopic esophageal cancer-bearing mice (median survival periods: control, 31 d; temsirolimus, 43 d; p = 0.0024). These data suggests that targeting mTOR by temsirolimus may become a therapeutic alternative for esophageal cancer, with a contribution to a better outcome.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Esophageal Neoplasms; Humans; Mice; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Iron deficiency (ID) is the most common nutrient deficiency worldwide, disproportionally affecting infants, children, and women of childbearing age. Although ID commonly occurs with anemia (IDA), nonanemic ID is 3 times more common than IDA in toddlers and also occurs in infants following gestational complications. Both conditions negatively affect motor, socio-emotional, and cognitive behaviors, suggesting that iron, apart from anemia, has a critical role in neurodevelopment. Here, the specific role of iron in regulation of mammalian target of rapamycin (mTOR) signaling (a kinase pathway that integrates metabolic supply and demand to regulate cell growth and morphology) was examined using 2 hippocampal, pyramidal cell-specific, nonanemic, genetic mouse models of ID: a CAMKIIα cre-loxP permanent knockout of divalent metal transporter-1 (DMT-1 CKO) and a CAMKIIα-tTA-driven reversible, overexpression of nonfunctional, dominant negative transferrin receptor-1 (DN TfR-1). In both models, mTOR activity, assessed by phosphorylation levels of key proteins, was upregulated during development by ID [S6K(Thr389) phosphorylation increased 87 and 57% in the DMT-1 CKO and DN TfR-1 models, respectively; P < 0.05]. This effect was shown to be iron-dependent, because iron repletion at postnatal d 21 normalized mTOR activity in the reversible DN TfR-1 model (62% reduction compared with unrepleted mice; P < 0.05). In the permanent DMT-1 CKO model, suppression of ID-induced mTOR hyperactivity by rapamycin administered during the sensitive period for iron improved Morris water maze performance despite ongoing ID (DMT-1 wild-type and DMT-1 CKO mice reached criterion in 3 d compared with 4 d necessary for vehicle-treated DMT-1 CKO mice; P < 0.05). Together, these findings implicate mTOR dysregulation as a cellular mechanism underlying the acute and persistent neurodevelopmental deficits that accompany early-life ID.

Topics: Animals; Animals, Newborn; Cation Transport Proteins; Disease Models, Animal; Hippocampus; Iron; Iron Deficiencies; Iron Metabolism Disorders; Maze Learning; Mice; Mice, Knockout; Mutation; Phosphorylation; Pyramidal Cells; Receptors, Transferrin; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Previous molecular studies showed that the mTOR inhibitor rapamycin prevents bladder smooth muscle hypertrophy in vitro. We investigated the effect of rapamycin treatment in vivo on bladder smooth muscle hypertrophy in a rat model of partial bladder outlet obstruction.. A total of 48 female Sprague-Dawley® rats underwent partial bladder outlet obstruction and received daily subcutaneous injections of rapamycin (1 mg/kg) or vehicle commencing 2 weeks postoperatively. A total of 36 rats underwent sham surgery and received rapamycin or vehicle. Rats were sacrificed 3, 6 and 12 weeks after surgery. Before sacrifice, voiding was observed in a metabolic cage for 24 hours. Bladder-to-body weight in gm bladder weight per kg body weight and post-void residual urine were assessed. We evaluated Col1a1, Col3a1, Eln and Mmp7 mRNA expression and histology. Two-factor ANOVA and the post hoc t test were applied.. Bladder outlet obstruction caused a significant increase in bladder weight in all obstructed groups. Three weeks postoperatively (1 week of treatment) there was no difference in the bladder-to-body weight ratio in the obstructed group. However, at 6 and 12 weeks (4 and 10 weeks of treatment, respectively) the bladder-to-body weight ratio of rats with obstruction plus rapamycin was significantly lower than that of rats with obstruction plus vehicle. Post-void residual urine volume after 6 and 12 weeks of obstruction was lower in obstructed rats with rapamycin compared to that in obstructed rats with vehicle. Rapamycin decreased the obstruction induced expression of Col1a1, Col3a1, Eln and Mmp7.. Rapamycin prevents mechanically induced hypertrophy in cardiovascular smooth muscle. In vivo mTOR inhibition may attenuate obstruction induced detrusor hypertrophy and help preserve bladder function.

Topics: Analysis of Variance; Animals; Biopsy, Needle; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Hypertrophy; Immunohistochemistry; Injections, Subcutaneous; Muscle Hypertonia; Muscle, Smooth; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Urinary Bladder; Urinary Bladder Neck Obstruction

Gastrointestinal cancers are frequently associated with chronic inflammation and excessive secretion of IL-6 family cytokines, which promote tumorigenesis through persistent activation of the GP130/JAK/STAT3 pathway. Although tumor progression can be prevented by genetic ablation of Stat3 in mice, this transcription factor remains a challenging therapeutic target with a paucity of clinically approved inhibitors. Here, we uncovered parallel and excessive activation of mTOR complex 1 (mTORC1) alongside STAT3 in human intestinal-type gastric cancers (IGCs). Furthermore, in a preclinical mouse model of IGC, GP130 ligand administration simultaneously activated mTORC1/S6 kinase and STAT3 signaling. We therefore investigated whether mTORC1 activation was required for inflammation-associated gastrointestinal tumorigenesis. Strikingly, the mTORC1-specific inhibitor RAD001 potently suppressed initiation and progression of both murine IGC and colitis-associated colon cancer. The therapeutic effect of RAD001 was associated with reduced tumor vascularization and cell proliferation but occurred independently of STAT3 activity. We analyzed the mechanism of GP130-mediated mTORC1 activation in cells and mice and revealed a requirement for JAK and PI3K activity but not for GP130 tyrosine phosphorylation or STAT3. Our results suggest that GP130-dependent activation of the druggable PI3K/mTORC1 pathway is required for inflammation-associated gastrointestinal tumorigenesis. These findings advocate clinical application of PI3K/mTORC1 inhibitors for the treatment of corresponding human malignancies.

Topics: Animals; Cytokine Receptor gp130; Disease Models, Animal; Everolimus; Female; Gastrointestinal Neoplasms; Gene Expression; Humans; Inflammation Mediators; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Multiprotein Complexes; Proteins; Signal Transduction; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

The deoxycorticosterone acetate (DOCA)-salt mouse model exhibits adverse cardiac remodeling in male mice and cardiac protection in female mice, even when blood pressure is normalized. We hypothesized that intact mammalian target of rapamycin (mTOR) signaling is necessary for cardiac protection in females. We first tested sex differences and intracellular signaling after mTOR targeting with rapamycin in wild-type mice. Radio-telemetric blood pressure was maintained at normal for 6 weeks. Rapamycin significantly reduced left ventricular hypertrophy, preserved ejection fraction, inhibited fibrosis, and maintained capillary structure in male mice. Decreased mTORC1 and increased mTORC2 activity were detected in rapamycin-treated male mice compared with vehicle controls. In contrast, female mice developed dilative left ventricular hypertrophy, cardiac fibrosis, and capillary loss similar to DOCA-salt females lacking the estrogen receptor β (ERβ(-/-)) that we described earlier. Because rapamycin downregulated ERβ in female mice, we next studied ERβ(-/-) normotensive DOCA-salt females. Vehicle-treated wild-type females maintained their high constitutive mTORC1 and mTORC2 in response to DOCA-salt. In contrast to males, both mTORCs were decreased by rapamycin, in particular mTORC2 by 60%. ERβ(-/-) DOCA-salt females showed similar mTORC1 and mTORC2 response patterns. We suggest that ERβ-dependent regulation involves sex-specific use of mTOR signaling branches. Maintenance of both mTORC1 and mTORC2 signaling seems to be essential for adaptive cardiac remodeling in females and supports a rationale for sex-specific therapeutic strategies in left ventricular hypertrophy.

Topics: Adaptation, Physiological; Animals; Blood Pressure; Capillaries; Desoxycorticosterone; Disease Models, Animal; Estrogen Receptor beta; Female; Heart; Hypertrophy, Left Ventricular; Male; Mice; Mice, Inbred C57BL; Mineralocorticoids; Sex Factors; Signal Transduction; Sirolimus; Stroke Volume; TOR Serine-Threonine Kinases

Pharmacological approaches can potentially improve fatty liver condition in alcoholic and non-alcoholic fatty liver diseases. The salutary effects of reducing lipid synthesis or promoting lipid oxidation have been well reported, but the benefits of increasing lipid degradation have yet to be well explored. Macroautophagy is a cellular degradation process that can remove subcellular organelles including lipid droplets. We thus investigated whether pharmacological modulation of macroautophagy could be an effective approach to alleviate fatty liver condition and liver injury.. C57BL/6 mice were given ethanol via intraperitoneal injection (acute) or by a 4-week oral feeding regime (chronic), or high fat diet for 12 weeks. An autophagy enhancer, carbamazepine or rapamycin, or an autophagy inhibitor, chloroquine, was given before sacrifice. Activation of autophagy, level of hepatic steatosis, and blood levels of triglycerides, liver enzyme, glucose and insulin were measured.. In both acute and chronic ethanol condition, macroautophagy was activated. Carbamazepine, as well as rapamycin, enhanced ethanol-induced macroautophagy in hepatocytes in vitro and in vivo. Hepatic steatosis and liver injury were exacerbated by chloroquine, but alleviated by carbamazepine. The protective effects of carbamazepine and rapamycin in reducing steatosis and in improving insulin sensitivity were also demonstrated in high fat diet-induced non-alcoholic fatty liver condition.. These findings indicate that pharmacological modulation of macroautophagy in the liver can be an effective strategy for reducing fatty liver condition and liver injury.

Topics: Animals; Autophagy; Biomarkers; Carbamazepine; Cells, Cultured; Chloroquine; Dietary Fats; Disease Models, Animal; Ethanol; Fatty Liver; Fatty Liver, Alcoholic; Hepatocytes; In Vitro Techniques; Lipid Metabolism; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Non-alcoholic Fatty Liver Disease; Sirolimus

The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4-6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4(+) T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4(+) T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Dexamethasone; Disease Models, Animal; Goblet Cells; Hypersensitivity; Immunoglobulin G; Inflammation; Interleukin-4; Mice; Pyroglyphidae; Sirolimus; TOR Serine-Threonine Kinases

pH-sensitive liposomes (HHG2C(18)-L and PEGHG2C(18)-L) based on zwitterionic oligopeptide lipids as anticancer drug carriers were developed and evaluated for effective intracellular delivery and enhanced antitumor activity. The amino acid-based lipids, 1,5-dioctadecyl-l-glutamyl 2-histidyl-hexahydrobenzoic acid (HHG2C(18)) and 1,5-distearyl N-(N-α-(4-mPEG2000) butanedione)-histidyl-l-glutamate (PEGHG2C(18)), were synthesized, which have the multistage pH-response to tumor microenvironmental pH (pH(e), pH 6.0-7.0) and endosomal/lysosomal pH (pH(i), pH 4.0-6.0) successively. HHG2C(18)-L contains HHG2C(18), while PEGHG2C(18)-L includes HHG2C(18) and PEGHG2C(18). Both of them displayed the capability of charge conversion to the surrounding pH. The zeta potentials of HHG2C(18)-L and PEGHG2C(18)-L were negative at pH 7.4, whereas positive at pH 6.5 and more positive at lower pH. Coumarin 6-loaded HHG2C(18)-L (C6/HHG2C(18)-L) and PEGHG2C(18)-L (C6/PEGHG2C(18)-L) showed higher tumor cellular uptake due to electrostatic absorptive endocytosis at pH(e) (pH 6.5), produced proton sponge effect for endo-lysosomal escape, and accumulated to the mitochondria based on stronger positive charge by the hydrolysis of a pH-sensitive linker at pH(i) (pH 5.5 and pH 4.5). Furthermore, temsirolimus (CCI-779)-loaded HHG2C(18)-L (CCI-779/HHG2C(18)-L) and PEGHG2C(18)-L (CCI-779/PEGHG2C(18)-L) had significantly higher antiproliferative and apoptosis inducing effects toward the human renal carcinoma (A498) cells at pH 6.5 relative to that at pH 7.4. The half maximal inhibitory concentration (IC50) of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L were about 3 μg/mL and 5 μg/mL at pH 6.5, 1.67-fold and 1.60-fold improved relative to that at pH 7.4, respectively. The total apoptotic ratio of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L increased from 9.90% and 7.78% at pH 7.4 to 19.53% and 12.10% at pH 6.5, respectively. In vivo, CCI-779/PEGHG2C(18)-L after intravenous administration presented remarkably higher bioavailability and blood persistence compared with unPEGylated CCI-779/HHG2C(18)-L, and had the strongest antitumor efficacy against xenograft renal cancer (Renca) tumor models. Accordingly, the results provide the feasibility of using pH-sensitive zwitterionic oligopeptide lipids to extend the applications of liposomes to efficient anticancer drug delivery in cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Delivery Systems; Endocytosis; Humans; Hydrogen-Ion Concentration; Lipids; Liposomes; Male; Mice; Mitochondria; Oligopeptides; Rats; Rats, Sprague-Dawley; Sirolimus; Tumor Microenvironment

Kaposi sarcoma originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi sarcoma-associated herpesvirus (KSHV) are endemic, Kaposi sarcoma is the most common cancer overall, but model systems for disease study are insufficient. Here, we report the development of a novel mouse model of Kaposi sarcoma, where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, and RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results show that mTOR inhibitors exert a direct anti-Kaposi sarcoma effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for Kaposi sarcoma and other cancers of endothelial origin.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Proliferation; Comparative Genomic Hybridization; Disease Models, Animal; DNA, Viral; Doxorubicin; Fluorescent Antibody Technique; Herpesvirus 8, Human; Humans; Immunoenzyme Techniques; Mice; Mice, SCID; Neovascularization, Pathologic; Phosphorylation; Real-Time Polymerase Chain Reaction; Sarcoma, Kaposi; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Tuberous sclerosis complex (TSC) is a hamartoma syndrome in which brain, renal, and lung tumors develop and cause both morbidity and death. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1. Rapamycin and related drugs have been shown to have clinical benefit for these tumors in patients with TSC and those with sporadic forms of TSC-related neoplasms. However, lifelong therapy seems to be required, as tumors are not eliminated by this treatment. We examined the potential benefit of MLN0128, a novel potent mTOR ATP-competitive inhibitor, as a therapeutic strategy for renal cystadenomas that develop in A/J Tsc2(+/-) mice. Rapamycin given by intraperitoneal injection at 3 mg/kg 3 times per week, and MLN0128 given by gavage at 0.75 mg/kg 5 times per week had equivalent effects in suppressing tumor development during a 4-week treatment period, with an approximate 99% reduction in microscopic tumor cell volume. Marked reduction in activation of mTOR complex (mTORC)1 and blockade of cell growth was seen with both drugs, whereas only MLN0128 treatment had effects in blocking mTORC2 and 4EBP1 phosphorylation. However, when either drug was discontinued and mice were observed for two additional months, there was dramatic recovery of tumor growth, with extensive proliferation. Hence, longlasting tumor growth control is not achieved with transient treatment with either drug, and MLN0128 and rapamycin have equivalent therapeutic benefit in this mouse model. Differences in side-effect profiles might make MLN0128 more attractive for treatment of patients with TSC-related tumors, but will require additional study in humans.

Topics: Animals; Benzoxazoles; Cell Growth Processes; Cystadenoma; Disease Models, Animal; Immunohistochemistry; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphorylation; Pyrimidines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis

We aimed to determine the effect of mechanistic target of rapamycin inhibitor everolimus on abdominal aortic aneurysm within the angiotensin II (A2)-infused apolipoprotein E-deficient mouse model.. Abdominal aortic aneurysm was induced via subcutaneous infusion of A2. Flow cytometry demonstrated increased circulating and aortic C-C chemokine receptor 2 (CCR2) monocytes during A2 infusion. The number of CCR2 monocytes present within the aorta was positively correlated with suprarenal aortic diameter. Simultaneous infusion of everolimus via a second subcutaneous osmotic micropump inhibited A2-induced aortic dilatation. Using flow cytometry and Western blot analysis, decreased aortic dilatation was associated with reduced development of CCR2 bone marrow monocytes, fewer numbers of circulating CCR2 monocytes, and lower aortic CCR2 concentration. In vitro, everolimus inhibited A2-stimulated production of interferon (IFN)-γ and IFNγ-induced CCR2 expression in apolipoprotein E-deficient mouse bone marrow monocytes. Further, everolimus diminished IFNγ/lipopolysaccharide-stimulated M1 polarization in apolipoprotein E-deficient mouse bone marrow monocyte-differentiated macrophages.. Systemic administration of everolimus limits aortic aneurysm in the A2-infused apolipoprotein E-deficient mouse model via suppressed development of bone marrow CCR2 monocytes and reduced egress of these cells into the circulation.

Topics: Angiotensin II; Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Apolipoproteins E; Cell Movement; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Everolimus; Flow Cytometry; Infusion Pumps, Implantable; Infusions, Subcutaneous; Interferon-gamma; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Protein Kinase Inhibitors; Receptor, Macrophage Colony-Stimulating Factor; Receptors, CCR2; Sirolimus; TOR Serine-Threonine Kinases

To investigate the effects of the potent immunosuppressive agents tacrolimus and rapamycin on the number of circulating mature endothelial cells and circulating endothelial progenitor cells in an experimental model.. It was an experimental study performed from December 2007 to January 2008 in which the effects of the immunosuppressive agents tacrolimus and rapamycin on endothelial progenitr cells and circulating mature endothelial cells were analysed on 24 male wistar albino rats in a controlled environment model. Circulating cell populations were measured by flow-cytometric analysis. Maun-Whitney U test and analysis of vartiance were used for statistical purposes.. Rapamycin increased the number of circulating mature endothelial cells approximately 2-fold compared to tacrolimus. The number of endothelial progenitor cells also was increased in the peripheral blood of rats treated with rapamycin compared to those treated with tacrolimus.. The study showed that treatment with rapamycin is associated with an increase in endothelial progenitor cells and circulating mature endothelial cells. This increase may be associated with endothelial cell damage and repair.

Topics: Animals; Cell Count; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Flow Cytometry; Graft Rejection; Immunosuppressive Agents; Kidney Transplantation; Male; Rats; Rats, Wistar; Sirolimus; Stem Cells; Tacrolimus

Neurofibromatosis type 1 (NF1) is an inherited disease predisposing affected patients to variable numbers of benign neurofibromas. To date there are no effective chemotherapeutic drugs available for this slow growing tumor. Molecularly targeted agents that aim to slow neurofibroma growth are being tested in clinical trials. So preclinical models for testing potential therapies are urgently needed to prioritize drugs for clinical trials of neurofibromas.. We used magnetic resonance imaging (MRI) to monitor neurofibroma development in the Nf1(flox/flox) ;DhhCre mouse model of GEM grade I neurofibroma. Based on studies implicating mTOR and Raf signaling in NF1 mutant cells, we tested the therapeutic effect of RAD001 and Sorafenib in this model. Mice were scanned to establish growth rate followed by 8 weeks of drug treatment, then re-imaged after the last dose of drug treatment. Tumor volumes were determined by volumetric measurement.. We found that rate of tumor growth varied among mice, as it does in human patients. RAD001 inhibited its predicted target pS6K, yet there was no significant decrease in the tumor volume in RAD001 treated mice compared to the vehicle control group. Sorafenib inhibited cyclinD1 expression and cell proliferation in tumors, and volumetric measurements identified significant decreases in tumor volume in some mice.. The data demonstrate that volumetric MRI analysis can be used to monitor the therapeutic effect in the preclinical neurofibroma drug screening, and suggest that Sorafenib might have clinical activity in some neurofibromas.

Topics: Animals; Antineoplastic Agents; Benzenesulfonates; Blotting, Western; Cell Proliferation; Chromatography, High Pressure Liquid; Disease Models, Animal; Drug Evaluation, Preclinical; Everolimus; Female; Hedgehog Proteins; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Integrases; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurofibroma, Plexiform; Neurofibromin 1; Niacinamide; Phenylurea Compounds; Pyridines; Signal Transduction; Sirolimus; Sorafenib; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tissue Distribution; Tumor Burden

Dietary restriction (DR) and rapamycin (Rapa) have been shown to increase the lifespan of a variety of organisms leading to the speculation that these interventions increase lifespan through related mechanisms. However, both these interventions have a detrimental effect in the G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). Our previous work indicated that different ALS SOD1 mutant mouse models differ in disease pathogenesis; therefore in this study we measured the effect of DR and Rapa in a second ALS mutant mouse model (the H46R/H48Q mutant). Interestingly, in mice expressing this mutant SOD1 protein, DR significantly delays disease onset and extends lifespan, while Rapa has no effect. These findings suggest that: (1) the effect of DR in ALS is not mediated through pathways common with Rapa, (2) the deleterious effect of DR and Rapa in the G93A ALS mouse model may not be universal to disease caused by all SOD1 mutations, and (3) the results reinforce our previous conclusions that the pathogenic mechanisms in G93A and H46R/H48Q mice are distinct.

Topics: Amyotrophic Lateral Sclerosis; Animals; Caloric Restriction; Disease Models, Animal; Humans; Immunosuppressive Agents; Mice; Mice, Transgenic; Sirolimus; Survival Analysis; Survival Rate; Treatment Outcome

Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro.. Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study, rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8), kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL).. SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03), regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies.. mTOR inhibition induces microcytosis and polyglobulia, but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells.

Topics: Anemia; Animals; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Erythrocyte Count; Erythrocyte Indices; Erythroid Precursor Cells; Erythropoiesis; Hematologic Diseases; Humans; Immunosuppressive Agents; Infusions, Parenteral; Kidney Transplantation; Leukocytes, Mononuclear; Male; Random Allocation; Rats; Rats, Wistar; Reference Values; Sirolimus; Statistics, Nonparametric; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that negatively regulates autophagy. Rapamycin, an inhibitor of mTOR signaling, can promote autophagy and exert neuroprotective effects in several diseases of the central nervous system (CNS). In the present study, we examined whether rapamycin treatment promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin significantly decreased the phosphorylation of the p70S6K protein and led to higher expression levels of LC3 and Beclin 1 in the injured spinal cord. In addition, neuronal loss and cell death in the injured spinal cord were significantly reduced in the rapamycin-treated mice compared to the vehicle-treated mice. Furthermore, the rapamycin-treated mice showed significantly higher locomotor function in Basso Mouse Scale (BMS) scores than did the vehicle-treated mice. These results indicate that rapamycin promoted autophagy by inhibiting the mTOR signaling pathway, and reduced neural tissue damage and locomotor impairment after SCI. The administration of rapamycin produced a neuroprotective function at the lesion site following SCI. Rapamycin treatment may represent a novel therapeutic strategy after SCI.

Topics: Animals; Autophagy; Blotting, Western; Disease Models, Animal; Female; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Sirolimus; Spinal Cord Injuries

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the po

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Carbon Radioisotopes; Disease Models, Animal; Everolimus; Glioblastoma; Humans; Magnetic Resonance Spectroscopy; Male; Neuroimaging; Rats; Rats, Nude; Sirolimus; Xenograft Model Antitumor Assays

Tuberous Sclerosis Complex (TSC) is a multiorgan genetic disease that prominently features brain malformations (tubers) with many patients suffering from epilepsy and autism. These malformations typically exhibit neuronal as well as glial cell abnormalities and likely underlie much of the neurological morbidity seen in TSC. Tuber pathogenesis remains poorly understood though upregulation of the mTORC1 signaling pathway in TSC has been consistently demonstrated. Here we address abnormal brain development in TSC by inactivating the mouse Tsc1 gene in embryonic neural progenitor cells. This strategy permits evaluation of the role of the Tsc1 gene in both neuronal as well as glial cell lineages. Tsc1(Emx1-Cre) conditional knockout (CKO) animals die by 25 days of life. Their brains have increased size and contain prominent large cells within the cerebral cortex that have greatly increased mTORC1 signaling and decreased mTORC2 signaling. Severe defects of cortical lamination, enlarged dysmorphic astrocytes and decreased myelination were also found. Tsc1(Emx1-Cre) CKO mice were then treated with rapamycin to see if the premature death and brain abnormalities can be rescued. Postnatal rapamycin treatment completely prevented premature death and largely reversed the glia pathology but not abnormal neuronal lamination. These findings support a model that loss of function of the TSC genes in embryonic neural progenitor cells causes cortical malformations in patients with TSC. The dramatic effect of rapamycin suggests that even with extensive multi-lineage abnormalities, a postnatal therapeutic window may exist for patients with TSC.

Topics: Animals; Brain; Cell Death; Disease Models, Animal; Mice; Mice, Knockout; Neuroglia; Neurons; Signal Transduction; Sirolimus; Transcription Factors; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Campylobacter jejuni is the worldwide leading cause of bacterial-induced enteritis. The molecular and cellular events that lead to campylobacteriosis are poorly understood. We identify mammalian target of rapamycin (mTOR) as a signaling pathway that leads to C jejuni-induced intestinal inflammation.. Germ-free (control) or conventionally derived Il10(-/-) mice that express enhanced green fluorescent protein (EGFP) under the control of nuclear factor κB (Il10(-/-); NF-κB(EGFP) mice) were infected with C jejuni (10(9) colony-forming units/mouse) for 12 days; their responses were determined using histologic, semiquantitative reverse-transcription polymerase chain reaction, fluorescence in situ hybridization, transmission electron microscopy, and tissue culture analyses. mTOR signaling was blocked by daily intraperitoneal injections of the pharmacologic inhibitor rapamycin (1.5 mg/kg). CD4(+) T cells were depleted by intraperitoneal injections of antibodies against CD4 (0.5 mg/mouse every 3 days). Bacterial survival in splenocytes was measured using a gentamycin killing assay.. C jejuni induced intestinal inflammation, which correlated with activation of mTOR signaling and neutrophil infiltration. The inflamed intestines of these mice had increased levels of interleukin-1β, Cxcl2, interleukin-17a, and EGFP; C jejuni localized to colons and extraintestinal tissues of infected Il10(-/-); NF-κB(EGFP) mice compared with controls. Rapamycin, administered before or after introduction of C jejuni, blocked C jejuni-induced intestinal inflammation and bacterial accumulation. LC3II processing and killing of C jejuni were increased in splenocytes incubated with rapamycin compared with controls.. mTOR signaling mediates C jejuni-induced colitis in Il10(-/-) mice, independently of T-cell activation. Factors involved in mTOR signaling might be therapeutic targets for campylobacteriosis.

Topics: Animals; Campylobacter Infections; Campylobacter jejuni; CD4-Positive T-Lymphocytes; Cells, Cultured; Chemokine CXCL2; Colitis; Colon; Disease Models, Animal; Enzyme Activation; Green Fluorescent Proteins; In Situ Hybridization, Fluorescence; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-10; Interleukin-17; Interleukin-1beta; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Electron, Transmission; Neutrophil Infiltration; Neutrophils; NF-kappa B; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.

Topics: Age Factors; Aging; Animals; Animals, Newborn; Cardiomyopathy, Hypertrophic; Cell Size; Cells, Cultured; Disease Models, Animal; Fibrosis; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Mutation; Myocardial Contraction; Myocytes, Cardiac; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Ventricular Function, Left

To evaluate the effect of a polymer-free Biolimus A9-eluting stent [BioFreedom (BF)], compared with that of a biodegradable polymer-based Biolimus A9-eluting stent [BioMatrix Flex (BMF)] and a bare metal stent (BMS) in balloon denuded and radiated hypercholesterolemic rabbit iliac arteries.. Rabbits were fed with 1% cholesterol diet (n = 14) for 14 days, both iliac arteries were balloon denuded and radiated, and then rabbits were switched to 0.15% cholesterol diet. After 4 weeks, BF (n = 8), BMF (n = 8), and BMS (n = 8) were deployed in denuded and radiated areas. Four weeks later animals were euthanized, arterial segments were processed for morphometry.. The neointimal area in vessels implanted with BF stents was significantly less than that seen in vessels implanted with BMS (0.90 mm(2) ± 0.14 vs. 1.29 mm(2) ± 0.23, P <0.01). Percent fibrin and fibrin score were higher with BMF stents compared to BMS (P <0.03 and <0.04) and giant cell number was significantly higher with both BMF and BF stents (P < 0.01 for both). Percent endothelialization was significantly higher and % uncovered struts were lower with BMS compared to either BMF or BF stents (P < 0.05 for both).. This study demonstrates that compared to BMS, BF stents significantly decreased neointimal hyperplasia.

Topics: Absorbable Implants; Angioplasty, Balloon; Animals; Atherosclerosis; Cardiovascular Agents; Constriction, Pathologic; Disease Models, Animal; Drug-Eluting Stents; Fibrin; Hypercholesterolemia; Hyperplasia; Iliac Artery; Inflammation; Male; Metals; Neointima; Plaque, Atherosclerotic; Polymers; Prosthesis Design; Rabbits; Sirolimus; Stents; Time Factors

We investigated microRNAs (miRs) associated with PTEN/mTOR signaling after spinal cord injury (SCI) and after hind limb exercise (Ex), a therapy implicated in promoting spinal cord plasticity. After spinalization, rats received cycling Ex 5 days/week. The expression of miRs, their target genes and downstream effectors were probed in spinal cord tissue at 10 and 31 days post injury. Ex elevated expression of miR21 and decreased expression of miR 199a-3p correlating with significant change in the expression of their respective target genes: PTEN mRNA decreased and mTOR mRNA increased. Western blotting confirmed comparable changes in protein levels. An increase in phosphorylated-S6 (a downstream effector of mTOR) within intermediate grey neurons in Ex rats was blocked by Rapamycin treatment. It thus appears possible that activity-dependent plasticity in the injured spinal cord is modulated in part through miRs that regulate PTEN and mTOR signaling and may indicate an increase in the regenerative potential of neurons affected by a SCI.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Exercise Therapy; Female; Gene Expression Regulation; MicroRNAs; Motor Activity; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Extensive efforts are underway to develop small-molecule inhibitors of the mammalian target of rapamycin (mTOR) kinase. It is hoped that these inhibitors will have widespread clinical impact in oncology because mTOR is a major downstream effector of PI3K signaling, one of the most frequently activated pathways in cancer. In cells, mTOR is the catalytic core subunit of two distinct complexes, mTORC1 and mTORC2, which are defined by unique mTOR-interacting proteins and have unique functions downstream of PI3K. Two classes of mTOR inhibitors are currently being evaluated as cancer therapeutics: rapamycin and its analogs, which partially inhibit mTORC1 and in some cell types mTORC2, and the recently described ATP-competitive inhibitors, which inhibit the kinase activity of both complexes. Although small molecules that selectively target mTORC2 do not yet exist, experiments using mouse genetics suggest that a theoretical mTORC2 inhibitor may have significant therapeutic value. Here, we discuss an approach to model mTOR complex specific inhibitors using mouse genetics and how it can be applied to other gene products involved in oncogenic signaling to which inhibitors do not exist.

Topics: Adenosine Triphosphate; Animals; Cell Proliferation; Disease Models, Animal; Drug Discovery; Gene Knockout Techniques; Humans; Male; Mice; Mice, Knockout; Prostatic Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Trans-Activators; Transcription Factors

Obesity is reaching pandemic proportions in Western society. It has resulted in increasing health care burden and decreasing life expectancy. Obesity is a complex, chronic disease, involving decades of pathophysiological changes and adaptation. Therefore, it is difficult ascertain the exact mechanisms for this long-term process in humans. To circumvent some of these issues, several surrogate models are available, including murine genetic loss-of-function mutations, transgenic gain-of-function mutations, polygenic models, and different environmental exposure models. The mouse model of diet-induced obesity has become one of the most important tools for understanding the interplay of high-fat Western diets and the development of obesity. The diet-induced obesity model closely mimics the increasingly availability of the high-fat/high-density foods in modern society over the past two decades, which are main contributors to the obesity trend in human. This model has lead to many discoveries of the important signalings in obesity, such as Akt and mTOR. The chapter describes protocols for diet induced-obesity model in mice and protocols for measuring insulin resistance and sensitivity.

Topics: Animals; Diet, High-Fat; Disease Models, Animal; Glucose; Humans; Insulin; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Obesity; Oncogene Protein v-akt; Sirolimus; TOR Serine-Threonine Kinases

Tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. In order to test roles for TSC1 and mTORC1 in β-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic β cells (Rip-Tsc1cKO mice). While obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function β-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of β-cell size and insulin production, but not β-cell number consistent with an important anabolic role for mTOR in β-cell function. Thus, mTOR promulgates a dominant signal to promote β-cell/islet size and insulin production, and this pathway is crucial for β-cell function and glycemic control. Here, we describe the methods of analyzing tissue-specific ablation of Tsc1 in pancreatic β cells.

Topics: Animals; Disease Models, Animal; Gene Expression Regulation; Gene Knockout Techniques; Humans; Insulin; Insulin-Secreting Cells; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Promoter Regions, Genetic; Proteins; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Obesity is an established risk and progression factor for postmenopausal breast cancer. Interventions to decrease caloric intake and/or increase energy expenditure beneficially impact tumor progression in normoweight humans and animal models. However, despite the increasingly high global prevalence of obesity, the effects and underlying mechanisms of these energy balance modulating interventions are poorly characterized in obese individuals. The goal of this study was to better characterize the mechanism(s) responsible for the link between energy balance and breast cancer progression in the postmenopausal obesity context. We compared the effects of calorie restriction (CR), treadmill exercise (EX), and mammalian target of rapamycin (mTOR inhibitor) treatment on body composition, serum biomarkers, cellular signaling, and mammary tumor growth in obese mice. Ovariectomized C57BL/6 mice were administered a diet-induced obesity regimen for 8 weeks, then randomized into three treatment groups: control (semipurified diet fed ad libitum, maintained the obese state); 30% CR (isonutrient relative to control except 30% reduction in carbohydrate calories); and EX (control diet fed ad libitum plus treadmill exercise). Mice were implanted with syngeneic MMTV-Wnt-1 mammary tumor cells at week 12. Rapamycin treatment (5  mg/kg every 48  h) started at week 14. Tumors were excised at week 18. CR and rapamycin (but not EX) significantly reduced final tumor weight compared to control. In follow-up analysis, constitutive activation of mTOR ablated the inhibitory effects of CR on Wnt-1 mammary tumor growth. We conclude that mTOR inhibition may be a pharmacologic strategy to mimic the anticancer effects of CR and break the obesity-breast cancer progression link.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Caloric Restriction; Cell Cycle; Cell Proliferation; Disease Models, Animal; Energy Intake; Female; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C57BL; Obesity; Ovariectomy; Physical Conditioning, Animal; Postmenopause; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Wnt1 Protein

Anorexia-cachexia syndrome (ACS) is a major determinant of cancer-related death that causes progressive body weight loss due to depletion of skeletal muscle mass and body fat. Here, we report the development of a novel preclinical murine model of ACS in which lymphomas harbor elevated Myc and activated mTOR signaling. The ACS phenotype in this model correlated with deregulated expression of a number of cytokines, including elevated levels of interleukin-10 which was under the direct translational control of mTOR. Notably, pharmacologic intervention to impair protein synthesis restored cytokine production to near-normal levels, delayed ACS progression, and extended host survival. Together, our findings suggest a new paradigm to treat ACS by strategies which target protein synthesis to block the production of procachexic factors.

Topics: Animals; Anorexia; Antineoplastic Agents; Body Weight; Cachexia; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Female; Harringtonines; Homoharringtonine; Humans; Interleukin-10; Kaplan-Meier Estimate; Lymphoma; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Protein Biosynthesis; Proto-Oncogene Proteins c-myc; Signal Transduction; Sirolimus; Syndrome; TOR Serine-Threonine Kinases

Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs with potent immunosuppressive, antiangiogenic, and antiproliferative properties. De novo or worsening proteinuria can occur during treatment with these agents, but the mechanism by which this occurs is unknown. We generated and characterized mice carrying a podocyte-selective knockout of the Mtor gene. Although Mtor was dispensable in developing podocytes, these mice developed proteinuria at 3 weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice exhibited an accumulation of the autophagosome marker LC3 (rat microtubule-associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, and damaged mitochondria. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors.

Topics: Animals; Autophagy; Cells, Cultured; Disease Models, Animal; Humans; Lysosomes; Mice; Mice, Knockout; Mice, Transgenic; Microtubule-Associated Proteins; Mitochondria; Podocytes; Proteinuria; Sirolimus; TOR Serine-Threonine Kinases

Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown.. To investigate mechanisms by which LRRK2 might cause PD.. We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing.. Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes.. These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.

Topics: Animals; Animals, Genetically Modified; Autophagy; Caenorhabditis elegans; Disease Models, Animal; Gene Expression Regulation; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Methionine; Mutation; Parkinson Disease; Protein Serine-Threonine Kinases; Sirolimus; tau Proteins; Valine

Approximately 30% of all epilepsy cases are acquired. At present there is no effective strategy to stop epilepsy development after the precipitating insult. Recent data from experimental models pointed to the mTOR pathway, which can be potently inhibited by rapamycin. However, data on the antiepileptic and antiepileptogenic properties of rapamycin are conflicting. Therefore, we tested whether rapamycin post-treatment influences epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy in rats. Animals were treated with rapamycin (6mg/kg) or vehicle daily for 2 wks, beginning 24h after stimulation. Sham-operated animals were treated with rapamycin or vehicle but were not stimulated. Animals were video-EEG monitored to detect spontaneous seizures. Animals were sacrificed 4 wks later and brains were collected for Timm staining. There were no significant differences in the number of stimulated rats developing epilepsy; latency to first spontaneous seizure; number of seizures, or seizure frequency in epileptic animals. The area occupied by mossy fibers was significantly increased in stimulated vs. sham-operated animals but was not different in animals treated with rapamycin vs. vehicle. Collectively, our data suggest that the antiepileptic or antiepileptogenic action of rapamycin is not a universal phenomenon and might be limited to certain experimental models or experimental conditions.

Topics: Amygdala; Animals; Disease Models, Animal; Epilepsy, Temporal Lobe; Male; Mossy Fibers, Hippocampal; Rats; Rats, Sprague-Dawley; Seizures; Sirolimus; TOR Serine-Threonine Kinases

Impaired endothelial recovery after the implantation of drug-eluting stents is a major concern because of the increased risk for late stent thrombosis. The disruption of the chemokine axis CXCL12/CXCR4 inhibits neointima formation by blocking the recruitment of smooth muscle progenitor cells. To directly compare a CXCR4-targeting treatment strategy with drugs that are currently used for stent coating, we studied the effects of the CXCR4 antagonist POL5551 and the drug sirolimus on neointima formation. Apolipoprotein E-deficient mice were treated with POL5551 or sirolimus continuously for 28 days after a carotid wire injury. POL5551 inhibited neointima formation by 63% (for a dosage of 2 mg/kg/day) and by 70% (for a dosage of 20 mg/kg/day). In comparison, sirolimus reduced the neointimal area by 69%. In contrast to treatment with POL5551 during the first three days after injury, injection of POL5551 (20 mg/kg) once per day for 28 days diminished neointimal hyperplasia by 53%. An analysis of the cellular composition of the neointima showed a reduction in the relative smooth muscle cell (SMC) and macrophage content in mice that had been treated with a high dose of POL5551. In contrast, the diminished SMC content after sirolimus treatment was associated with a neointimal enrichment of macrophages. Furthermore, endothelial recovery was impaired by sirolimus, but not by POL5551. Therefore, the inhibition of CXCR4 by POL5551 is equally effective in preventing neointima formation as sirolimus, but POL5551 might be more beneficial because treatment with it results in a more stable lesion phenotype and because it does not impair re-endothelialisation.

Topics: Angioplasty; Animals; Apolipoproteins E; Blood Vessel Prosthesis Implantation; Carotid Arteries; Cell Movement; Coronary Restenosis; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Humans; Lysophospholipids; Macrophages; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Neointima; Postoperative Complications; Proteins; Receptors, CXCR4; Sirolimus

The ischemia-reperfusion injury (IRI) remains a major problem in transplantation. The objective of this study was to evaluate the effects of preconditioning a donor group with rapamycin and another donor group with tacrolimus to prevent IRI. Twelve hours before nephrectomy, donor Wistar rats received immunosuppressive drugs. The sample was divided into four experimental groups: a sham group, an untreated control group, a group treated with rapamycin (2 mg/kg) and a group treated with tacrolimus (0.3 mg/kg). Left kidneys were removed and, after three hours of cold ischemia, grafts were transplanted. Twenty-four hours later, the transplanted organs were recovered for histological analysis and evaluation of cytokine expression. The pre-conditioning treatment with rapamycin or tacrolimus significantly reduced donor blood urea nitrogen and creatinine levels compared with control group (BUN: p < 0.001 vs. control and creatinine: p < 0.001 vs. control). Acute tubular necrosis was significantly lower in donors treated with immunosuppressant drugs compared with the control group (p < 0.001). Finally, inflammatory cytokines such as TNF-a, IL-6 and rIL-21 showed lower levels in the graft of pre-treated animals. This exploratory experimental study shows that preconditioning donors with rapamycin and tacrolimus in different groups improves clinical outcome and pathology in recipients and reduces in situ pro-inflammatory cytokines associated with Th17 differentiation, creating a favorable environment for the differentiation of regulatory T cells (Tregs).

Topics: Animals; Cytokines; Disease Models, Animal; Immunosuppression Therapy; Immunosuppressive Agents; Inflammation; Inflammation Mediators; Kidney Transplantation; Living Donors; Male; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus; Transplantation Conditioning; Tumor Necrosis Factor-alpha

Current treatments used in Multiple Sclerosis (MS) are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS. One possible explanation is that these therapies are unable to target the inflammatory component most active during the progressive phase of the disease, and compartmentalized behind the blood-brain barrier. Our findings show that Rapamycin ameliorates clinical and histological signs of chronic EAE when administered during ongoing disease. Moreover, Rapamycin significantly reduced the hyperalgesia observed before clinical development of EAE which, in turn, is completely abolished by the administration of the drug.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Hyperalgesia; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Pain Threshold; Peptide Fragments; Pertussis Toxin; RNA, Messenger; Sirolimus; T-Lymphocytes; Time Factors

Mounting epidemiological evidence supports a role for insulin-signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin-induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo.. The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Diabetes Mellitus, Experimental; Disease Models, Animal; Fatty Acids; Hyperinsulinism; Immunoblotting; Lipogenesis; Liver Neoplasms; Male; Mice; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Rats, Inbred Lew; Sensitivity and Specificity; Sirolimus; Streptozocin; Thymoma; TOR Serine-Threonine Kinases; Transfection

Non stent based delivery of antiproliferative agents using drug coated balloon catheters may offer additional flexibility and efficacy in a broad range of applications. The lipophilic antiproliferative drug zotarolimus makes it a potential candidate for balloon delivery. The aim of the present study was to evaluate the safety and efficacy of a prototype zotarolimus coated balloon (ZCB) catheter in comparison to a zotarolimus eluting stent (ZES) in the porcine coronary overstretch model.. Eighty-four stents (diameters 3.0 and 3.5 mm; length 15 mm) were implanted in LAD and Cx of 42 domestic pigs: control (TriMaxx, Abbott, polymer coated stent without drug, implanted with uncoated PCI catheter, n = 56); ZES (ZoMaxx, Abbott, stent coated with zotarolimus in polymer, implanted with uncoated PCI catheter, n = 14); ZCB (TriMaxx, Abbott, polymer coated stent without drug, implanted with zotarolimus coated PCI catheter, n = 14). Drug content of the vessel wall (n = 9) was measured about 10-30 min post intervention with ZCB in additional pigs.. Immediately after ZCB treatment 101 ± 31 μg of zotarolimus was detected in the coronary arteries. After 28 days ZES led to a reduction of neointimal area from 4.32 ± 1.45 to 3.32 ± 1.11 mm2 (P = 0.019 vs. control). The effect of neointimal inhibition was more pronounced with the novel ZCB (2.79 ± 1.43 mm², P = 0.001 vs. control). Inflammation score was significantly reduced in vessels treated with the ZCB (0.75 ± 0.86 compared to control (1.45 ± 0.94, P = 0.013) and ZES (1.65 ± 0.90, P = 0.012).. Zotarolimus coated balloons and stents were found to effectively reduce neointimal proliferation in the porcine coronary model. Inflammation scores were significantly reduced after treatment with the coated balloon. Zotarolimus balloon coating might be a novel option in preventing and treating restenosis.

Topics: Angioplasty, Balloon, Coronary; Animals; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Hyperplasia; Neointima; Sirolimus; Swine

Hyperthyroidism is characterized in rats by increased energy expenditure and marked hyperphagia. Alterations of thermogenesis linked to hyperthyroidism are associated with dysregulation of hypothalamic AMPK and fatty acid metabolism; however, the central mechanisms mediating hyperthyroidism-induced hyperphagia remain largely unclear. Here, we demonstrate that hyperthyroid rats exhibit marked up-regulation of the hypothalamic mammalian target of rapamycin (mTOR) signalling pathway associated with increased mRNA levels of agouti-related protein (AgRP) and neuropeptide Y (NPY), and decreased mRNA levels of pro-opiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC), an area where mTOR co-localizes with thyroid hormone receptor-α (TRα). Central administration of thyroid hormone (T3) or genetic activation of thyroid hormone signalling in the ARC recapitulated hyperthyroidism effects on feeding and the mTOR pathway. In turn, central inhibition of mTOR signalling with rapamycin in hyperthyroid rats reversed hyperphagia and normalized the expression of ARC-derived neuropeptides, resulting in substantial body weight loss. The data indicate that in the hyperthyroid state, increased feeding is associated with thyroid hormone-induced up-regulation of mTOR signalling. Furthermore, our findings that different neuronal modulations influence food intake and energy expenditure in hyperthyroidism pave the way for a more rational design of specific and selective therapeutic compounds aimed at reversing the metabolic consequences of this disease.

Topics: Agouti-Related Protein; AMP-Activated Protein Kinases; Animals; Disease Models, Animal; Eating; Feeding Behavior; Hyperphagia; Hyperthyroidism; Hypothalamus; Male; Neural Pathways; Neuropeptide Y; Phosphorylation; Pro-Opiomelanocortin; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sirolimus; Thyroid Hormone Receptors alpha; Time Factors; TOR Serine-Threonine Kinases; Triiodothyronine; Weight Loss

Rapamycin, which is employed in the management of patients undergoing liver surgery, induces the synthesis of heme oxygenase-1 (HO-1) in some non-liver cell types. The aim was to investigate whether rapamycin can induce HO-1 expression in the liver, and to test the effects of rapamycin on liver function in the early phase of ischemia reperfusion (IR) injury.. Isolated rat hepatocytes and a rat model of segmental hepatic ischemia and reperfusion were employed. Bile flow was measured gravimetrically or by using indocyanine green. mRNA and protein (by quantitative PCR and Western blot, respectively) and blood concentrations of rapamycin, bilirubin, and liver marker enzymes were measured.. In isolated hepatocytes, rapamycin induced a 6-fold increase in HO-1, comparable to that induced by cobalt proporphyrin (CoPP), and a 2-fold increase in peroxiredoxin-1. Pretreatment of rats with rapamycin resulted in a small increase in liver HO-1 expression, a 20% inhibition of the basal rate of bile flow, and a 50% inhibition in the rate of bile flow recovery after ischemia. CoPP increased basal bile flow by 20% and inhibited bile flow recovery by 50%. These effects were associated with small increases in the blood concentrations of bilirubin and liver marker enzymes.. Rapamycin, through HO-1 induction, has the potential to protect the liver against damage in the late phase of IR. The inhibition by rapamycin of bile flow indicates that its actions on liver function in the acute phase of IR injury are complex.

Topics: Animals; Anti-Bacterial Agents; Bile; Bilirubin; Disease Models, Animal; Female; Gene Expression Regulation, Enzymologic; Heme Oxygenase-1; Hepatocytes; Liver; Liver Transplantation; Male; Primary Cell Culture; Protoporphyrins; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus

Stent-based delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus is a promising strategy for the treatment of coronary artery disease. We studied potential adverse effects associated with mTOR inhibition.. Macrophages in culture were either treated with everolimus or starved to inhibit mTOR. Everolimus led to inhibition of protein translation, activation of p38 MAPK, and the release of proinflammatory cytokines (eg, IL-6, TNFα) and chemokines (eg, MCP1, Rantes) before induction of autophagic death. These effects were also observed with rapamycin, but not after starvation. Everolimus-induced cytokine release was similar in macrophages lacking the essential autophagy gene Atg7 but was inhibited when macrophages were cotreated with p38 MAPK inhibitor SB202190 or the glucocorticoid clobetasol. Combined stent-based delivery of clobetasol and everolimus in rabbit plaques downregulated TNFα expression as compared with everolimus-treated plaques but did not affect the ability of everolimus to induce macrophage clearance.. mTOR inhibition by everolimus triggers cytokine release in macrophages through inhibition of protein translation and p38 activation. These findings provide a rationale for combined local treatment of atherosclerotic plaques with everolimus and an anti-inflammatory agent.

Topics: Animals; Autophagy; Blotting, Western; Cell Survival; Cells, Cultured; Coronary Artery Disease; Cytokines; Disease Models, Animal; Drug-Eluting Stents; Enzyme-Linked Immunosorbent Assay; Everolimus; Glucocorticoids; Humans; Immunohistochemistry; Macrophages; Mice; Microscopy, Electron, Transmission; Prosthesis Design; Rabbits; Sirolimus

The mammalian target of rapamycin (mTOR) plays a central role in regulating the proliferation of cancer cells, and mTOR-specific inhibitors such as rapamycin analogs are considered as a promising therapy for malignant glioma. In this study, we investigated the possibility of using mTOR inhibitors to treat gliomas. We used a molecular marker, phosphorylation of S6 protein, to monitor biological effects of mTOR inhibitors within xenografts. Phosphorylation was decreased more in U87MG glioma after treatment with high doses of rapamycin or its analog, torisel (10 mg/kg or 25 mg/kg), but only slightly after a low dose of rapamycin (3 mg/kg). This effect correlated with enhanced survival of rats after weekly peritoneal injections of both drugs at the highest two doses but not at the low dose. High doses of both drugs caused weight loss in rats. Clinical trial data indicates that low doses of Torisel (<3 mg/kg) were not efficacious in recurrent GBM. It is concluded that systemic administration of rapamycin analogues may not be a treatment option for patients with malignant glioma due to the intolerability of high doses that might otherwise be effective. The present study underscores the need for better pre-clinical evaluation of drugs with respect to therapeutic window.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glioma; Humans; Immunosuppressive Agents; Male; Neoplasm Transplantation; Phosphorylation; Protein Kinases; Rats; Rats, Nude; Sirolimus; Time Factors; Xenograft Model Antitumor Assays

Mammalian target of rapamycin (mTOR) inhibition has been associated with gonadal dysfunction. The aim of this study was to characterize the effect of sirolimus (SRL) on male gonadal function in an experimental model.. Male Wistar rats were treated with intraperitoneal administration of vehicle or SRL. Vehicle group was treated for 12 weeks. Rats treated with SRL were killed at 4, 8, and 12 weeks. A group of rats was treated with SRL for 4 weeks and then observed during 8 weeks to analyze the possible reversibility of the effect of mTOR inhibition. Body and testicular weight, testosterone, follicle-stimulating hormone level, and luteinizing hormone level were measured and testicular histology was analyzed including proliferation and apoptosis analysis.. Testicular weight was significantly lower in all SRL groups. After SRL withdrawal testicular weight had partially recovered. The expression of steroidogenic acute regulatory protein decreased during SRL treatment, which could explain the reduction of testosterone levels, because steroidogenic acute regulatory protein is crucial for testosterone synthesis. Spermatogenesis was blocked on the spermatogonial level by SRL treatment. Withdrawal of SRL treatment led to complete recovery.. mTOR inhibition in healthy animals produces sexual hormone dysfunction, seminiferous tubule dystrophy and spermatogenesis blockade. Furthermore, the spermatogenesis blockade produced by SRL is reversible.

Topics: Animals; Apoptosis; Cell Proliferation; Disease Models, Animal; Follow-Up Studies; Immunosuppressive Agents; Injections, Intraperitoneal; Male; Organ Size; Phosphoproteins; Rats; Rats, Wistar; Sirolimus; Spermatogenesis; Testicular Diseases; Testis; Testosterone; TOR Serine-Threonine Kinases

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 12; Caspase 3; Cells, Cultured; Cerebral Cortex; Cinnamates; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Mice; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sirolimus; Thiourea; Transcription Factor CHOP

Recurrent excitatory circuits and abnormal recurrent excitatory inputs are essential in epileptogenesis. Studies in temporal lobe epilepsy have shown that mossy fiber sprouting, which represents synaptic reorganization, renders the formation of abnormal recurrent excitatory circuits and inputs. The mammalian target of rapamycin (mTOR) pathway has recently been proved important in mossy fiber sprouting. In the present study, rapamycin, a mTOR inhibiter, was injected into the mouse of temporal lobe epilepsy. Electrophysiological and histological properties of the hippocampus were investigated by whole cell patch clamp, extracellular recording and Timm staining. Following the development of epilepsy, frequency of spontaneous excitatory postsynaptic currents (EPSCs) and amplitude of antidromically evoked EPSCs in granule cells were remarkably increased, as well as the epileptiform activity and mossy fiber sprouting were detected, which indicated the formation of abnormal recurrent excitatory circuits. By the use of rapamycin, frequency of spontaneous EPSCs, amplitude of antidromically evoked EPSCs, the epileptiform activity and mossy fiber sprouting were all remarkably suppressed. Our findings suggested an anti-epileptogenic role of rapamycin by suppressing the recurrent excitatory circuits of dentate gyrus.

Topics: Animals; Anticonvulsants; Dentate Gyrus; Disease Models, Animal; Epilepsy, Temporal Lobe; Excitatory Postsynaptic Potentials; Male; Mice; Mice, Inbred C57BL; Nerve Fibers; Sirolimus; TOR Serine-Threonine Kinases

Proliferation signal inhibitors/mTOR-inhibitors have been shown to reduce de novo development of hypercholesterolemic atherosclerosis in animal models. However, their effect on pre-existing atherosclerosis has not yet been studied.. Feeding LDL-R-KO mice a high cholesterol diet for 12 weeks resulted in formation of moderate fibroatheroma (induction phase). Sixty mice received either everolimus (1 or 5 mg/kg) or no everolimus for further 12 weeks (treatment phase). Everolimus significantly enhanced hypercholesterolemia (plasma cholesterol +45%, p<0.001). Atherosclerosis progressed obstructively in treated and non-treated mice. Everolimus (5 mg/kg) tended to reduced progression in aortic root lesions (0.28±0.02 vs. 0.33±0.03 mm(2), p=ns) and brachiocephalic lesions (0.044±0.006 vs. 0.066±0.012 mm(2), p=ns) but without significance. Everolimus (5mg/kg) resulted in an arrest of CD68 positive plaque area (p=0.03) and nearly halved CD68 fraction (p=0.05) in aortic root lesions but not in brachiocephalic lesions. Taken together, despite a trend to reduced progression and inflammatory cell content there was less conclusive net effect of everolimus treatment than expected.. A higher potential of everolimus in the treatment of atherosclerosis might be obscured by its concomitant hypercholesterolemia. Considering stronger effects in previous studies we suggest that everolimus might exert more potent anti-atherogenic properties in earlier stages of atherogenesis than in advanced atherosclerosis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Aorta; Atherosclerosis; Biomarkers; Brachiocephalic Trunk; Cholesterol; Disease Models, Animal; Everolimus; Hypercholesterolemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Plaque, Atherosclerotic; Protein Kinase Inhibitors; Receptors, LDL; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Topics: Animals; Autophagy; Disease Models, Animal; Humans; Immunosuppressive Agents; Osteoarthritis; Sirolimus; TOR Serine-Threonine Kinases

Brain aggregates (BrnAggs) derived from fetal mouse brains contain mature neurons and glial cells. We determined that BrnAggs are consistently infected with Rocky Mountain Laboratory scrapie strain prions and produce increasing levels of the pathogenic form of the prion protein (PrP). Their abundant dendrites undergo degeneration shortly after prion infection. Treatment of prion-infected BrnAggs with drugs, such as a γ-secretase inhibitors and quinacrine (Qa), which stop PrP formation and dendritic degeneration, mirrors the results from rodent studies. Because PrP is trafficked into lysosomes by endocytosis and autophagosomes by phagocytosis in neurons of prion strain-infected BrnAggs, we studied the effects of drugs that modulate subcellular trafficking. Rapamycin (Rap), which activates autophagy, markedly increased light-chain 3-II (LC3-II)-positive autophagosomes and cathepsin D-positive lysosomes in BrnAggs but could not eliminate the intracellular PrP within them. Adding Qa to Rap markedly reduced the number of LC3-II-positive autolysosomes. Rap + Qa created a competition between Rap increasing and Qa decreasing LC3-II. Rapamycin + Qa decreased total PrP by 56% compared with that of Qa alone, which reduced PrP by 37% relative to Rap alone. We conclude that the decrease was dominated by the ability of Qa to decrease the formation of PrP. Therefore, BrnAggs provide an efficient in vitro tool for screening drug therapies and studying the complex biology of prions.

Topics: Alanine; Animals; Azepines; Brain; Cathepsin D; Dendrites; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Female; Gene Expression Regulation; Immunosuppressive Agents; In Vitro Techniques; Lysosomes; Male; Mice; Mice, Knockout; Microglia; Microscopy, Confocal; Microtubule-Associated Proteins; Nerve Degeneration; Nerve Tissue Proteins; Pregnancy; Prion Diseases; Prion Proteins; Prions; Protein Transport; PrPSc Proteins; Quinacrine; Sirolimus; Subcellular Fractions; Time Factors

Autophagy, the bulk intracellular degradation of cytoplasmic constituents, can be a pro-survival or a pro-death mechanism depending on the context. A recent study showed that autophagy was activated in the phase of early brain injury following subarachnoid hemorrhage (SAH). However, whether autophagy activation after SAH is protective or harmful is still elusive. This study was undertaken to determine the potential role of autophagy pathway activation in early brain injury following SAH. The rats were pretreated with intracerebral ventricular infusion of either the autophagy inducer rapamycin (RAP) or inhibitor 3-methyladenine (3-MA) before SAH onset. The results from electron microscopic examinations showed that RAP administration caused the formation of autophagosomal vacuoles, and 3-MA induced neuronal apoptosis. RAP treatment significantly increased the expression of autophagic proteins Atg5 and Beclin 1, the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I and reduced caspase-3 activity, the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells, brain edema and neurological deficits after SAH. Conversely, 3-MA treatment exacerbated early brain injury. RAP treatment significantly increased the expression of the autophagic proteins Atg5 and Beclin 1, the ratio of LC3-II to LC3-I and reduced caspase-3 activity, the number of TUNEL-positive cells, brain edema and neurological deficits after SAH. Conversely, 3-MA treatment reversed these changes and exacerbated early brain injury. To further clarify the mechanism of autophagy protection, we investigated the expression levels of key apoptosis-related molecules. The results showed that RAP administration decreased Bax translocation to the mitochondria and downstream cytochrome c release from the mitochondria to the cytosol. Taken together, our study indicates that activation of autophagic pathways reduces early brain injury after SAH. This neuroprotective effect is likely exerted by anti-apoptotic mechanisms.

Topics: Adenine; Animals; Apoptosis; Autophagy; Blotting, Western; Disease Models, Animal; In Situ Nick-End Labeling; Male; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Sirolimus; Subarachnoid Hemorrhage

Psychiatric disorders are fairly common comorbidities of epilepsy in humans. Following pilocarpine-induced status epilepticus (SE), experimental animals not only developed spontaneous recurrent seizures, but also exhibited significantly elevated levels of aggressive behavior. The cellular and molecular mechanism triggering these behavioral alterations remains unclear. In the present study, we found that aggression is positively correlated with development of spontaneous seizures. Treatment with rapamycin, a potent mTOR (mammalian target of rapamycin pathway)-pathway inhibitor, markedly diminished aggressive behavior. Therefore, the mTOR pathway may have significance in the underlying molecular mechanism leading to aggression associated with epilepsy.

Topics: Aggression; Animals; Behavior, Animal; Disease Models, Animal; Immunosuppressive Agents; Physical Stimulation; Pilocarpine; Rats; Rats, Sprague-Dawley; Sirolimus; Status Epilepticus; Touch

Tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 (hamartin) or the TSC2 (tuberin) gene. Among the multisystemic manifestations of TSC, the neurodevelopmental features cause the most morbidity and mortality, presenting a considerable clinical challenge. Hamartin and tuberin form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) kinase, a major cellular regulator of protein translation, cell growth and proliferation. Hyperactivated mTORC1 signaling, an important feature of TSC, has prompted a number of preclinical and clinical studies with the mTORC1 inhibitor rapamycin. Equally exciting is the prospect of treating TSC in the perinatal period to block the progression of brain pathologies and allow normal brain development to proceed. We hypothesized that low-dose rapamycin given prenatally and/or postnatally in a well-established neuroglial (Tsc2-hGFAP) model of TSC would rescue brain developmental defects. We developed three treatment regimens with low-dose intraperitoneal rapamycin (0.1 mg/kg): prenatal, postnatal and pre/postnatal (combined). Combined rapamycin treatment resulted in almost complete histologic rescue, with a well-organized cortex and hippocampus almost identical to control animals. Other treatment regimens yielded less complete, but significant improvements in brain histology. To assess how treatment regimens affected cognitive function, we continued rapamycin treatment after weaning and performed behavioral testing. Surprisingly, the animals treated with the combined therapy did not perform as well as postnatally-treated animals in learning and memory tasks. These results have important translational implications in the optimization of the timing and dosage of rapamycin treatment in TSC affected children.

Topics: Animals; Brain; Cognition; Disease Models, Animal; Humans; Learning; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Neuroglia; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

HIV-associated nephropathy (HIVAN) is characterized by proliferative phenotype in the form of collapsing glomerulopathy and microcystic dilatation of tubules. Recently, epithelial mesenchymal transition (EMT) of renal cells has been demonstrated to contribute to the pathogenesis of proliferative HIVAN phenotype. We hypothesized that sirolimus would modulate HIVAN phenotype by attenuating renal cell EMT. In the present study, we evaluated the effect of sirolimus on the development of renal cell EMT as well as on display of HIVAN phenotype in a mouse model of HIVAN (Tg26). Tg26 mice receiving normal saline (TgNS) showed enhanced proliferation of both glomerular and tubular cells when compared to control mice-receiving normal saline (CNS); on the other hand, Tg26 mice receiving sirolimus (TgS) showed attenuated renal cell proliferation when compared with TgNS. TgNS also showed increased number of α-SMA-, vimentin-, and FSP1-positive cells (glomerular as well as tubular) when compared with CNS; however, TgS showed reduced number of SMA, vimentin, and FSP1+ve renal cells when compared to TgNS. Interestingly, sirolimus preserved renal epithelial cell expression of E-cadherin in TgS. Since sirolimus attenuated renal cell ZEB expression (a repressor of E-cadherin transcription), it appears that sirolimus may be attenuating renal cell EMT by preserving epithelial cell E-cadherin expression.

Topics: Actins; AIDS-Associated Nephropathy; Animals; Anti-HIV Agents; Cadherins; Calcium-Binding Proteins; Cell Proliferation; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Kidney Glomerulus; Kidney Tubules; Male; Mice; Mice, Transgenic; S100 Calcium-Binding Protein A4; Sirolimus; Transcription Factors; Vimentin; Zinc Finger E-box-Binding Homeobox 1

Previous studies have shown that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin prevents epileptogenesis after pharmacologically induced status epilepticus (SE) in rat models of temporal lobe epilepsy. Because rapamycin is also known for its immunosuppressant properties we hypothesized that one of the mechanisms by which it exerts this effect could be via suppression of brain inflammation, a process that has been suggested to play a major role in the development and progression of epilepsy.. Rats were treated with rapamycin or vehicle once daily for 7 days (6 mg/kg/day, i.p.) starting 4 h after the induction of SE, which was evoked by electrical stimulation of the angular bundle. Hereafter rapamycin was administered every other day until rats were sacrificed, 6 weeks after SE. Video-electroencephalography was used to monitor the occurrence of seizures. Neuronal death, synaptic reorganization, and microglia and astrocyte activation were assessed by immunohistologic staining. Fluorescein was administered to quantify blood-brain barrier leakage.. Rapamycin treatment did not alter SE severity and duration compared to vehicle treatment rats. Rapamycin-treated rats developed hardly (n = 9) or no (n = 3) seizures during the 6-week treatment, whereas vehicle-treated rats showed a progressive increase of seizures starting 1 week after SE (mean 8 ± 2 seizures per day during the sixth week). Cell loss and sprouting that normally occur after SE were prominent but on average significantly less in rapamycin-treated rats versus vehicle-treated rats. Nevertheless, various inflammation markers (CD11b/c and CD68) were dramatically upregulated and not significantly different between post-SE groups. Of interest, blood-brain barrier leakage was barely detected in the rapamycin-treated group, whereas it was prominent in the vehicle-treated group.. mTOR inhibition led to strong reduction of seizure development despite the presence of microglia activation, suggesting that effects of rapamycin on seizure development are not due to a control of inflammation. Whether the effects on blood-brain barrier leakage in rapamycin-treated rats are a consequence of seizure suppressing properties of the drug, or contribute to a real antiepileptogenic effect still needs to be determined.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood-Brain Barrier; CD11b Antigen; Cytokines; Disease Models, Animal; Drug Administration Schedule; Electric Stimulation; Electroencephalography; Immunosuppressive Agents; Male; Microglia; Monocytes; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Seizures; Signal Transduction; Sirolimus; Statistics as Topic; Statistics, Nonparametric; Status Epilepticus; Time Factors; TOR Serine-Threonine Kinases; Vimentin

Delayed endothelialization contributes to stent thrombosis of current drug-eluting stents. The asymmetrical coating technique provides an anti-proliferative effect abluminally without affecting luminal endothelialization. Layer-by-layer self-assembled chitosan/heparin (C/H LBL) has been proved to promote re-endothelialization. A novel stent system, C/H LBL coated luminally and sirolimus released abluminally (C/H LBL-SES), was fabricated.. Bare metal stents (BMS), traditionally circumferential sirolimus-eluting stents (SES), and C/H LBL-SES were implanted into porcine coronary arteries. At the 7, 14 and 28 days follow-up (FU), angiography, intravascular ultrasound (IVUS), vasomotor function induced by acetylcholine (Ach), scanning-electron microscopy and histopathology were performed. Remodeling index (RI) was based on IVUS and defined as cross-sectional area (CSA) of vessel at in-stent segment divided by CSA of reference vessel and expressed as a percentage with a normal range from 0.95 to 1.05.. Thirty-eight mini pigs were enrolled and 74 stents (BMS = 23, C/H LBL = 28, SES = 23) were implanted in this study. At 28 days after implantation, the diameter stenosis of C/H LBL-SES by quantitative coronary angiography was 18.8 ± 2.5 %, the area stenosis by histomorphometry was 24.2 ± 2.9 %, which were comparable to that of SES and superior to BMS. At 14 days, re-endothelialization of C/H LBL-SES was almost completed, while only about 50 % of surface of SES was covered by endothelium. At 7, 14 and 28 days FU, although C/H LBL-SES suffered a greater vasoconstriction induced by Ach infusion than BMS (P < 0.05), it behaved better than SES (P < 0.01). No sign of stent malapposition was detected, while RI was within the normal range by IVUS. No acute or subacute thrombotic events occurred in all three groups.. The asymmetrically designed C/H LBL-SES successfully inhibited neointima hyperplasia, while diminishing vasoconstriction after Ach-stress. Endothelialization of C/H LBL-SES was less affected compared with traditionally circumferentially coated SES.

Topics: Acetylcholine; Animals; Cardiovascular Agents; Chitosan; Coated Materials, Biocompatible; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Heparin; Hyperplasia; Metals; Microscopy, Electron, Scanning; Neointima; Percutaneous Coronary Intervention; Prosthesis Design; Sirolimus; Swine; Swine, Miniature; Time Factors; Ultrasonography, Interventional; Vasoconstriction

The everolimus-eluting XIENCE side-branch access (SBA) stent has been the focus of numerous recent publications. Most of the information available on this device comes from the preclinical studies performed in ovine models as well as perfused synthetic heart models. It has now become available in Europe as part of a limited test launch. Delivered via a low-profile, dual-lumen, single-tip catheter, a single inflation device deploys the stent in the main branch and expands a portal opening into the ostium of the side branch to allow for scaffolding and entry into the side branch. This case report describes the first-in-man experience with this novel device.

Topics: Angioplasty, Balloon, Coronary; Animals; Cattle; Coronary Angiography; Coronary Artery Disease; Disease Models, Animal; Dogs; Drug-Eluting Stents; Equipment Safety; Europe; Follow-Up Studies; Humans; Imaging, Three-Dimensional; Male; Prosthesis Design; Sirolimus; Treatment Outcome; Ultrasonography, Interventional

Diabetes remains a significant risk factor for restenosis/thrombosis following stenting. Although vascular healing responses following drug-eluting stent (DES) treatment have been characterized previously in healthy animals, comparative assessments of different DES in a large animal model with isolated features of diabetes remains limited. We aimed to comparatively assess the vascular response to paclitaxel-eluting (PES) and everolimus-eluting (EES) stents in a porcine coronary model of streptozotocin (STZ)-induced type I diabetes.. Twelve Yucatan swine were induced hyperglycemic with a single STZ dose intravenously to ablate pancreatic β-cells. After two months, each animal received one XIENCE V® (EES) and one Taxus Liberte (PES) stent, respectively, in each coronary artery. After three months, vascular healing was assessed by angiography and histomorphometry. Comparative in vitro effects of everolimus and paclitaxel (10-5 M-10-12 M) after 24 hours on carotid endothelial (EC) and smooth muscle (SMC) cell viability under hyperglycemic (42 mM) conditions were assayed by ELISA. Caspase-3 fluorescent assay was used to quantify caspase-3 activity of EC treated with everolimus or paclitaxel (10-5 M, 10-7 M) for 24 hours.. After 3 months, EES reduced neointimal area (1.60 ± 0.41 mm, p < 0.001) with trends toward reduced % diameter stenosis (11.2 ± 9.8%, p = 0.12) and angiographic late-loss (0.28 ± 0.30 mm, p = 0.058) compared to PES (neointimal area: 2.74 ± 0.58 mm, % diameter stenosis: 19.3 ± 14.7%, late loss: 0.55 ± 0.53 mm). Histopathology revealed increased inflammation scores (0.54 ± 0.21 vs. 0.08 ± 0.05), greater medial necrosis grade (0.52 ± 0.26 vs. 0.0 ± 0.0), and persistently elevated fibrin scores (1.60 ± 0.60 vs. 0.63 ± 0.41) with PES compared to EES (p < 0.05). In vitro, paclitaxel significantly increased (p < 0.05) EC/SMC apoptosis/necrosis at high concentrations (≥ 10-7 M), while everolimus did not affect EC/SMC apoptosis/necrosis within the dose range tested. In ECs, paclitaxel (10-5 M) significantly increased caspase-3 activity (p < 0.05) while everolimus had no effect.. After 3 months, both DES exhibited signs of delayed healing in a STZ-induced diabetic swine model. PES exhibited greater neointimal area, increased inflammation, greater medial necrosis, and persistent fibrin compared to EES. Differential effects of everolimus and paclitaxel on vascular cell viability may potentially be a factor in regulating delayed healing observed with PES. Further investigation of molecular mechanisms may aid future development of stent-based therapies in treating coronary artery disease in diabetic patients.

Topics: Animals; Apoptosis; Cardiovascular Agents; Cells, Cultured; Coronary Angiography; Coronary Artery Disease; Coronary Restenosis; Coronary Vessels; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Disease Models, Animal; Dose-Response Relationship, Drug; Drug-Eluting Stents; Endothelial Cells; Everolimus; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Necrosis; Neointima; Paclitaxel; Percutaneous Coronary Intervention; Prosthesis Design; Sirolimus; Swine; Time Factors; Wound Healing

Guanosine is an extracellular signaling molecule implicated in the modulation of glutamatergic transmission and neuroprotection. The present study evaluated the antidepressant-like effect of guanosine in the forced swimming test (FST) and in the tail suspension test (TST) in mice. The contribution of NMDA receptors as well as l-arginine-NO-cGMP and PI3K-mTOR pathways to this effect was also investigated. Guanosine administered orally produced an antidepressant-like effect in the FST (0.5-5 mg/kg) and TST (0.05-0.5 mg/kg). The anti-immobility effect of guanosine in the TST was prevented by the treatment of mice with NMDA (0.1 pmol/site, i.c.v.), d-serine (30 μg/site, i.c.v., a co-agonist of NMDA receptors), l-arginine (750 mg/kg, i.p., a substrate for nitric oxide synthase), sildenafil (5 mg/kg, i.p., a phosphodiesterase 5 inhibitor), LY294002 (10 μg/site, i.c.v., a reversible PI3K inhibitor), wortmannin (0.1 μg/site, i.c.v., an irreversible PI3K inhibitor) or rapamycin (0.2 nmol/site, i.c.v., a selective mTOR inhibitor). In addition, the administration of ketamine (0.1 mg/kg, i.p., a NMDA receptor antagonist), MK-801 (0.001 mg/kg, i.p., another NMDA receptor antagonist), 7-nitroindazole (50 mg/kg, i.p., a neuronal nitric oxide synthase inhibitor) or ODQ (30 pmol/site i.c.v., a soluble guanylate cyclase inhibitor) in combination with a sub-effective dose of guanosine (0.01 mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. None of the treatments affected locomotor activity. Altogether, results firstly indicate that guanosine exerts an antidepressant-like effect that seems to be mediated through an interaction with NMDA receptors, l-arginine-NO-cGMP and PI3K-mTOR pathways.

Topics: Analysis of Variance; Animals; Antidepressive Agents; Arginine; Cyclic GMP; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agents; Exploratory Behavior; Female; Freezing Reaction, Cataleptic; Guanosine; Hindlimb Suspension; Immunosuppressive Agents; Male; Mice; N-Methylaspartate; Nitric Oxide; Phosphatidylinositol 3-Kinases; Receptors, N-Methyl-D-Aspartate; Serine; Signal Transduction; Sirolimus; Swimming; TOR Serine-Threonine Kinases

Aging is, by far, the greatest risk factor for most neurodegenerative diseases. In non-diseased conditions, normal aging can also be associated with declines in cognitive function that significantly affect quality of life in the elderly. It was recently shown that inhibition of Mammalian TOR (mTOR) activity in mice by chronic rapamycin treatment extends lifespan, possibly by delaying aging {Harrison, 2009 #4}{Miller, 2011 #168}. To explore the effect of chronic rapamycin treatment on normal brain aging we determined cognitive and non-cognitive components of behavior throughout lifespan in male and female C57BL/6 mice that were fed control- or rapamycin-supplemented chow. Our studies show that rapamycin enhances cognitive function in young adult mice and blocks age-associated cognitive decline in older animals. In addition, mice fed with rapamycin-supplemented chow showed decreased anxiety and depressive-like behavior at all ages tested. Levels of three major monoamines (norepinephrine, dopamine and 5-hydroxytryptamine) and their metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid) were significantly augmented in midbrain of rapamycin-treated mice compared to controls. Our results suggest that chronic, partial inhibition of mTOR by oral rapamycin enhances learning and memory in young adults, maintains memory in old C57BL/6J mice, and has concomitant anxiolytic and antidepressant-like effects, possibly by stimulating major monoamine pathways in brain.

Topics: Aging; Analysis of Variance; Animals; Avoidance Learning; Behavior, Animal; Biogenic Monoamines; Brain; Cognition Disorders; Disease Models, Animal; Female; Gene Expression Regulation; Hindlimb Suspension; Immunosuppressive Agents; Male; Maze Learning; Memory; Mice; Mice, Inbred C57BL; Sex Factors; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Sequential treatment with targeted agents is standard of care for patients with metastatic renal cell carcinoma (mRCC). However, clinical data directly comparing treatment outcomes with a mammalian target of rapamycin inhibitor or a vascular endothelial growth factor-targeted agent in the second-line setting are lacking. We evaluated sequential treatment in a syngeneic, orthotopic mouse model of mRCC.. BALB/c mice were orthotopically implanted with murine RCC (RENCA) cells expressing luciferase and randomized to vehicle, sunitinib, sunitinib followed by sorafenib, or sunitinib followed by everolimus. Tumor growth and metastases were assessed by in vivo (whole body) and ex vivo (primary tumor, lung, liver) luciferase activity and necropsies, performed on day 20 or 46 for vehicle and treatment groups, respectively.. Sunitinib followed by everolimus was associated with reduced luciferase activity and primary tumor weight and volume compared with sunitinib, and sunitinib followed by sorafenib.. Sequential therapy with sunitinib followed by everolimus demonstrated significant antitumor and anti-metastatic effects.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Models, Animal; Drug Administration Schedule; Everolimus; Female; Indoles; Kidney Neoplasms; Luciferases; Luminescent Measurements; Mice; Mice, Inbred BALB C; Pyrroles; Random Allocation; Sirolimus; Sunitinib

Chondrosarcomas are the second most frequent primary malignant type of bone tumor. No effective systemic treatment has been identified in advanced or adjuvant phases for chondrosarcoma. The aim of the present study was to determine the antitumor effects of doxorubicin and everolimus, an mTOR inhibitor on chondrosarcoma progression.. Doxorubin and/or everolimus were tested in vivo as single agent or in combination in the rat orthotopic Schwarm chondrosarcoma model, in macroscopic phase, as well as with microscopic residual disease. Response to everolimus and/or doxorubicin was evaluated using chondrosarcoma volume evolution (MRI). Histological response was evaluated with % of tumor necrosis, tumor proliferation index, metabolism quantification analysis between the treated and control groups. Statistical analyses were performed using chi square, Fishers exact test. Doxorubicin single agent has no effect of tumor growth as compared to no treatment; conversely, everolimus single agent significantly inhibited tumor progression in macroscopic tumors with no synergistic additive effect with doxorubicin. Everolimus inhibited chondrosarcoma proliferation as evaluated by Ki67 expression did not induce the apoptosis of tumor cells; everolimus reduced Glut1 and 4EBP1 expression. Importantly when given in rats with microscopic residual diseases, in a pseudo neoadjuvant setting, following R1 resection of the implanted tumor, everolimus significantly delayed or prevented tumor recurrence.. MTOR inhibitor everolimus blocks cell proliferation, Glut1 expression and HIF1a expression, and prevents in vivo chondrosarcoma tumor progression in both macroscopic and in adjuvant phase post R1 resection. Taken together, our preclinical data indicate that mTOR inhibitor may be effective as a single agent in treating chondrosarcoma patients. A clinical trial evaluating mTOr inhibitor as neo-adjuvant and adjuvant therapy in chondrosarcoma patients is being constructed.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Proliferation; Chondrosarcoma; Disease Models, Animal; Doxorubicin; Everolimus; Humans; Male; Neoplasm Recurrence, Local; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

Lymphangioleiomyomatosis (LAM) is an interstitial lung disease characterized by invasion and proliferation of abnormal smooth muscle (ASM) cells in lung parenchyma and axial lymphatics. LAM cells bear mutations in tuberous sclerosis (TSC) genes. TSC2(-/-) ASM cells, derived from a human renal angiomyolipoma, require epidermal growth factor (EGF) for proliferation. Blockade of EGF receptors (EGFR) causes cell death. TSC2(-/-) ASM cells, previously labeled with PKH26-GL dye, were endonasally administered to 5-week-old immunodeficient female nude mice, and 4 or 26 weeks later anti-EGFR antibody or rapamycin was administered twice a week for 4 consecutive weeks. TSC2(-/-) ASM cells infiltrated lymph nodes and alveolar lung walls, causing progressive destruction of parenchyma. Parenchymal destruction was efficiently reversed by anti-EGFR treatment and partially by rapamycin treatment. Following TSC2(-/-) ASM cell administration, lymphangiogenesis increased in lungs as indicated by more diffuse LYVE1 expression and high murine VEGF levels. Anti-EGFR antibody and rapamycin blocked the increase in lymphatic vessels. This study shows that TSC2(-/-) ASM cells can migrate and invade lungs and lymph nodes, and anti-EGFR antibody is more effective than rapamycin in promoting lung repair and reducing lymphangiogenesis. The development of a model to study metastasis by TSC cells will also help to explain how they invade different tissues and metastasize to the lung.

Topics: Administration, Intranasal; Animals; Antibodies; Cell Count; Cell Movement; Cell Proliferation; Cell Survival; Disease Models, Animal; ErbB Receptors; Female; Humans; Lung; Lymph Nodes; Lymphangioleiomyomatosis; Mice; Mice, Nude; Myocytes, Smooth Muscle; Phosphorylation; Physical Conditioning, Animal; Receptors, Estrogen; Receptors, Progesterone; Ribosomal Protein S6 Kinases; Sirolimus; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Although the prognosis for clinically localized prostate cancer is now favorable, there are still no curative treatments for castration-resistant prostate cancer (CRPC) and, therefore, it remains fatal. In this study, we investigate a new therapeutic approach for treatment of CRPC, which involves dual targeting of a major signaling pathway that is frequently deregulated in the disease. We found that dual targeting of the Akt and mTOR signaling pathways with their respective inhibitors, MK-2206 and ridaforolimus (MK-8669), is highly effective for inhibiting CRPC in preclinical studies in vivo using a refined genetically engineered mouse model of the disease. The efficacy of the combination treatment contrasts with their limited efficacy as single agents, since delivery of MK-2206 or MK-8669 individually had a modest impact in vivo on the overall tumor phenotype. In human prostate cancer cell lines, although not in the mouse model, the synergistic actions of MK-2206 and ridaforolimus (MK-8669) are due in part to limiting the mTORC2 feedback activation of Akt. Moreover, the effects of these drugs are mediated by inhibition of cellular proliferation via the retinoblastoma (Rb) pathway. Our findings suggest that dual targeting of the Akt and mTOR signaling pathways using MK-2206 and ridaforolimus (MK-8669) may be effective for treatment of CRPC, particularly for patients with deregulated Rb pathway activity.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Male; Mice; Mice, Transgenic; Orchiectomy; Phenotype; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

A novel self-expanding, drug-eluting stent (DES) was designed to slowly release everolimus in order to prevent restenosis after percutaneous peripheral intervention. The purpose of this experimental animal study was to test the hypothesis that long-term local, stent-mediated delivery of everolimus would reduce neointimal hyperplasia in porcine iliac arteries.. The iliac arteries of 24 Yucatan mini-swine were percutaneously treated with overlapping 8- × 28-mm self-expanding nitinol stents loaded with everolimus (225 μg/cm2 stent surface area) formulated in a poly(ethylene-co-vinyl alcohol) copolymer intended to deliver the drug in a sustained fashion over about 6 months (DES). Bare nitinol self-expanding stents (bare metal stent [BMS]) were implanted in an identical fashion on the contralateral side to serve as controls. After 3, 6, or 12 months, the animals were sacrificed and the stented arteries perfusion-fixed for histomorphometric analysis.. The chronic presence of everolimus in arterial tissue reduced stent-induced inflammation after 3 months (inflammation score: BMS 2.29±0.44 vs DES 0.17±0.17; P=.001) and 6 months (BMS 2.06±0.43 vs DES 0.50±0.5; P=.007), although some late inflammation was observed after drug exhaustion (BMS 1.00±0.25 vs DES 2.56±0.62 after 12 months; P=not significant [NS]). Treatment with locally delivered everolimus significantly reduced neointimal hyperplasia after 3 months (neointimal thickness: BMS 0.79±0.20 vs DES 0.37±0.04 mm; P=.03) and 6 months (BMS 0.73±0.14 vs DES 0.41±0.08 mm; P=.05), although the effect had dissipated after 12 months (BMS 0.68±0.11 vs DES 0.67±0.11 mm; P=NS). Remarkably, stent-induced neoatherosclerosis, characterized by the histologic presence of foamy macrophages and cholesterol clefts, was significantly attenuated by treatment with everolimus (atherogenic change scores at 3 months: BMS 0.56±0.15 vs DES 0.04±0.04; P=.003; 6 months: BMS 0.84±0.23 vs DES 0.00±0.00; P=.004; and 12 months: BMS 0.09±0.10 vs DES 0.19±0.19; P=NS).. In this experimental animal model, local arterial stent-mediated delivery of everolimus inhibited the formation of neointimal hyperplasia and neoatherosclerosis during the first 6 months. The effect was transient, however, as arterial morphology and histology appeared similar to control stented arteries after 12 months.

Topics: Animals; Atherosclerosis; Disease Models, Animal; Drug-Eluting Stents; Endovascular Procedures; Everolimus; Graft Occlusion, Vascular; Hyperplasia; Iliac Artery; Immunosuppressive Agents; Neointima; Sirolimus; Swine; Swine, Miniature

Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7-knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.

Topics: Acute Kidney Injury; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 7; Biomarkers; Blood Urea Nitrogen; Cells, Cultured; Chloroquine; Cisplatin; Creatinine; Cytoprotection; Disease Models, Animal; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Kidney Tubules, Proximal; Mice; Mice, Inbred C57BL; Mice, Knockout; Microtubule-Associated Proteins; Reperfusion Injury; Signal Transduction; Sirolimus; Time Factors; Tumor Suppressor Protein p53

To assess the efficacy of rapamycin treatment in chemoprevention and chemotherapy of tumorigenesis in a genetically defined mouse model of head and neck squamous cell carcinoma (HNSCC).. Knockdown of Tgfbr1 and/or Pten using siRNA-mediated RNA interference was carried out in human HNSCC cell lines to analyze molecular changes in the mTOR pathway. Tgfbr1(flox/flox); Pten(flox/flox); K14-CreER(tam) mice were treated with oral gavage of tamoxifen for the conditional deletion of Tgfbr1 and Pten in oral mucosa, resulting in HNSCC. Tgfbr1 and Pten conditonal deletion (2cKO) mice were treated with rapamycin before or after the onset of HNSCC, and the efficacy of this treatment was assessed by determining tumor burden, longevity, and molecular analysis of the mTOR pathway. Molecular changes observed in human HNSCC cell lines and 2cKO mice were compared to identify key alterations in the mTOR pathway.. Knockdown of Tgfbr1 and/or Pten in human HNSCC cell lines resulted in activation of mTOR activity complex 1 and increased levels of survivin. Furthermore, we observed similar changes in HNSCC of the 2cKO mouse. In the human HNSCC tissue array, a loss of Tgfbr1 expression correlated with increased survivin levels. Chemopreventive rapamycin treatment significantly delayed the onset of the HNSCC tumors and prolonged survival in 2cKO mice. In addition, we also found that rapamycin had a therapeutic effect on squamous cell carcinomas in these mice. In 2cKO HNSCC tongue tumors, rapamycin treatment induced apoptosis, inhibited cell proliferation and phosphorylation of Akt and S6, and decreased survivin expression.. These findings indicate that tumorigenesis in 2cKO HNSCC is associated with activation of the Akt/mTOR/survivin pathway, and inhibition of this pathway by rapamycin treatment successfully ameliorates the onset and progression of tumorigenesis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Head and Neck Neoplasms; Humans; Inhibitor of Apoptosis Proteins; Mice; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Sirolimus; Survivin; TOR Serine-Threonine Kinases

Activation of the mammalian target of rapamycin (mTOR) signaling pathway is aberrant in autosomal-dominant polycystic kidney disease (ADPKD). The mTOR inhibitors, such as rapamycin, ameliorate PKD in rodent models, but clinical trials have not shown benefit, possibly as a result of low tissue concentrations of rapamycin at clinically tolerable doses. To overcome this limitation, we synthesized a folate-conjugated form of rapamycin (FC-rapa) that is taken up by folate receptor-mediated endocytosis and cleaved intracellularly to reconstitute the active drug. We found that renal cyst-lining cells highly express the folate receptor in ADPKD and mouse models. In vitro, FC-rapa inhibited mTOR activity in a dose- and folate receptor-dependent manner. Treatment of a PKD mouse model with FC-rapa inhibited mTOR in the target tissue, strongly attenuated proliferation and growth of renal cysts and preserved renal function. Furthermore, FC-rapa inhibited mTOR activity in the kidney but not in other organs. In summary, these results suggest that targeting the kidney using FC-rapa may overcome the significant side effects and lack of renal efficacy observed in clinical trials with mTOR inhibitors in ADPKD.

Topics: Animals; Cell Line; Disease Models, Animal; Endocytosis; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Kidney; Mice; Polycystic Kidney, Autosomal Dominant; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

We have previously shown that pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) provide permanent donor-specific protection of islet allografts. The efficacy of donor ECDI-SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI-SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b(+) IDO(+) cells in the cardiac allograft, and that the presence of this population is dependent on Gr1(+) cells. Consequently, depletion of Gr1(+) cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI-SPs infusions. In addition, T cells from ECDI-SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI-SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI-SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.

Topics: Analysis of Variance; Animals; Antigen-Presenting Cells; CD11b Antigen; Cell Transplantation; Disease Models, Animal; Fluorescent Antibody Technique; Graft Rejection; Graft Survival; Heart Transplantation; Immunohistochemistry; Indoleamine-Pyrrole 2,3,-Dioxygenase; Isoantigens; Male; Mice; Mice, Inbred BALB C; Random Allocation; Sirolimus; Spleen; Statistics, Nonparametric; Transplantation Tolerance; Transplantation, Homologous

Rapamycin, an mTOR inhibitor and immunosuppressive agent in clinic, has protective effects on traumatic brain injury and neurodegenerative diseases. But, its effects on transient focal ischemia/reperfusion disease are not very clear. In this study, we examined the effects of rapamycin preconditioning on mice treated with middle cerebral artery occlusion/reperfusion operation (MCAO/R). We found that the rapamycin preconditioning by intrahippocampal injection 20 hr before MCAO/R significantly improved the survival rate and longevity of mice. It also decreased the neurological deficit score, infracted areas and brain edema. In addition, rapamycin preconditioning decreased the production of NF-κB, TNF-α, and Bax, but not Bcl-2, an antiapoptotic protein in the ischemic area. From these results, we may conclude that rapamycin preconditioning attenuate transient focal cerebral ischemia/reperfusion injury and inhibits apoptosis induced by MCAO/R in mice.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Edema; Brain Infarction; Disease Models, Animal; Female; Gene Expression Regulation; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred BALB C; Nervous System Diseases; Neurologic Examination; NF-kappa B; Reperfusion Injury; Sirolimus; Survival Rate; Tumor Necrosis Factor-alpha

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

Topics: Adenoma; AMP-Activated Protein Kinases; Animals; Cervix Uteri; Disease Models, Animal; Endometrial Neoplasms; Endometrium; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Mesenchymal Stem Cells; Mice; Multiprotein Complexes; Ovary; Oviducts; Protein Serine-Threonine Kinases; Proteins; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Microenvironment; Tumor Suppressor Proteins

Accumulated evidence indicates that metabotropic glutamate 5 (mGlu5) receptor blockade exerts antidepressant-like and anxiolytic-like effects in several animal models. The novelty-suppressed feeding (NSF) test is used to measure anxiety-induced hypophagia in rodents. Anxiogenic-like behavior can be counteracted by acute treatment with anxiolytics or chronic treatment with antidepressants. The objective of the present study was to investigate the effect of an mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), using the NSF test and to investigate the mechanisms underlying the effects of MPEP. The administration of MPEP at 1 h prior to testing significantly shortened the latency period until feed (an acute effect), and this effect lasted for 24 h (a sustained effect), similar to the results observed using the N-methyl-D-aspartate receptor antagonist ketamine. Pretreatment with a protein synthesis inhibitor, anisomycin, blocked the sustained, but not the acute, effects of MPEP, suggesting the involvement of new protein synthesis in the sustained effect of MPEP. In addition, the sustained effect of MPEP in the NSF test was partially abolished by pretreatment with a mammalian target of rapamycin (mTOR) antagonist, rapamycin. In contrast, a tropomyosin-related kinase, the tyrosine kinase inhibitor K252a, did not counteract the sustained effects of MPEP in this test. Taken together, these results are the first report to demonstrate that the blockade of the mGlu5 receptor exerted acute and sustained effects in the NSF test and that new protein synthesis may contribute to the sustained effects of MPEP, which may not mediate brain-derived neurotrophic factor-mTOR signaling.

Topics: Analysis of Variance; Animals; Anti-Anxiety Agents; Anxiety; Carbazoles; Diazepam; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; Exploratory Behavior; Feeding Behavior; Fluvoxamine; Immunosuppressive Agents; Indole Alkaloids; Inhibition, Psychological; Male; Mice; Mice, Inbred C57BL; Pyridines; Reaction Time; Sirolimus; Time Factors

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Topics: Acute Disease; Animals; Antineoplastic Agents; Child; Disease Models, Animal; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulins; Janus Kinase 1; Janus Kinase 2; Mice; Molecular Targeted Therapy; Nitriles; Philadelphia Chromosome; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Receptors, Cytokine; Signal Transduction; Sirolimus; STAT5 Transcription Factor; Survival Rate; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Autophagy is a cell survival response to nutrient deprivation that delivers cellular components to lysosomes for digestion. In recent years, autophagy has also been shown to assist in the degradation of misfolded proteins linked to neurodegenerative disease (Ross and Poirier, 2004). In support of this, rapamycin, an autophagy inducer, improves the phenotype of several animal models of neurodegenerative disease. Our Tg(PrP-A116V) mice model Gerstmann-Sträussler-Scheinker disease (GSS), a genetic prion disease characterized by prominent ataxia and extracellular PrP amyloid plaque deposits in brain (Yang et al., 2009). To determine whether autophagy induction can mitigate the development of GSS, Tg(PrP-A116V) mice were chronically treated with 10 or 20 mg/kg rapamycin intraperitoneally thrice weekly, beginning at 6 weeks of age. We observed a dose-related delay in disease onset, a reduction in symptom severity, and an extension of survival in rapamycin-treated Tg(PrP-A116V) mice. Coincident with this response was an increase in the autophagy-specific marker LC3II, a reduction in insoluble PrP-A116V, and a near-complete absence of PrP amyloid plaques in the brain. An increase in glial cell apoptosis of unclear significance was also detected. These findings suggest autophagy induction enhances elimination of misfolded PrP before its accumulation in plaques. Because ataxia persisted in these mice despite the absence of plaque deposits, our findings also suggest that PrP plaque pathology, a histopathological marker for the diagnosis of GSS, is not essential for the GSS phenotype.

Topics: Animals; Disease Models, Animal; Female; Gerstmann-Straussler-Scheinker Disease; Male; Mice; Mice, Knockout; Mice, Transgenic; Plaque, Amyloid; Prions; Random Allocation; Sirolimus; Time Factors

The dentate gyrus is hypothesized to function as a "gate," limiting the flow of excitation through the hippocampus. During epileptogenesis, adult-generated granule cells (DGCs) form aberrant neuronal connections with neighboring DGCs, disrupting the dentate gate. Hyperactivation of the mTOR signaling pathway is implicated in driving this aberrant circuit formation. While the presence of abnormal DGCs in epilepsy has been known for decades, direct evidence linking abnormal DGCs to seizures has been lacking. Here, we isolate the effects of abnormal DGCs using a transgenic mouse model to selectively delete PTEN from postnatally generated DGCs. PTEN deletion led to hyperactivation of the mTOR pathway, producing abnormal DGCs morphologically similar to those in epilepsy. Strikingly, animals in which PTEN was deleted from ≥ 9% of the DGC population developed spontaneous seizures in about 4 weeks, confirming that abnormal DGCs, which are present in both animals and humans with epilepsy, are capable of causing the disease.

Topics: Animals; Animals, Newborn; Carrier Proteins; Cation Transport Proteins; Dentate Gyrus; Disease Models, Animal; Disks Large Homolog 4 Protein; Electroencephalography; Epilepsy; Gene Expression Regulation; Gliosis; Green Fluorescent Proteins; Guanylate Kinases; Immunosuppressive Agents; Kruppel-Like Transcription Factors; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mossy Fibers, Hippocampal; Neurons; Olfactory Pathways; Phosphopyruvate Hydratase; PTEN Phosphohydrolase; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Zinc Finger Protein GLI1

  Rapamycin (RAP) has certain antiepileptogenic features. However, it is unclear whether these effects can be explained by the anticonvulsant action of RAP, which has not been studied. To address this question, we tested potential anticonvulsant effects of RAP in immature and adult rats using different seizure models and treatment paradigms. In addition, we studied changes in the expression of neuropeptide Y (NPY) induced by RAP, which may serve as an indirect target of the RAP action..   A complex approach was adopted to evaluate the anticonvulsant potential of RAP: We used flurothyl-, pentylenetetrazole (PTZ)-, N-methyl-D-aspartate (NMDA)-, and kainic acid (KA)-induced seizures to test the effects of RAP using different pretreatment protocols in immature and adult rats. We also evaluated expression of NPY within the primary motor cortex, hippocampal CA1, and dentate gyrus (DG) after different pretreatments with RAP in immature rats..   We found the following: (1) RAP administered with short-term pretreatment paradigms has a weak anticonvulsant potential in the seizure models with compromised inhibition. (2) Lack of RAP efficacy correlates with decreased NPY expression in the cortex, CA1, and DG. Specifically in immature rats, a single dose of RAP (3 mg/kg) 4 or 24 h before seizure testing had anticonvulsant effects against PTZ-induced seizures. In the flurothyl seizure model only the 4-h pretreatment with RAP was anticonvulsant in the both age groups. Short-term pretreatments with RAP had no effects against NMDA- and KA-induced seizures tested in immature rats. Long-term pretreatments with RAP over 8 days did not show beneficial effect in all tested seizure models in developing rats. Moreover, the long-term pretreatment with RAP had a slight proconvulsant effect on KA-induced seizures. In immature rats, any lack of anticonvulsant effect (including proconvulsant effect of multiple doses of RAP) was associated with downregulation of NPY expression in the cortex and DG. In immature animals, after a single dose of RAP with 24 h delay, we found a decrease of NPY expression in DG, and CA1 as well..   Our data show weak age-, treatment paradigm-, and model-specific anticonvulsant effects of RAP as well as loss of those effects after long-term RAP pretreatment associated with downregulation of NPY expression. These findings suggest that RAP is a poor anticonvulsant and may have beneficial effects only against epileptogenesis. In addition, our data present new insights into mechanisms of RAP action on seizures indicating a possible connection between mammalian target of rapamycin (mTOR) signaling and NPY system.

Topics: Age Factors; Animals; Animals, Newborn; Anticonvulsants; Disease Models, Animal; Gene Expression Regulation; Male; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Seizures; Sirolimus; Treatment Outcome

The efficacy and action mechanism of everolimus in the treatment of experimental autoimmune uveoretinitis (EAU) was analyzed. Disease was induced in B10.RIII mice by immunization with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180). Everolimus was administered by oral gavage (5 mg/kg/d) beginning either two days before or 14 days after immunization. Everolimus significantly reduced the histopathological uveitis score compared to sham-treated mice. To examine the effect on the antigen-specific immune response, proliferation ([(3)H]-thymidine test) and delayed-type hypersensitivity (DTH) response were measured. Furthermore, content of T-helper-1, -2, and -17 cytokines were analyzed intraocularly (Bead Array) and in cell culture supernatants from splenocytes (sandwich ELISA). To study the effect on the humoral immune response the presence of antigen-specific serum antibodies was tested (indirect ELISA). The DTH, the humoral immune response, the proliferation of splenocytes and the intraocular Th1, Th2, Th17 cytokine content and in vitro production of Th1 and Th17 cytokines were impaired after everolimus treatment. The study of CD4+CD25+FoxP3+ regulatory T cells (T(reg)) in peripheral blood, draining lymph nodes, and spleen by flow cytometry showed an increased number of splenic T(reg) in mice of the everolimus therapy group. Furthermore the T(reg) of these mice had a higher suppressive capacity than cells from sham-treated mice. These results indicate that the immunosuppressive effect of everolimus on EAU was associated with the suppression of pathogenic effector responses and induction of regulatory T cells.

Topics: Animals; Antibodies; Autoimmune Diseases; Cell Proliferation; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Everolimus; Eye Proteins; Flow Cytometry; Forkhead Transcription Factors; Hypersensitivity, Delayed; Immunosuppressive Agents; Mice; Retinitis; Retinol-Binding Proteins; Sirolimus; Spleen; T-Lymphocytes, Regulatory; Uveitis, Posterior

We investigated the effects of bevacizumab and rapamycin on central corneal opacity and apoptotic keratocyte number after photorefractive keratectomy (PRK) followed by ultraviolet B (UV-B) irradiation.. A total of 60 right eyes of Sprague-Dawley rats in four groups (n = 15 each) underwent PRK ablation to 80 μm with a 3-mm zone. Sponges soaked with 0.02% mitomycin C (MMC), 2.5% bevacizumab, 0.01% rapamycin, and balanced saline solution were applied for 2 minutes to these eyes in the MMC, bevacizumab, rapamycin, and control groups, respectively. At 3 weeks after PRK, all right eyes were exposed to 100 mJ/cm(2) UV-B irradiation. Biomicroscopy was used to determine the amount of haze, and TUNEL staining for apoptosis and histology were performed at 3, 6, and 12 weeks.. Contrary to the results at 3 weeks, central corneal haze, and apoptotic keratocyte and keratocyte number decreased significantly in the MMC, bevacizumab, and rapamycin groups compared to the control group, and the keratocyte number was lower in the MMC group than the bevacizumab and rapamycin groups at 6 weeks (all P < 0.05). At 12 weeks, the apoptotic keratocyte number was lower in the MMC, bevacizumab, and rapamycin groups than the control group, and the keratocyte number was significantly lower in the MMC than the rapamycin and control groups (all P < 0.05).. Intraoperative bevacizumab and rapamycin administration decreases central corneal haze and apoptotic keratocyte number after PRK. Bevacizumab and rapamycin may be safe alternatives to MMC during refractive surgery to prevent postoperative corneal opacity less affecting the keratocyte number.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Bevacizumab; Cell Count; Corneal Keratocytes; Corneal Opacity; Disease Models, Animal; Immunosuppressive Agents; In Situ Nick-End Labeling; Microscopy, Acoustic; Photorefractive Keratectomy; Rats; Rats, Sprague-Dawley; Sirolimus; Treatment Outcome; Vascular Endothelial Growth Factor A

Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results.. BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation.. Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies.. The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleolus; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Letrozole; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Receptor, ErbB-3; Receptors, Estrogen; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Triazoles; Xenograft Model Antitumor Assays

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response.As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; beta-Defensins; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cyclosporine; Disease Models, Animal; Elafin; Gene Expression Regulation; Humans; Inflammation; Injections, Intraperitoneal; Interleukin-17; Keratinocytes; Keratins; Ki-67 Antigen; L-Selectin; Membrane Glycoproteins; Mice; Mice, SCID; Sirolimus; Skin; Skin Transplantation; Transplantation, Heterologous

Novel vascular scaffolds aim at equipoise between safety and efficacy. Intravascular optical coherence tomography (OCT) allows in-vivo serial assessment of stent-vessel interactions with high resolution and frequent sampling and may complement histology assessment. We investigated the vascular response to a novel absorbable coating sirolimus-eluting stent (AC-SES) by means of serial OCT and histology evaluation in a porcine model.. One AC-SES and one bare-metal stent (BMS) were implanted in separate coronary arteries of three Yucatan mini-swine. Serial OCT was performed post procedure and at 3-, 28-, 90-, and 180-day follow-up. Normalized optical density (NOD) was used for the assessment of tissue response over time. Histological evaluation was performed at day 180.. A total of 6408 stent struts were analyzed. OCT revealed 100% of struts covered at 28 days, and a significant difference in NOD from 3 to 28 days (0.64 ± 0.07 vs 0.71 ± 0.05, respectively; P<.001) in the AC-SES group. Neointimal thickness was 0.14 ± 0.08 mm, 0.17 ± 0.11 mm, and 0.16 ± 0.09 mm in the AC-SES group and 0.18 ± 0.10 mm, 0.14 ± 0.09 mm, and 0.10 ± 0.08 mm in the BMS group, while rates of uncovered struts were 0%, 0%, and 3.1% and 1.4%, 7.8%, and 21.5%, respectively, at 28, 90, and 180 days. Minimal inflammation and a mature endothelialization were demonstrated in both groups by histology.. OCT serial assessment of vascular response suggested NIH maturation 28 days following AC-SES implantation in pigs. These findings, coupled with histological demonstration of low inflammation scores and complete endothelial coverage as measured at 180 days, suggest a satisfactory healing response to AC-SES.

Topics: Absorbable Implants; Animals; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Follow-Up Studies; Neointima; Percutaneous Coronary Intervention; Sirolimus; Swine; Swine, Miniature; Time Factors; Tomography, Optical Coherence

Chronic pancreatitis is a disease that involves the lymphocytic inflammation of the pancreatic gland, the destruction and fibrous transformation of the endocrine and ductal structures. An involvement of the immune system in the disease progression is assumed and possibly allows immune modulation as a novel treatment strategy. We used a new model of experimental chronic pancreatitis to examine the effect of immune modulation with the mTOR-inhibitor rapamycin on clinical, chemical and histological parameters of chronic pancreatitis. Pancreatitis was induced by injecting 8 mg/kg bodyweight DBTC intravenously in male Sprague Dawley rats. 24 and 72 hours later, 20 µg/kg bodyweight cerulein was injected intraperitoneally to simulate recurrent attacks of pancreatitis typical for the clinical course. 48 hours after the DBTC injection, rats were randomly allocated to placebo or sirolimus (1.5 mg/kg bw i.p.). The treatment was repeated every 24hours for 5 days. The rats were sacrificed 7, 14, 21 and 35 days after DBTC injection. Histologic examination revealed a reduced acute pancreatic damage in the treatment group in the first week and less chronic changes in the further course. ALT and amylase increased in Placebo animals over the observation period and was lower in sirolimus treated animals. Oral glucose tolerance test showed that all placebo animals were diabetic four weeks after DBTC while sirolimus treated animals were normoglycemic. An early, limited treatment with immunomodulatory and antifibrotic agents like sirolimus can positively influence the detrimental course of experimental chronic pancreatitis and may offer a treatment alternative in humans.

Topics: Animals; Disease Models, Animal; Disease Progression; Glucose Tolerance Test; Immunomodulation; Immunosuppressive Agents; Male; Pancreatitis, Chronic; Random Allocation; Rats; Rats, Sprague-Dawley; Sirolimus; Treatment Outcome

Impairment of reciprocal social interaction is a core symptom of autism spectrum disorder. Genetic disorders frequently accompany autism spectrum disorder, such as tuberous sclerosis complex caused by haploinsufficiency of the TSC1 and TSC2 genes. Accumulating evidence implicates a relationship between autism spectrum disorder and signal transduction that involves tuberous sclerosis complex 1, tuberous sclerosis complex 2 and mammalian target of rapamycin. Here we show behavioural abnormalities relevant to autism spectrum disorder and their recovery by the mammalian target of rapamycin inhibitor rapamycin in mouse models of tuberous sclerosis complex. In Tsc2(+/-) mice, we find enhanced transcription of multiple genes involved in mammalian target of rapamycin signalling, which is dependent on activated mammalian target of rapamycin signalling with a minimal influence of Akt. The findings indicate a crucial role of mammalian target of rapamycin signalling in deficient social behaviour in mouse models of tuberous sclerosis complex, supporting the notion that mammalian target of rapamycin inhibitors may be useful for the pharmacological treatment of autism spectrum disorder associated with tuberous sclerosis complex and other conditions that result from dysregulated mammalian target of rapamycin signalling.

Topics: Animals; Disease Models, Animal; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Sirolimus; Social Behavior; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Mammalian target of rapamycin (mTOR) is an important mediator for cross talk between nutritional signals and metabolic signals of insulin by downregulating insulin receptor substrate proteins. Therefore, mTOR inhibition could become a therapeutic strategy in insulin-resistant states, including insulin resistance induced by burn. We tested this hypothesis in the rat model of 30% TBSA full thickness burn, using the mTOR inhibitor rapamycin. Rapamycin (0.4 mg/kg, i.p.) was injected 2 h before euglycemic-hyperinsulinemic glucose clamps at 4 days after burn. IRS-1, phospho-serine³⁰⁷, phospho-tyrosine of IRS-1 and phospho-mTOR in muscle tissue were determined by immunoprecipitation and Western blot analysis or immunohistochemistry. Plasma TNF-α, insulin and C-peptide were determined before and after euglycemic-hyperinsulinemic glucose clamps. Our data showed that TNF-α, insulin and C-peptide significantly increased in the early stage after burn (P < 0.01). The infused rates of total 10% glucose (GIR, mg/kg min) significantly decreased at 4 days after burn. The level of IRS-1 serine³⁰⁷ phosphorylation in muscle in vivo significantly increased after burn (P < 0.01), while insulin-induced tyrosine phosphorylation of IRS-1 significantly decreased (P < 0.01). Inhibition of mTOR by rapamycin inhibited the phosphorylation of mTOR, reduced serine³⁰⁷ phosphorylation, elevated tyrosine phosphorylation and partly prevented the decrease of GIR after burn. However, TNF-α, insulin and C-peptide were not decreased by rapamycin treatment postburn. Taken together, these results indicate that the mTOR pathway is an important modulator of the signals involved in the acute regulation of insulin-stimulated glucose metabolism, and at least, partly contributes to burn-induced insulin resistance. mTOR inhibition may become a therapeutic strategy in insulin-resistant states after burn.

Topics: Animals; Anti-Bacterial Agents; Blotting, Western; Burns; C-Peptide; Disease Models, Animal; Glucose Clamp Technique; Immunohistochemistry; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Muscle, Skeletal; Phosphorylation; Phosphoserine; Phosphotyrosine; Rats; Rats, Sprague-Dawley; Serine; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

Topics: Animals; Brain; Cell Size; Codon, Nonsense; Disease Models, Animal; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Hepatocytes; Humans; Liver; Mutant Proteins; Neurons; Sirolimus; Transcription Factors; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Zebrafish

Repeated cocaine exposure induces locomotor sensitization, which is mediated by adaptive changes in synaptic transmission in the mesolimbic dopamine pathway. The molecular mechanisms underlying this adaptation remain poorly understood. One pathway that may play a role is the mammalian target of rapamycin (mTOR) which is implicated in synaptic plasticity. In the present study, we found that cocaine exposure stimulates mTOR activity in rat brain. Furthermore, inhibition of mTOR by rapamycin blocked the induction as well as the expression of cocaine-induced locomotor sensitization in rats. These data elucidate a novel mechanism by which the mTOR pathway mediates cocaine-induced behavioral changes and could suggest a new interventional strategy for drug abuse.

Topics: Animals; Anti-Bacterial Agents; Cocaine; Cocaine-Related Disorders; Disease Models, Animal; Female; Rats; Rats, Sprague-Dawley; Sirolimus; TOR Serine-Threonine Kinases

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Enzyme Inhibitors; Female; Furans; Humans; In Vitro Techniques; Mechanistic Target of Rapamycin Complex 1; Melanoma; Mice; Mice, Nude; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteins; Pyridines; Pyrimidines; Signal Transduction; Sirolimus; Skin Neoplasms; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors; Transplantation, Heterologous

Animal models are instrumental in understanding disease pathophysiology and mechanisms of therapy action and resistance in vivo.. To establish and characterize a panel of mouse models of renal cell carcinoma (RCC) derived from patients undergoing radical nephrectomy.. In vivo and in vitro animal experiments.. Tumor tissues obtained during surgery were implanted into the subcutaneous space of female BALB/c nude mice and serially passaged into new mice. Tumors were characterized by histology, short tandem repeat (STR) fingerprinting, von Hippel-Lindau (VHL) gene sequencing, and single nucleotide polymorphism (SNP) analysis. Tumor-bearing mice were treated with sunitinib or everolimus. Primary cell cultures were derived from patient tumors and transfected with a lentivirus carrying the luciferase gene. Four subcutaneous xenograft mouse models were developed, representing papillary type 1, papillary type 2, clear cell, and clear cell with sarcomatoid features RCC.. RCC mouse models were established from four patients with distinct histologies of RCC. Tumor growth was dependent on histologic type, the size of the implanted tumor chip, and the passage number. Mouse tumors accurately represented their respective original patient tumors, as STR fingerprints were matching, histology was comparable, and SNP profiles and VHL mutation status were conserved with multiple passages. Bioluminescence imaging results were commensurate with subcutaneous xenograft growth patterns. Mice treated with sunitinib and everolimus exhibited an initial response, followed by a later stage of resistance to these agents, which mimics the clinical observations in patients with RCC.. We developed four mouse xenograft models of RCC with clear-cell and papillary histologies, with stable histologic and molecular characteristics. These models can be used to understand the basic biology of RCC as well as response and resistance to therapy.

Topics: Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Disease Models, Animal; DNA Fingerprinting; Everolimus; Female; Humans; Immunosuppressive Agents; Indoles; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Polymorphism, Single Nucleotide; Pyrroles; Sirolimus; Sunitinib; Transfection; Tumor Cells, Cultured; von Hippel-Lindau Disease; Xenograft Model Antitumor Assays

Pregnancy in women with diabetes is associated with a higher risk of perinatal complications. In particular, infants of diabetic mothers frequently suffer from respiratory distress syndrome (RDS), which is a leading cause of death in preterm infants and is considered to be primarily due to hyperinsulinemia in infants in response to maternal hyperglycemia. To elucidate the mechanism of how insulin signaling induces RDS, bronchoalveolar epithelium-specific Akt1 transgenic (TG) mice were generated. Akt1 overexpression in fetal lung epithelium resulted in RDS in preterm infants born by Caesarean section at embryonic day 18.5 (E18.5). The expression levels of hypoxia-inducible factor 2α (HIF-2α) and its target vascular endothelial growth factor (VEGF) were downregulated in the lung of Akt1 TG mice. Inhibition of the Akt-mammalian target of rapamycin (mTOR) signaling axis by rapamycin restored the expression of VEGF and improved the lung pathology of Akt1 TG pups. Rapamycin also attenuated the RDS phenotype in wild-type mice delivered preterm at E17.5. In cultured lung epithelial cells, insulin reduced VEGF expression and transcriptional activity of HIF-2 on VEGF promoter in an mTOR-dependent manner. Thus, aberrant activation of the Akt-mTOR pathway in lung epithelium plays a causal role in the pathogenesis of infant RDS, presumably through downregulation of HIF-2-dependent VEGF expression in the lung.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Disease Models, Animal; Female; Humans; Infant, Newborn; Insulin; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pregnancy; Proto-Oncogene Proteins c-akt; Respiratory Distress Syndrome, Newborn; Respiratory Mucosa; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factors

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.

Topics: Adaptor Proteins, Signal Transducing; Amyotrophic Lateral Sclerosis; Animals; Autophagy; bcl-2-Associated X Protein; Caspase 3; Disease Models, Animal; Heat-Shock Proteins; Immunosuppressive Agents; Mice; Mitochondria; Motor Neurons; Neurodegenerative Diseases; Poly(ADP-ribose) Polymerases; Sequestosome-1 Protein; Sirolimus; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; TOR Serine-Threonine Kinases

The human condition autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of cysts in the kidneys that increase renal volume and lead to kidney failure. Mice studies are performed for treatment development monitored with imaging. The analysis of the imaging data is typically manual, which is costly and potentially biased. This paper presents a reliable and reproducible method for the automated segmentation of polycystic mouse kidneys.. Treated and untreated mice have been imaged longitudinally with high field anatomic MRI. The region of interest (ROI) of the kidneys in the images is identified and restored for artifacts. It is then analyzed statistically and geometric models are estimated for each kidney. The statistical and geometric information are provided to the graph cuts algorithm that delineates the kidneys.. The accuracy of the analysis has been demonstrated by showing consistency with results obtained with previous methods as well as by comparing with manual segmentations.. The method developed can accelerate and improve the accuracy of kidney volumetry in preclinical treatment trials for ADPKD.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Immunosuppressive Agents; Magnetic Resonance Imaging; Mice; Morpholines; Organ Size; Polycystic Kidney, Autosomal Dominant; Reproducibility of Results; Sirolimus; Spiro Compounds; Treatment Outcome

The identification of mammalian target of rapamycin (mTOR) as a major mediator of neurofibromatosis-1 (NF1) tumor growth has led to the initiation of clinical trials using rapamycin analogs. Previous studies from our laboratory have shown that durable responses to rapamycin treatment in a genetically engineered mouse model of Nf1 optic glioma require 20 mg/kg/day, whereas only transient tumor growth suppression was observed with 5 mg/kg/day rapamycin despite complete silencing of ribosomal S6 activity. To gain clinically relevant insights into the mechanism underlying this dose-dependent effect, we used Nf1-deficient glial cells in vitro and in vivo. First, there was an exponential relationship between blood and brain rapamycin levels. Second, we show that currently used biomarkers of mTOR pathway inhibition (phospho-S6, phospho-4EBP1, phospho-STAT3, and Jagged-1 levels) and tumor proliferation (Ki67) do not accurately reflect mTOR target inhibition or Nf1-deficient glial growth suppression. Third, the incomplete suppression of Nf1-deficient glial cell proliferation in vivo following 5 mg/kg/day rapamycin treatment reflects mTOR-mediated AKT activation, such that combined 5 mg/kg/day rapamycin and PI3-kinase (PI3K) inhibition or dual PI3K/mTOR inhibition recapitulates the growth suppressive effects of 20 mg/kg/day rapamycin. These new findings argue for the identification of more accurate biomarkers for rapamycin treatment response and provide reference preclinical data for comparing human rapamycin levels with target effects in the brain.

Topics: Animals; Antibiotics, Antineoplastic; Astrocytes; Brain; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Silencing; Ki-67 Antigen; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurofibromatosis 1; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin suppression by rapamycin inhibits tumor growth and neovascularization via cyclooxygenase-2 (COX-2) downregulation with no effect on lung metastases. We hypothesize that combining a selective COX-2 antagonist (celecoxib) with rapamycin would decrease lung metastases.. Ewing sarcoma cells (SK-NEP-1) were surgically implanted into the left kidney of athymic mice (n = 40). The mice were divided into 4 treatment groups (control, rapamycin only, celecoxib only, and combination) and then killed at 6 weeks. Primary tumors were weighed. Vasculature was examined using lectin angiography and immunohistochemistry, and lung metastases were examined using H&E and CD99 immunostaining. Tumor weight and lung metastases were analyzed.. Mean primary tumor weights were significantly reduced in the rapamycin-treated groups but not in the celecoxib-only group. Lectin angiography and endothelial markers immunostaining showed markedly decreased vascularity in the rapamycin-treated groups but not in the celecoxib-only group. Celecoxib-treated groups showed significantly fewer mice with lung metastases than non-celecoxib-treated groups.. Celecoxib prevents lung metastasis in a murine model of Ewing sarcoma with no effect on tumor size or neovascularization. Cyclooxygenase-2 may represent a future potential target for metastatic disease prevention.

Topics: Angiogenesis Inhibitors; Animals; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Down-Regulation; Drug Therapy, Combination; Female; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Pyrazoles; Sarcoma, Ewing; Sirolimus; Sulfonamides

Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.

Topics: Androstadienes; Animals; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Formaldehyde; Gene Expression Regulation, Enzymologic; Hyperalgesia; Inflammation; Male; Nerve Tissue Proteins; Pain Measurement; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reaction Time; Signal Transduction; Sirolimus; Spinal Cord; Statistics, Nonparametric; Substance P; Time Factors; TOR Serine-Threonine Kinases; Wortmannin

Temporal lobe epilepsy is prevalent and can be difficult to treat effectively. Granule cell axon (mossy fiber) sprouting is a common neuropathological finding in patients with mesial temporal lobe epilepsy, but its role in epileptogenesis is unclear and controversial. Focally infused or systemic rapamycin inhibits the mammalian target of rapamycin (mTOR) signaling pathway and suppresses mossy fiber sprouting in rats. We tested whether long-term systemic treatment with rapamycin, beginning 1 d after pilocarpine-induced status epilepticus in mice, would suppress mossy fiber sprouting and affect the development of spontaneous seizures. Mice that had experienced status epilepticus and were treated for 2 months with rapamycin displayed significantly less mossy fiber sprouting (42% of vehicle-treated animals), and the effect was dose dependent. However, behavioral and video/EEG monitoring revealed that rapamycin- and vehicle-treated mice displayed spontaneous seizures at similar frequencies. These findings suggest mossy fiber sprouting is neither pro- nor anti-convulsant; however, there are caveats. Rapamycin treatment also reduced epilepsy-related hypertrophy of the dentate gyrus but did not significantly affect granule cell proliferation, hilar neuron loss, or generation of ectopic granule cells. These findings are consistent with the hypotheses that hilar neuron loss and ectopic granule cells might contribute to temporal lobe epileptogenesis.

Topics: Analysis of Variance; Animals; Anticonvulsants; Cation Transport Proteins; Diazepam; Disease Models, Animal; Drug Administration Schedule; Electroencephalography; Epilepsy, Temporal Lobe; Hippocampus; Immunosuppressive Agents; Mice; Mossy Fibers, Hippocampal; Neurons; Seizures; Sirolimus; Videotape Recording

Autophagy is activated in cardiomyocytes in ischaemic heart disease, but its dynamics and functional roles remain unclear after myocardial infarction. We observed the dynamics of cardiomyocyte autophagy and examined its role during postinfarction cardiac remodelling.. Myocardial infarction was induced in mice by ligating the left coronary artery. During both the subacute and chronic stages (1 and 3 weeks postinfarction, respectively), autophagy was found to be activated in surviving cardiomyocytes, as demonstrated by the up-regulated expression of microtubule-associated protein-1 light chain 3-II (LC3-II), p62 and cathepsin D, and by electron microscopic findings. Activation of autophagy, specifically the digestion step, was prominent in cardiomyocytes 1 week postinfarction, especially in those bordering the infarct area, while the formation of autophagosomes was prominent 3 weeks postinfarction. Bafilomycin A1 (an autophagy inhibitor) significantly aggravated postinfarction cardiac dysfunction and remodelling. Cardiac hypertrophy was exacerbated in this group and was accompanied by augmented ventricular expression of atrial natriuretic peptide. In these hearts, autophagic findings (i.e. expression of LC3-II and the presence of autophagosomes) were diminished, and activation of AMP-activated protein kinase was enhanced. Treatment with rapamycin (an autophagy enhancer) brought about opposite outcomes, including mitigation of cardiac dysfunction and adverse remodelling. A combined treatment with bafilomycin A1 and rapamycin offset each effect on cardiomyocyte autophagy and cardiac remodelling in the postinfarction heart.. These findings suggest that cardiomyocyte autophagy is an innate mechanism that protects against progression of postinfarction cardiac remodelling, implying that augmenting autophagy could be a therapeutic strategy.

Topics: AMP-Activated Protein Kinases; Analysis of Variance; Animals; Atrial Natriuretic Factor; Autophagy; Blotting, Western; Cathepsin D; Disease Models, Animal; Enzyme Activation; Fluorescent Antibody Technique; Heart Ventricles; Hypertrophy, Left Ventricular; Macrolides; Mice; Microscopy, Electron; Microtubule-Associated Proteins; Myocardial Infarction; Myocytes, Cardiac; Phosphorylation; Sirolimus; Time Factors; Ventricular Function, Left; Ventricular Remodeling

To investigate the entire process of autophagy in the left ventricle of septic mice, and the functional significance of autophagy by using pharmacological agents.. Myocardial dysfunction is a common feature in sepsis and contributes to an increased risk of developing multiple organ failure. Autophagy functions predominantly as a prosurvival pathway in the heart during cellular stress. A dynamic process of autophagy that involves the complete activation of autophagy from autophagosome formation to fusion with lysosomes has driven the development of new approaches to detecting autophagy.. Male mice were subjected to cecal ligation and puncture (CLP) or sham operation. At 1 hour after CLP operation, mice received either rapamycin (induction of autophagy), bafilomycin A1 (inhibition of autophagosomal degradation), or vehicle.. The formation of autophagosomes was increased whereas the degradation of autophagosomes was decreased in the left ventricle at 24 hours after CLP. This was consistent with the morphologic finding that septic hearts revealed an increase in autophagosomes but few autolysosomes, indicating incompletion of the autophagic process. Rapamycin, which induced complete activation of autophagy, restored CLP-induced depressed cardiac performances. This cardioprotective effect was also seen in increased ATP levels, and decreased inflammatory responses. Bafiomycin A1, which resulted in incompletion of the autophagic process, did not show any above beneficial effects in CLP mice.. Incompletion of the autophagic process may contribute to sepsis-induced cardiac dysfunction. Treatment with rapamycin may serve a cardioprotective role in sepsis, possibly through the effect of complete induction of autophagy.

Topics: Animals; Autophagy; Disease Models, Animal; Heart Ventricles; Immunosuppressive Agents; Male; Mice; Mice, Inbred C3H; Sepsis; Sirolimus; Ventricular Dysfunction, Left

Topics: Animals; Clinical Trials, Phase III as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Everolimus; Humans; Indoles; Mice; Neuroendocrine Tumors; Pancreatic Neoplasms; Pyrroles; Signal Transduction; Sirolimus; Sunitinib; Survival Rate; Translational Research, Biomedical

Rapamycin has been reported to inhibit hepatic fibrosis, lung fibrosis, renal fibrosis, and subglottic stenosis. Fibrosis is also involved in urethral stricture. Therefore, we investigated the effect of rapamycin on the inhibition of urethral stricture formation in a rabbit model. First, models of urethral stricture were successfully established by electrocoagulation of the bulbar urethra in adult New Zealand male rabbits. Forty-six model rabbits were randomly assigned to four groups: high-dose rapamycin (R(H), 1.0 mg/day), low-dose rapamycin (R(L), 0.1 mg/day), dimethyl sulfoxide (DMSO) alone (DMSO, solvent control), and normal saline (NS). Urethral stricture was assessed by a retrograde urethrogram and video-urethroscopy. Urethra pathology was evaluated by hematoxylin and eosin and Sirius red staining. After 28 days of treatment, lumen reduction in the R(H), R(L), DMSO, and NS groups was 36.0, 56.5, 69.1, and 82.9, respectively. Comparison of the rapamycin groups (R(H) and R(L)) and control groups (DMSO and NS) indicated significantly less restriction in the rapamycin groups. Histopathological analysis confirmed the presence of fibroblasts and an increase in collagen at the stricture site in the two control groups but not in the R(H) or R(L) groups. These results indicate that rapamycin inhibits experimentally induced urethral stricture formation in rabbits. This effect may be due to its inhibition of fibroblast proliferation and collagen expression.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Pilot Projects; Rabbits; Random Allocation; Sirolimus; Urethral Stricture

Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α-synuclein (SNCA). A loss in lysosomal acid-β-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk.. We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols.. We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (≤40%; 10μM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice.. Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders.

Topics: alpha-Synuclein; Animals; Cathepsin D; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gaucher Disease; Gene Expression Regulation; Glucosylceramidase; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Lewy Body Disease; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Mutation; Parkinson Disease; Rats; Sirolimus; Transfection

Infantile spasms are seizures manifesting within a spectrum of epileptic encephalopathies of infancy that often lead to cognitive impairment. Their current therapies, including adrenocorticotropic hormone (ACTH), high dose steroids, or vigabatrin, are not always effective and may be associated with serious side effects. Overactivation of the TORC1 complex of the mTOR pathway is implicated in the pathogenesis of certain genetic and acquired disorders that are linked with infantile spasms, like tuberous sclerosis. Here, we tested the therapeutic potential of rapamycin, a TORC1 inhibitor, as a potential treatment for infantile spasms in the multiple-hit rat model of ACTH-refractory symptomatic infantile spasms, which is not linked to tuberous sclerosis. Rapamycin or vehicle was given after spasms appeared. Their effects on spasms, other seizures, performance in Barnes maze, and expression of the phosphorylated S6 ribosomal protein (pS6: a TORC1 target) in the cortex, using immunofluorescence, were compared. Rapamycin suppressed spasms dose-dependently and improved visuospatial learning, although it did not reduce the frequency of other emerging seizures. High-dose pulse rapamycin effected acute and sustained suppression of spasms and improved cognitive outcome, without significant side effects. Therapeutically effective rapamycin doses normalized the pS6 expression, which was increased in perilesional cortical regions of pups with spasms. These findings support that pathological overactivation of TORC1 may be implicated in the pathogenesis of infantile spasms, including those that are not linked to tuberous sclerosis. Furthermore, a high-dose, pulse rapamycin treatment is a promising, well tolerated and disease-modifying new therapy for infantile spasms, including those refractory to ACTH.

Topics: Animals; Animals, Newborn; Cognition Disorders; Disease Models, Animal; Drug Administration Schedule; Humans; Infant, Newborn; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Spasms, Infantile; Transcription Factors

To evaluate antitumor efficacy of the generic mammalian target of rapamycin (mTOR) inhibitor sirolimus in preclinical animal models of head and neck squamous cell carcinoma (HNSCC) and compare its effects with those of the patented analogue temsirolimus.. In vivo study.. To develop xenograft established tumor model (ETM) of HNSCC, FaDu cells were injected subcutaneously into nude mice. When tumors reached 50 to 60 mm(3), mice were randomized into five groups and treated daily intraperitoneally with sirolimus at various doses for 5 days per week for 3 weeks. Tumor volumes were measured. The results were compared with historical data on temsirolimus effects. In the minimal residual disease (MRD) model, surgical wounds were created and FaDu cells implanted. After 72 hours, animals were randomized into two groups and were injected intraperitoneally with 0 or 5 mg/kg sirolimus for 5 days per week for 30 days.. In the ETM, sirolimus significantly inhibited tumor growth (P < .01), although there was no overall significant difference in tumor growth inhibition between sirolimus and temsirolimus. In the MRD model, sirolimus significantly suppressed growth of tumors (P < .001) and improved survival compared with controls (P < .01). There was a significant decrease in pS6 expression, indicating mTOR inhibition.. In this study, we demonstrate that the generic mTOR inhibitor sirolimus shows potent antitumor activity in HNSCC and produces comparable effects to the patent drug temsirolimus. Sirolimus has the potential of serving as an economic and comparative targeted agent to temsirolimus in the treatment of HNSCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Disease Models, Animal; Head and Neck Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Sirolimus

Adenovirus-mediated cytotoxic T-lymphocyte-associated antigen-4 immunoglobulin (AdCTLA4Ig) gene transfer has been reported to enhance both organ and composite tissue grafts survival in rodent transplantation models. The authors tested the efficacy of local expression of CTLA4Ig gene on the survival of rat free flap allografts.. Brown Norway rat groin flaps were transplanted in Lewis rat recipients. The donor flaps were perfused ex vivo with AdCTLA4Ig via the afferent artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the flaps were assessed by immunohistochemical staining and semiquantitative reverse transcriptase polymerase chain reaction after transplantation. A mixed lymphocyte reaction was performed to test the antigen-specific immune response. Flow cytometry was used to detect the variations of CD4+25+Foxp3+ T cells in recipients' spleens.. Immunohistochemical staining and reverse transcriptase polymerase chain reaction demonstrated expression of CTLA4Ig transgene in AdCTLA4Ig-perfused free flap allografts. AdCTLA4Ig-perfused free flap allografts survived significantly longer compared with survival of enhanced green fluorescent protein adenovirus-perfused free flap allografts. When combined with a short course of rapamycin, the survival time of AdCTLA4Ig-perfused free flap allografts was remarkably prolonged. The mixed lymphocyte reaction results indicate that ex vivo transfer of AdCTLA4Ig induces antigen-specific unresponsiveness. AdCTLA4Ig perfusion did not change the proportion of CD4+25+Foxp3+ T cells in recipients' spleens.. The authors demonstrated that a singular ex vivo perfusion of AdCTLA4Ig gene induced efficient transduction of the flaps and, when combined with a short course of rapamycin, promoted the remarkably longer survival of flap allografts.

Topics: Abatacept; Animals; Disease Models, Animal; DNA; Free Tissue Flaps; Gene Expression; Gene Transfer Techniques; Graft Rejection; Graft Survival; Groin; Immunoconjugates; Immunohistochemistry; Immunosuppressive Agents; Male; Polymerase Chain Reaction; Rats; Rats, Inbred BN; Rats, Inbred Lew; Rats, Sprague-Dawley; Sirolimus; Skin Transplantation; Transplantation, Homologous

Nanoparticles possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded nanoparticles(NPs) can reduce neointima formation in a rat model of vein graft disease.. Poly(lactic-co-glycolic acid) (PLGA) NPs containing rapamycin was prepared using an oil/water solvent evaporation technique. Nanoparticle size and morphology were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA (RPLGA) NPs was studied using MTT Assay. Excised rat jugular vein was treated ex vivo with blank-NPs, or rapamycin-loaded NPs, then interposed back into the carotid artery position using a cuff technique. Grafts were harvested at 21 days and underwent morphometric analysis as well as immunohistochemical analysis.. Rapamycin was efficiently loaded in PLGA nanoparticles with an encapsulation efficiency was 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/ml significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts shown that the concentration of rapamycin in vein grafts at 3 weeks after grafting were 0.9 ± 0.1 μg/g. In grafted veins without treatment intima-media thickness was 300.4 ± 181.5 μm after grafting 21 days. Whereas, Veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). CD-31 staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization in 21 days vein grafts with no significant effect of blank or rapamycin-loaded NPs group.. We conclude that sustained-release rapamycin from rapymycin loaded NPs inhibits vein graft thickening without affecting the reendothelialization in rat carotid vein-to-artery interposition grafts and this may be a promising therapy for the treatment of vein graft disease.

Topics: Animals; Anti-Bacterial Agents; Disease Models, Animal; Endothelium, Vascular; Graft Survival; Hyperplasia; Jugular Veins; Male; Microscopy, Electron, Transmission; Muscle, Smooth, Vascular; Nanoparticles; Neointima; Rats; Rats, Sprague-Dawley; Sirolimus

Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections.. Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII.. A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed.. The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-β1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction.. The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

Topics: Adoptive Transfer; Animals; Antibodies; CD4-Positive T-Lymphocytes; Cells, Cultured; Coagulants; CTLA-4 Antigen; Disease Models, Animal; Drug Administration Schedule; Factor VIII; Forkhead Transcription Factors; Hemophilia A; Humans; Immune Tolerance; Immunosuppressive Agents; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Recombinant Proteins; Sirolimus; Time Factors; Transforming Growth Factor beta1

Etiologic factors for pancreatic cancer, the 4th deadliest malignant neoplasm in the United States, include obesity and abnormal glucose metabolism. Calorie restriction (CR) and rapamycin each affect energy metabolism and cell survival pathways via inhibition of mammalian target of rapamycin (mTOR) signaling. By using a Panc02 murine pancreatic cancer cell transplant model in 45 male C57BL/6 mice, we tested the hypothesis that rapamycin mimics the effects of CR on pancreatic tumor growth. A chronic regimen of CR, relative to an ad libitum-fed control diet, produced global metabolic effects such as reduced body weight (20.6 ± 1.6 g vs. 29.3 ± 2.3 g; P < 0.0001), improved glucose responsiveness, and decreased circulating levels of insulin-like growth factor (IGF)-1 (126 ± 8 ng/mL vs. 199 ± 11 ng/mL; P = 0.0006) and leptin (1.14 ± 0.2 ng/mL vs. 5.05 ± 1.2 ng/mL; P = 0.01). In contrast, rapamycin treatment (2.5 mg/kg intraperitoneal every other day, initiated in mice following 20 weeks of ad libitum control diet consumption), relative to control diet, produced no significant change in body weight, IGF-1 or leptin levels, but decreased glucose responsiveness. Pancreatic tumor volume was significantly reduced in the CR group (221 ± 107 mm(3); P < 0.001) and, to a lesser extent, the rapamycin group (374 ± 206 mm(3); P = 0.04) relative to controls (550 ± 147 mm(3)), and this differential inhibition correlated with expression of the proliferation marker Ki-67. Both CR and rapamycin decreased phosphorylation of mTOR, p70/S6K, and S6 ribosomal protein, but only CR decreased phosphorylation of Akt, GSK-3β, extracellular signal regulated kinase/mitogen-activated protein kinase, and STAT3(TYR705). These findings suggest that rapamycin partially mimics the anticancer effects of CR on tumor growth in a murine model of pancreatic cancer.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Body Weight; Caloric Restriction; Carcinoma, Pancreatic Ductal; Combined Modality Therapy; Disease Models, Animal; Glucose; Glucose Tolerance Test; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunoenzyme Techniques; Insulin-Like Growth Factor I; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition-like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Differentiation; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Activation; Glomerular Basement Membrane; Glomerular Mesangium; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Multiprotein Complexes; Phosphorylation; Podocytes; Protein Processing, Post-Translational; Proteins; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

The podocyte plays a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis. Inhibitors of mammalian target of rapamycin (mTOR) have variably decreased or caused proteinuria and sclerosis in human disease and experimental settings. In this issue of the JCI, two interesting studies of podocyte-specific manipulation of the mTOR system shed light on the complexity of this pathway in the podocyte.

Topics: Adaptor Proteins, Signal Transducing; Adult; Animals; Carrier Proteins; Child; Diabetic Nephropathies; Disease Models, Animal; Genetic Predisposition to Disease; Humans; Kidney Glomerulus; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Podocytes; Proteins; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors

The cellular and molecular mechanisms and safety after drug-eluting stent (DES) implantation in diabetic patients are still poorly understood; therefore, in this study, we evaluated the pathologic responses of the sirolimus-eluting stent (SES) or paclitaxel-eluting stent (PES) in a type I diabetes mellitus (DM) rat model.. The type I DM rat model was manipulated by intra-peritoneal streptozotocin injection. Two weeks later, DES was implanted in the aorta of rats with hyperglycemia or not as a control. Four weeks after DES implantation, the stented aorta was isolated and histomorphometric analysis was performed.. On histomorphometric analysis, increased thrombus, inflammatory cell infiltration, and neointimal hyperplasia (NIH) without change of the smooth muscle cell number after DES implantation were observed in DM rats compared with non-DM (NDM) rats. Furthermore, delayed coverage of mature endothelial cells defined as a von Willebrand factor expression and increased immature endothelial cells as a c-kit expression after DES implantation were observed in DM rats compared with NDM rats. Increased fibrin deposition and decreased hyaluronic acid accumulation at NIH after DES implantation were also observed in DM rats compared with NDM rats.. In conclusion, the main mechanism of restenosis after DES implantation under hyperglycemic conditions was initial thrombus with changes of the extracellular matrix rather than SMC proliferation. These results provided a therapeutic clue for the selection of DES and application of combination therapy using anti-thrombotic and anti-inflammatory drugs in diabetic patients.

Topics: Animals; Anti-Inflammatory Agents; Aorta; Body Weight; Coronary Restenosis; Diabetes Mellitus, Type 1; Disease Models, Animal; Drug-Eluting Stents; Fibrin; Humans; Hyaluronic Acid; Hyperglycemia; Inflammation; Male; Paclitaxel; Rats; Rats, Sprague-Dawley; Sirolimus; Thrombosis

Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM(-/-) mice, despite having fewer naïve CD8⁺ T cells, effectively clear the virus. However, aberrant CD8⁺ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM(-/-) splenic CD8⁺ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM(-/-) mice. Altogether, these results show that CD8⁺T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value.

Topics: Animals; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; CD8-Positive T-Lymphocytes; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Enzyme Activation; Immunologic Memory; Interleukin-15; Lymphocyte Activation; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; Receptors, Antigen, T-Cell; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

To determine the efficacy of sirolimus-eluting bioabsorbable magnesium alloy stents (SEBMAS) in restenosis prevention.. A balloon-expandable bioabsorbable magnesium alloy stent (BMAS) was created and coated with biodegradable poly(lactic acid-co-trimethylene carbonate) that contained the antiproliferative drug sirolimus (140 ± 40 µg/cm²). Both the uncoated BMAS and the coated SEBMAS were deployed 2 cm apart in balloon-injured infrarenal abdominal aortas of 20 New Zealand white rabbits. The stented aortic segments were removed at 30, 60, 90, and 120 days (5 rabbits per interval) after implantation. The average stent strut sectional area of each group was measured to evaluate the degree of magnesium corrosion and to forecast the biodegradation time profile of the magnesium stent. Histology and histopathology of the sectioned stented aortic segments were performed to evaluate neointima formation, endothelialization, and inflammation.. The SEBMAS degraded gradually after being implanted into the rabbit aorta, and total biocorrosion occurred after ~120 days. In all groups, the lumen area was significantly greater, but the neointimal area was significantly smaller in SEBMAS segments compared with the uncoated BMAS segments (p < 0.05) at all time points. There was no significant difference in the injury or inflammation scores between the groups. Endothelialization was delayed at 30 days in the SEBMAS segments vs. the uncoated BMAS segments.. SEBMAS further reduces intimal hyperplasia and improves the lumen area when compared to uncoated BMAS; however, it delays vascular healing and endothelialization.

Topics: Alloys; Angioplasty; Animals; Aorta, Abdominal; Aortic Diseases; Arterial Occlusive Diseases; Cardiovascular Agents; Cell Proliferation; Coated Materials, Biocompatible; Constriction, Pathologic; Dioxanes; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Hyperplasia; Lactic Acid; Magnesium; Male; Polyesters; Polymers; Prosthesis Design; Rabbits; Secondary Prevention; Sirolimus; Time Factors; Wound Healing

Hemophagocytic lymphohistiocytosis (HLH) is an immunodysregulatory disorder for which more effective treatments are needed. The macrolide rapamycin has immunosuppressive properties, making it an attractive candidate for controlling the aberrant T cell activation that occurs in HLH. To investigate its therapeutic potential, we used rapamycin to treat Lymphocytic Choriomeningitis Virus (LCMV)-infected perforin-deficient (Prf1(-/-)) mice according to a well-established model of HLH. At the regimens tested, rapamycin did not improve weight loss, splenomegaly, hemophagocytosis, cytopenias, or proinflammatory cytokine production in LCMV-infected Prf1(-/-) animals. Thus, single agent rapamycin appears ineffective in treating the clinical and laboratory manifestations of LCMV-induced HLH.

Topics: Animals; Arenaviridae Infections; Cell Separation; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immunosuppressive Agents; Lymphocytic choriomeningitis virus; Lymphohistiocytosis, Hemophagocytic; Mice; Mice, Inbred C57BL; Mice, Knockout; Perforin; Sirolimus

As in other diseases associated with mental retardation, dendrite morphology and synaptic plasticity are impaired in Down's syndrome (DS). Both these features of neurons are critically influenced by BDNF, which regulates local dendritic translation through phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (mTOR) and Ras-ERK signaling cascades. Here we show that the levels of BDNF and phosphorylated Akt-mTOR (but not Ras-ERK) pathway proteins are augmented in hippocampal dendrites of Ts1Cje mice, a DS model. Consequently, the rate of local dendritic translation is abnormally high and the modulatory effect of exogenous BDNF is lost. Interestingly, rapamycin (a Food and Drug Administration-approved drug) restores normal levels of phosphorylated Akt-mTOR proteins and normal rates of local translation in Ts1Cje neurons, opening new therapeutic perspectives for DS. The NMDAR inhibitors APV, MK-801, and memantine also restore the normal levels of phospho-mTOR in dendrites of Ts1Cje hippocampal neurons. We propose a model to explain how BDNF-mediated regulation of local translation is lost in the Ts1Cje hippocampus through the establishment of a glutamatergic positive-feedback loop. Together, these findings help elucidate the mechanisms underlying altered synaptic plasticity in DS.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cells, Cultured; Dendrites; Disease Models, Animal; Down Syndrome; Hippocampus; Immunohistochemistry; Mice; Mice, Transgenic; Neurons; Phosphorylation; Protein Biosynthesis; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Statistics, Nonparametric; TOR Serine-Threonine Kinases

Deep infiltrating endometriosis (DIE) is a particular clinical and histological entity of endometriosis responsible for chronic pelvic pain and infertility. Here we characterize the proliferative phenotype of DIE cells, to explore the cellular and molecular mechanisms that could explain their aggressive potential. In addition, the inhibition of mTOR/AKT pathway was tested, as a potential treatment of DIE. Included were 22 patients with DIE and 12 control patients without endometriosis. Epithelial and stromal cells were extracted from biopsies of eutopic endometrium and deep infiltrating endometriotic nodules from patients with DIE. Cell proliferation was determined by thymidine incorporation. Oxidative stress was assayed by spectrofluorometry. The ERK and mTOR/AKT pathways were analyzed in vitro by Western blot and for AKT in vivo in a mouse model of DIE. The proliferation rate of eutopic endometrial cells and of deep infiltrating endometriotic cells from DIE patients was higher than that of endometrial cells from controls. The hyperproliferative phenotype of endometriotic cells was associated with an increase in endogenous oxidative stress, and with activation of the ERK and mTOR/AKT pathways. mTOR/AKT inhibition by temsirolimus decreased endometriotic cell proliferation both in vitro and in vivo in a mouse model of DIE. Blocking the mTOR/AKT pathway offers new prospects for the treatment of DIE.

Topics: Adult; Animals; Biopsy; Cell Proliferation; Disease Models, Animal; Endometriosis; Endometrium; Female; Humans; Mice; Mice, Nude; Middle Aged; Oxidative Stress; Phenotype; Protein Kinase Inhibitors; Reactive Oxygen Species; Sirolimus; Spectrometry, Fluorescence; TOR Serine-Threonine Kinases

Topics: Animals; Calcium; Calcium Signaling; Disease Models, Animal; Glomerular Filtration Rate; Humans; Kidney Diseases; Kidney Glomerulus; Models, Biological; Mutation; Rats; Signal Transduction; Sirolimus; Transcription Factors

Molecular deregulations underlying epithelioid sarcoma (ES) progression are poorly understood yet critically needed to develop new therapies. Epidermal growth factor receptor (EGFR) is overexpressed in ES; using preclinical models, we examined the ES EGFR role and assessed anti-ES EGFR blockade effects, alone and with mTOR inhibition.. EGFR and mTOR expression/activation was examined via tissue microarray (n = 27 human ES specimens; immunohistochemistry) and in human ES cell lines (Western blot and quantitative reverse transcriptase PCR). Cell proliferation, survival, migration, and invasion effects of EGFR and mTOR activation treated with erlotinib (anti-EGFR small-molecule inhibitor) alone and combined with rapamycin were assessed in cell culture assays. In vivo growth effects of erlotinib alone or with rapamycin were evaluated using severe combined immunodeficient mouse ES xenograft models.. EGFR was expressed and activated in ES specimens and cell lines. EGFR activation increased ES cell proliferation, motility, and invasion and induced cyclin D1, matrix metalloproteinase (MMP) 2, and MMP9 expression. EGFR blockade inhibited these processes and caused significant cytostatic ES growth inhibition in vivo. mTOR pathway activation at varying levels was identified in all tissue microarray-evaluable ES tissues; 88% of samples had no or reduced PTEN expression. Similarly, both ES cell lines showed enhanced mTOR activity; VAESBJ cells exhibited constitutive mTOR activation uncoupled from EGFR signaling. Most importantly, combined erlotinib/rapamycin resulted in synergistic anti-ES effects in vitro and induced superior tumor growth inhibition in vivo versus single agent administration.. EGFR and mTOR signaling pathways are deregulated in ES. Preclinical ES model-derived insights suggest that combined inhibition of these targets might be beneficial, supporting evaluations in clinical trials.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Invasiveness; Quinazolines; Sarcoma; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

In order to evaluate the effectiveness of everolimus vs. rapamycin in the treatment of diabetic nephropathy, 8-week old diabetic (db/db) mice received everolimus (2 mg/kg every day) or rapamycin (2 mg/kg every day) for 4 weeks or 12 weeks respectively. Blood and 24-h urine samples were collected for biochemical tests. One kidney from each mouse was homogenized for protein analysis and the other was removed for histological analysis. The expression levels of transforming growth factor-β1 (TGF-β1)and phospho-p70s6k were detected by using ELISA and Western blot, respectively in the renal tissue as well as in mesengial cell culture samples. Everolimus was significantly more effective than rapamycin in improving indexes of renal function and glomerular hypertrophy, and in decreasing accumulation and expansion of the extracellular matrix. However, everolimus inhibited TGF-β1 secretion and p70s6k phosphorylation induced by high glucose in vitro less efficiently than rapamycin at the same dose. Everolimus was more effective than rapamycin in preventing diabetic nephropathy in vivo, which may be contributed to the fact that everolimus has better bioavailability and a higher oral absorption rate.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Everolimus; Male; Mice; Sirolimus

Topics: Animals; Bleomycin; Disease Models, Animal; Humans; Immunosuppressive Agents; Lung Diseases, Interstitial; Lymphangioleiomyomatosis; Mesenchymal Stem Cell Transplantation; Pulmonary Fibrosis; Sirolimus

In temporal lobe epilepsy many somatostatin interneurons in the dentate gyrus die. However, some survive and sprout axon collaterals that form new synapses with granule cells. The functional consequences of γ-aminobutyric acid (GABA)ergic synaptic reorganization are unclear. Development of new methods to suppress epilepsy-related interneuron axon sprouting might be useful experimentally.. Status epilepticus was induced by systemic pilocarpine treatment in green fluorescent protein (GFP)-expressing inhibitory nerurons (GIN) mice in which a subset of somatostatin interneurons expresses GFP. Beginning 24 h later, mice were treated with vehicle or rapamycin (3 mg/kg intraperitoneally) every day for 2 months. Stereologic methods were then used to estimate numbers of GFP-positive hilar neurons per dentate gyrus and total length of GFP-positive axon in the molecular layer plus granule cell layer. GFP-positive axon density was calculated. The number of GFP-positive axon crossings of the granule cell layer was measured. Regression analyses were performed to test for correlations between GFP-positive axon length versus number of granule cells and dentate gyrus volume.. After pilocarpine-induced status epilepticus, rapamycin- and vehicle-treated mice had approximately half as many GFP-positive hilar neurons as did control animals. Despite neuron loss, vehicle-treated mice had over twice the GFP-positive axon length per dentate gyrus as controls, consistent with GABAergic axon sprouting. In contrast, total GFP-positive axon length was similar in rapamycin-treated mice and controls. GFP-positive axon length correlated most closely with dentate gyrus volume.. These findings suggest that rapamycin suppressed axon sprouting by surviving somatostatin/GFP-positive interneurons after pilocarpine-induced status epilepticus in GIN mice. It is unclear whether the effect of rapamycin on axon length was on interneurons directly or secondary, for example, by suppressing growth of granule cell dendrites, which are synaptic targets of interneuron axons. The mammalian target of rapamycin (mTOR) signaling pathway might be a useful drug target for influencing GABAergic synaptic reorganization after epileptogenic treatments, but additional side effects of rapamycin treatment must be considered carefully.

Topics: Animals; Axons; Dentate Gyrus; Disease Models, Animal; Epilepsy, Temporal Lobe; Female; Green Fluorescent Proteins; Interneurons; Male; Mice; Pilocarpine; Sirolimus; Somatostatin; Status Epilepticus; Synapses

Epileptic seizures, particularly infantile spasms, are often seen in infants with tuberous sclerosis complex (TSC) soon after birth. It is feared that there are long-term developmental and cognitive consequences from ongoing, frequent epilepsy. In addition, the hallmark brain pathology of TSC, cortical tubers and giant cells are fully developed at late gestational ages. These observations have led us to examine the benefit of prenatal rapamycin in a new fetal brain model of TSC. In this Tsc1(cc) Nes-cre(+) mouse model, recombination and loss of Tsc1 in neural progenitor cells leads to brain enlargement, hyperactivation of mTOR, and neonatal death on P0 due to reduced pup-maternal interaction. A single dose of prenatal rapamycin given to pregnant dams (1 mg/kg, subcutaneous) rescued the lethality of mutant mice. This one dose of prenatal rapamycin treatment reduced hyperactivation of the mTOR pathway in the mutant brain without causing apparent pregnancy loss. Continued postnatal rapamycin beginning at day 8 extended the survival of these mice to a median of 12 days with complete suppression of hyperactive mTOR. However, the rapamycin-treated mutants developed enlarged brains with an increased number of brain cells, displaying marked runting and developmental delay. These observations demonstrate the therapeutic benefit and limitations of prenatal rapamycin in a prenatal-onset brain model of TSC. Our data also suggest the possibility and limitations of this approach for TSC infants and mothers.

Topics: Animals; Animals, Newborn; Brain; Cell Count; Cell Size; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Integrases; Intermediate Filament Proteins; Mice; Nerve Tissue Proteins; Nestin; Neurons; Phenotype; Pregnancy; Signal Transduction; Sirolimus; Survival Analysis; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis

Growing evidence has indicated that the blockade of group II metabotropic glutamate (mGlu2/3) receptor exerts antidepressant-like effects in several animal models of depression. However, the molecular mechanisms underlying the action of mGlu2/3 receptor antagonists are not well understood. Here, we investigated the involvement of mammalian target of rapamycin (mTOR) signaling in the acute and sustained antidepressant-like effects of mGlu2/3 receptor antagonists such as (1R, 2R, 3R, 5R, 6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039) and (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). Mice were subjected to a tail suspension test (TST) to assess the acute and sustained antidepressant-like effects. We evaluated the effect of rapamycin, an mTOR antagonist, on the acute and sustained antidepressant-like effects of mGlu2/3 receptor antagonists. Both MGS0039 and LY341495 exerted antidepressant-like effects, as evaluated using the TST; these effects were sustained for 24 h. Pretreatment with rapamycin blocked the sustained, but not the acute, antidepressant-like effects of mGlu2/3 receptor antagonists, as observed in ketamine. The present result suggests that the blockade of the mGlu2/3 receptor may activate mTOR signaling, and that the activation of mTOR signaling may contribute to the sustained antidepressant-like effects of mGlu2/3 receptor antagonists.

Topics: Amino Acids; Analysis of Variance; Animals; Bridged Bicyclo Compounds; Depression; Dicarboxylic Acids; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Hindlimb Suspension; Ketamine; Male; Mice; Mice, Inbred ICR; Receptors, Metabotropic Glutamate; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Xanthenes

Stroke is a major cause of mortality and disability. The management with thrombolytic therapy has to be initiated within 3-4 h and is associated with limitations like increased risk of intracranial hemorrhage and progression of cerebral injury. Immunophilin inhibitors such as cyclosporine A and tacrolimus have been shown to afford neuroprotection by improving neurological functions and infarct volume in models of ischemic stroke. In the present study, the effect of rapamycin in middle cerebral artery occlusion (MCAo) model of ischemic stroke was evaluated. Ischemic stroke was induced in rats by occluding the MCA using the intraluminal thread. After 1 h of MCAo, animals were administered rapamycin (50, 150, 250 μg/kg, i.p.). After 2 h of occlusion, reperfusion was done. Thirty minutes after reperfusion, animals were subjected to diffusion-weighted magnetic resonance imaging for assessment of protective effect of rapamycin. Twenty-four hours after MCAo, motor performance was assessed, the animals were euthanized and the brains were removed for estimation of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. Significant improvement was observed with rapamycin 150 and 250 μg/kg in percent infarct area, apparent diffusion coefficient and signal intensity as compared to vehicle treated group. Rapamycin treatment ameliorated motor impairment associated with MCAo and significantly reversed the changes in levels of malondialdehyde, glutathione, nitric oxide and myeloperoxidase. The results of the present study indicate neuroprotective effect of rapamycin in MCAo model of stroke. Therefore, rapamycin might be considered as a therapeutic strategy for stroke management.

Topics: Animals; Brain; Brain Ischemia; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione; Hand Strength; Humans; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Neuroimaging; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Rotarod Performance Test; Sirolimus

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Neuroendocrine; Disease Models, Animal; Everolimus; Humans; Indoles; Mice; Pancreatic Neoplasms; Pyrroles; Sirolimus; Sunitinib; TOR Serine-Threonine Kinases

Prosthetic mesh is used frequently in abdominal wall hernia reconstruction but is prone to postoperative adhesion formation. Complications resulting from intra-abdominal adhesions represent a considerable clinical and cost burden. We, herein, investigate the antiproliferative and antiadhesiogenic properties of sirolimus and hydrogel-impregnated, drug-eluting mesh to decrease such complications in a mouse model of abdominal wall hernia repair.. A 1 × 1cm(2) polypropylene mesh from 1 of 3 groups (group 1, plain control; group 2, hydrogel [2% agarose]; and group 3, hydrogel + 10 mcg sirolimus) was implanted operatively into the peritoneal cavity of BALB/c mice and followed for up to 4 weeks. Adhesions were scored by percent surface area of mesh (range, 0-100%), severity (range, 0-3), and tenacity (range, 0-4). Representative samples were assessed by scanning electron microscopy.. Mesh impregnated with the combination of hydrogel and sirolimus led to a significant decrease in adhesion formation. The percent surface area of adhesional attachment to mesh was decreased from 100.0 ± 0% in the plain mesh control group versus 18 ± 8% (P < .001) in the combined impregnated mesh group. Similarly, adhesion severity scores were decreased from a score of 2.9 ± 0.1 (plain mesh) versus 1.4 ± 0.1 (sirolimus/hydrogel-impregnated mesh) (P < .001). Scores for tenacity were also decreased markedly from 3.5 ± 0.2 (plain mesh) versus 1.5 ± 0.1 (sirolimus/hydrogel-impregnated mesh (P < .001).. Creation of a sirolimus drug-eluting and hydrogel-impregnated polypropylene mesh resulted in marked decrease of adhesion formation in this mouse model, was well tolerated without side effects, and has potential for clinical application.

Topics: Abdominal Wall; Abdominal Wound Closure Techniques; Animals; Biocompatible Materials; Disease Models, Animal; Hernia, Abdominal; Hydrogel, Polyethylene Glycol Dimethacrylate; Immunosuppressive Agents; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Polypropylenes; Sirolimus; Surgical Mesh; Tissue Adhesions

Induction of allogeneic hematopoietic chimerism is a promising strategy to induce tolerance to donor islets for treating type 1 diabetes. Successful induction of chimerism requires overcoming host alloimmunity. In diabetes-prone nonobese diabetic (NOD) mice, this is challenging due to their general tolerance resistance. Although the adaptive alloimmunity of NOD mice is a known barrier to allogeneic chimerism, whether NOD natural killer (NK) cells are an additional barrier has not been examined. Because NOD NK cells exhibit functional defects, they may not inhibit chimerism generation.. Antibody depletion of NK cells in vivo, or transplantation of F1 hybrid donor cells to eliminate the "missing-self" trigger of NK cells, was preformed to test the NK-mediated rejection of donor bone marrow cells. We also studied the capacity of rapamycin to block the NK cell response against allogeneic cells in vivo.. Depleting NK cells or rendering them unresponsive to the donor greatly improved the level of chimerism obtained in NOD mice. Rapamycin significantly reduced the resistance to allogeneic chimerism mounted by NOD NK cells; however, it was much less effective than NK cell depletion by antibodies.. Contrary to the view that NOD NK cells are defective, we found these cells to be a substantial barrier to allogeneic chimerism in the presence or absence of adaptive immunity. Moreover, rapamycin will need to be combined with other approaches to fully overcome the NK cell barrier.

Topics: Adaptive Immunity; Animals; Chimerism; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Graft Rejection; Hematopoietic Stem Cells; Immunosuppressive Agents; Islets of Langerhans Transplantation; Killer Cells, Natural; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred NOD; Sirolimus; Transplantation Chimera; Transplantation, Homologous

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Topics: Animals; Autophagy; Burkholderia cenocepacia; Burkholderia Infections; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Down-Regulation; Interleukin-1beta; Intracellular Space; Lysosomes; Macrophages; Mice; Mice, Inbred C57BL; Microbial Viability; Microtubule-Associated Proteins; Mutation; Phagosomes; Pneumonia; RNA, Small Interfering; Sirolimus; Vacuoles

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1(G93A) rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1(G93A) mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1(G93A) mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anterior Horn Cells; Astrocytes; Cell Differentiation; Cell Proliferation; Cell Survival; Cervical Vertebrae; Cyclosporine; Disease Models, Animal; Female; Humans; Immunosuppression Therapy; Male; Mice; Mutation; Neuroglia; Neurons; Oligodendroglia; Pregnancy; Sirolimus; Spinal Cord; Stem Cell Transplantation; Stem Cells; Superoxide Dismutase; Superoxide Dismutase-1; Tacrolimus

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.

Topics: Animals; Asthma; Blotting, Western; Bronchial Hyperreactivity; Cell Separation; Cytokines; Disease Models, Animal; Female; Flow Cytometry; Goblet Cells; Hypersensitivity; Immunoglobulin E; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Pyroglyphidae; Real-Time Polymerase Chain Reaction; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain involvement causes epilepsy, intellectual disability, and autism. The hallmark pathological finding in TSC is the cerebral cortical tuber and its unique constituent, giant cells. However, an animal model that replicates giant cells has not yet been described. Here, we report that mosaic induction of Tsc1 loss in neural progenitor cells in Tsc1(cc) Nestin-rtTA(+) TetOp-cre(+) embryos by doxycycline leads to multiple neurological symptoms, including severe epilepsy and premature death. Strikingly, Tsc1-null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated giant cells had multiple signs of organelle dysfunction, including markedly increased mitochondria, aberrant lysosomes, and elevated cellular stress. We found similar vacuolated giant cells in human tuber specimens. Postnatal rapamycin treatment completely reversed these phenotypes and rescued the mutants from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR complex 1 activation in the developing brain. This TSC brain model provides insights into the pathogenesis and organelle dysfunction of giant cells, as well as epilepsy control in patients with TSC.

Topics: Animals; Blotting, Western; Cell Survival; Disease Models, Animal; Humans; Mice; Microscopy, Electron; Microscopy, Fluorescence; Neurons; Polymerase Chain Reaction; Sirolimus; Stem Cells; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Dentate granule cell axon (mossy fiber) sprouting creates an aberrant positive-feedback circuit that might be epileptogenic. Presumably, mossy fiber sprouting is initiated by molecular signals, but it is unclear whether they are expressed transiently or persistently. If transient, there might be a critical period when short preventative treatments could permanently block mossy fiber sprouting. Alternatively, if signals persist, continuous treatment would be necessary. The present study tested whether temporary treatment with rapamycin has long-term effects on mossy fiber sprouting.. Mice were treated daily with 1.5 mg/kg rapamycin or vehicle (i.p.) beginning 24 h after pilocarpine-induced status epilepticus. Mice were perfused for anatomic evaluation immediately after 2 months of treatment ("0 delay") or after an additional 6 months without treatment ("6-month delay"). One series of sections was Timm-stained, and an adjacent series was Nissl-stained. Stereologic methods were used to measure the volume of the granule cell layer plus molecular layer and the Timm-positive fraction. Numbers of Nissl-stained hilar neurons were estimated using the optical fractionator method.. At 0 delay, rapamycin-treated mice had significantly less black Timm staining in the granule cell layer plus molecular layer than vehicle-treated animals. However, by 6-month delay, Timm staining had increased significantly in mice that had been treated with rapamycin. Percentages of the granule cell layer plus molecular layer that were Timm-positive were high and similar in 0 delay vehicle-treated, 6-month delay vehicle-treated, and 6-month delay rapamycin-treated mice. Extent of hilar neuron loss was similar among all groups that experienced status epilepticus and, therefore, was not a confounding factor. Compared to naive controls, average volume of the granule cell layer plus molecular layer was larger in 0 delay vehicle-treated mice. The hypertrophy was partially suppressed in 0 delay rapamycin-treated mice. However, 6-month delay vehicle- and 6-month delay rapamycin-treated animals had similar average volumes of the granule cell layer plus molecular layer that were significantly larger than those of all other groups.. Status epilepticus-induced mossy fiber sprouting and dentate gyrus hypertrophy were suppressed by systemic treatment with rapamycin but resumed after treatment ceased. These findings suggest that molecular signals that drive mossy fiber sprouting and dentate gyrus hypertrophy might persist for >2 months after status epilepticus in mice. Therefore, prolonged or continuous treatment might be required to permanently suppress mossy fiber sprouting.

Topics: Animals; Critical Period, Psychological; Disease Models, Animal; Epilepsy, Temporal Lobe; Hippocampus; Immunosuppressive Agents; Mice; Mossy Fibers, Hippocampal; Muscarinic Agonists; Neurons; Pilocarpine; Sirolimus; Time Factors

Pulmonary fibrosis is a devastating disease with a poor prognosis. Although the diagnosis and pathophysiology of this disease have been better characterized over the past few years, there is no effective therapy for the disease. The aim of this study was to evaluate the anti-inflammatory and anti-fibrotic effects of sirolimus (SRL), which is a potential anti-fibrotic agent, by using bleomycin (BLM)-induced pulmonary fibrosis model in rats.. A single intra-tracheal injection of BLM (2.5 U/kg) was administered and sirolimus (2.5 mg/kg/day) was given orally, beginning either one day before (early SRL) or nine days after (late SRL) the BLM administration. The effect of SRL on fibrosis was studied by analysis of cytokine levels in BAL fluid, measurement of lung tissue hydroxyproline (HPL) content and histopathological examination.. Both early and late SRL administrations caused a decrease in the levels of IL-13, PDGF-A and TGF-β1 (p=0.001) and an increase in IFN-γ levels (p=0.001) in BAL fluid. Early and late SRL also caused a decrease in HPL content (p=0.001). Early sirolimus caused a significant decrease in fibrosis score (p=0.001), while late SRL did not.. Sirolimus was effective in BLM-induced pulmonary fibrosis model, especially in the early phases of the disease.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Disease Models, Animal; Injections, Intravenous; Interferon-gamma; Interleukin-13; Lung; Male; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Sirolimus; Transforming Growth Factor beta1

The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF), a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK), which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1), was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI) in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group) with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001) in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

Topics: Animals; Catheterization; Disease Models, Animal; Enzyme Activation; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Infusions, Intra-Arterial; Lipoproteins; Mitogen-Activated Protein Kinases; Models, Biological; Monocytes; Muscle, Smooth, Vascular; Paclitaxel; Phosphorylation; PPAR gamma; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rosiglitazone; Sirolimus; Thiazolidinediones; Thromboplastin; Umbilical Arteries

L-DOPA-induced dyskinesia, the rate-limiting side effect in the therapy of Parkinson's disease, is mediated by activation of mammalian target of rapamycin (mTOR) signaling in the striatum. We found that Ras homolog enriched in striatum (Rhes), a striatal-specific protein, binds to and activates mTOR. Moreover, Rhes(-/-) mice showed reduced striatal mTOR signaling and diminished dyskinesia, but maintained motor improvement on L-DOPA treatment, suggesting a therapeutic benefit for Rhes-binding drugs.

Topics: Adaptor Proteins, Signal Transducing; Adrenergic Agents; Animals; Antiparkinson Agents; Cell Line, Transformed; Corpus Striatum; Culture Media, Serum-Free; Disability Evaluation; Disease Models, Animal; Dyskinesia, Drug-Induced; Functional Laterality; Gene Expression Regulation; GTP-Binding Proteins; Humans; Immunosuppressive Agents; Levodopa; Mice; Mice, Knockout; Movement; Mutation; Neurons; Neurotoxicity Syndromes; Oxidopamine; Phosphorylation; Protein Binding; Radioligand Assay; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection

Bronchiolitis obliterans, the pathological hallmark of chronic pulmonary rejection, severely impacts long-term survival following lung transplantation. However, experimental reproduction of this pathophysiological phenomenon has not been achieved with contemporary in vivo models. Here, a model of chronic rejection is described, with sensitised recipients receiving unilateral orthotopic rat lung transplants. Lewis rats, sensitised with skin from brown Norway rats 7 days before receiving left lung transplants from donors that were Lewis x brown Norway F(1) hybrids, were analysed during day 21-84. The development of chronic rejection was modulated by a treatment with rapamycin and cyclosporin, and characterised histologically, immunohistochemically and by reverse transcriptase PCR. Characteristic histopathological changes leading to chronic rejection were induced over time by an initial treatment with cyclosporin in the presence of continuous rapamycin application. At day 84, fibrotic lesions replaced the respiratory epithelium within small bronchioles, with strong expression of smooth muscle alpha-actin and upregulation of mRNA for T-helper cell type-1 cytokines, smooth muscle alpha-actin, transforming growth factor-beta and CC chemokine ligand 5, but decreased forkhead box protein P3 gene expression. A reproducible and clinically relevant experimental set-up for progressive chronic rejection in rat pulmonary allografts is described. This model will permit better understanding of the pathological changes of small airways during the development of bronchiolitis obliterans, and may serve as an in vivo set-up for testing the efficacy of novel therapeutic interventions.

Topics: Actins; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Bronchiolitis Obliterans; CD3 Complex; Chemokine CCL5; Chronic Disease; Cyclosporine; Disease Models, Animal; Fluorescent Antibody Technique; Forkhead Transcription Factors; Graft Rejection; Immunosuppressive Agents; Lung Transplantation; Macrophages, Alveolar; Male; Rats; Rats, Inbred BN; Rats, Inbred Lew; Sirolimus; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta; Transplantation, Homologous

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

Topics: Animals; Benzamides; Disease Models, Animal; Dose-Response Relationship, Drug; Fibroblasts; Fibrosis; Imatinib Mesylate; Kidney; Kidney Diseases; Male; Piperazines; Proto-Oncogene Proteins c-abl; Pyrimidines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Smad2 Protein; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; Ureteral Obstruction

Though stents are deployed in diseased arteries drug distribution has only been quantified in intact, non-diseased vessels. We correlated steady-state arterial drug distribution with tissue ultrastructure and composition in abdominal aortae from atherosclerotic human autopsy specimens and rabbits with lesions induced by dietary manipulation and controlled injury. Paclitaxel, everolimus, and sirolimus depositions in the human aortae were maximal in the media and scaled inversely with lipid content. Net tissue paclitaxel and everolimus levels were indistinguishable in mildly injured rabbit arteries independent of diet. Yet, serial sectioning of cryopreserved arterial segments demonstrated a differential transmural deposition pattern that was amplified with disease and correlated with the expression of their intracellular targets, tubulin and FKBP-12. Tubulin distribution and paclitaxel binding increased with vascular injury and macrophage infiltration, and were reduced with lipid content. Sirolimus analogs and their specific binding target FKBP-12 were less sensitive to alterations of diet in mildly injured arteries, presumably reflecting a faster transient response of FKBP-12 to injury. The data demonstrate that disease-induced changes in the distribution of drug-binding proteins and interstitial lipid alter the distribution of these drugs, forcing one to consider how disease might affect the evaluation and efficacy of the local release of these and like compounds.

Topics: Animals; Aorta, Abdominal; Atherosclerosis; Cholesterol; Disease Models, Animal; Drug Delivery Systems; Drug-Eluting Stents; Everolimus; Humans; Male; Paclitaxel; Protein Binding; Rabbits; Sirolimus; Tissue Distribution; Tunica Intima

Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the expansion of the polyglutamine repeat region within the ataxin-3 protein. The mutant protein forms intracellular aggregates in the brain. However, the cellular mechanisms causing toxicity are still poorly understood and there are currently no effective treatments. In this study we show that administration of a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor performance in a transgenic mouse model of spinocerebellar ataxia type 3. Temsirolimus inhibits mammalian target of rapamycin and hence upregulates protein degradation by autophagy. Temsirolimus reduces the number of aggregates seen in the brains of transgenic mice and decreases levels of cytosolic soluble mutant ataxin-3, while endogenous wild-type protein levels remain unaffected. Temsirolimus is designed for long-term use in patients and therefore represents a possible therapeutic strategy for the treatment of spinocerebellar ataxia type 3. Using this disease model and treatment paradigm, we employed a microarray approach to investigate transcriptional changes that might be important in the pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin specific peptidase-15, which showed expression changes at both the messenger ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were also changed in mice expressing another mutant polyglutamine protein, huntingtin. In total we identified 16 transcripts that were decreased in transgenic ataxin-3 mice that were normalized following temsirolimus treatment. In this mouse model with relatively mild disease progression, the number of transcripts changed was low and the magnitude of these changes was small. However, the importance of these transcriptional alterations in the pathogenesis of spinocerebellar ataxia type 3 remains unclear.

Topics: Animals; Ataxin-3; Autophagy; Cells, Cultured; Disease Models, Animal; Humans; Machado-Joseph Disease; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nuclear Proteins; Rats; Rats, Sprague-Dawley; Sirolimus; Transcription Factors

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

Topics: Adaptor Proteins, Signal Transducing; Animals; CA1 Region, Hippocampal; Carrier Proteins; Cell Cycle Proteins; Cognition Disorders; Disease Models, Animal; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factors; Excitatory Postsynaptic Potentials; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Expression Regulation; Immunoprecipitation; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Receptors, Metabotropic Glutamate; Serine; Signal Transduction; Sirolimus

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Cell Division; Disease Models, Animal; Fibrosis; Gene Expression; Humans; Immunosuppressive Agents; Intermediate Filament Proteins; Intracellular Signaling Peptides and Proteins; Kidney Tubules, Collecting; Kidney Tubules, Distal; Mice; Mosaicism; Nerve Tissue Proteins; Nestin; Phenotype; Polycystic Kidney Diseases; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Most genetically engineered mouse (GEM) models for colon cancer are based on tissuewide or germline gene modification, resulting in tumors predominantly of the small intestine. Several of these models involve modification of the adenomatous polyposis coli (Apc) gene and are excellent models for familial cancer predisposition syndromes. We have developed a stochastic somatic mutation model for sporadic colon cancer that presents with isolated primary tumors in the distal colon and recapitulates the entire adenoma-carcinoma-metastasis axis seen in human colon cancer. Using this model, we have analyzed tumors that are either solely mutant in the Apc gene or in combination with another colon cancer-associated mutant gene, the Kras G12D allele. Because of the restricted location in the distal colon, the natural history of the tumors can be analyzed by serial colonoscopy. As the mammalian target of rapamycin (mTOR) pathway is a critical component of the complex signaling network in colon cancer, we used this model to assess the efficacy of mTOR blockade through rapamycin treatment of mice with established tumors. After treatment, Apc mutant tumors were more than 80% smaller than control tumors. However, tumors that possessed both Apc and Kras mutations did not respond to rapamycin treatment. These studies suggest that mTOR inhibitors should be further explored as potential colorectal cancer therapies in patients whose tumors do not have activating mutations in KRAS.

Topics: Animals; Antibiotics, Antineoplastic; Colonic Neoplasms; Disease Models, Animal; Disease Progression; Genes, APC; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mutation; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

We report that rapamycin, an allosteric inhibitor of certain but not all actions of the key cellular kinase mammalian target of rapamycin (mTOR), protects neurons from death in both cellular and animal toxin models of Parkinson's disease (PD). This protective action appears to be attributable to blocked translation of RTP801/REDD1/Ddit4, a protein that is induced in cell and animal models of PD and in affected neurons of PD patients and that causes neuron death by leading to dephosphorylation of the survival kinase Akt. In support of this mechanism, in PD models, rapamycin spares phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity. The capacity of rapamycin to provide neuroprotection in PD models appears to arise from its selective suppression of some but not all actions of mTOR, as indicated by the contrasting finding that Torin1, a full catalytic mTOR inhibitor, is not protective and induces Akt dephosphorylation and neuron death.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Analysis of Variance; Animals; Cell Death; Cycloheximide; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Nerve Growth Factor; Neurons; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Parkinsonian Disorders; PC12 Cells; Protein Serine-Threonine Kinases; Rats; Serine; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Tyrosine 3-Monooxygenase

Cholangiocyte proliferation is necessary for biliary recovery from cold ischemia and reperfusion injury (CIRI), but there are few studies on its intracellular mechanism. In this process, the role of rapamycin, a new immunosuppressant used in liver transplantation, is still unknown. In order to determine whether rapamycin can depress cholangiocyte regeneration by inhibiting signal transducer and activator of transcription 3 (STAT3) activation, rapamycin (0.05 mg/kg) was administered to rats for 3 days before orthotopic liver transplantation. The results indicated that cholangiocytes responded to extended cold preservation (12 hours) with severe bile duct injures, marked activation of the interleukin-6 (IL-6)/STAT3 signal pathway, and increased expression of cyclin D1 until 7 days after transplantation, and this was followed by compensatory cholangiocyte regeneration. However, rapamycin treatment inhibited STAT3 activation and resulted in decreased cholangiocyte proliferation and delayed biliary recovery after liver transplantation. On the other hand, rapamycin showed no effect on the expression of IL-6. We conclude that the IL-6/STAT3 signal pathway is involved in initiating cholangiocytes to regenerate and repair CIRI. Rapamycin represses cholangiocyte regeneration by inhibiting STAT3 activation, which might have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation. Insights gained from this study will be helpful in designing therapy using rapamycin in clinical patients after liver transplantation.

Topics: Animals; Bile Ducts, Intrahepatic; Cell Division; Cryopreservation; Cyclin D1; Disease Models, Animal; Graft Rejection; Immunosuppressive Agents; Interleukin-6; Liver Regeneration; Liver Transplantation; Male; Phosphorylation; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; STAT3 Transcription Factor

Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026.. These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.

Topics: Animals; Cysts; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Liver Diseases; Mice; Polycystic Kidney, Autosomal Dominant; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels; Vascular Endothelial Growth Factor A

Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor disorder characterized by the growth of hamartomas in various organs including the kidney, brain, skin, lungs, and heart. Rapamycin has been shown to reduce the size of kidney angiomyolipomas associated with TSC; however, tumor regression is incomplete and kidney angiomyolipomas regrow after cessation of treatment. Mouse models of TSC2 related tumors are useful for evaluating new approaches to drug therapy for TSC.. In cohorts of Tsc2+/- mice, we compared kidney cystadenoma severity in A/J and C57BL/6 mouse strains at both 9 and 12 months of age. We also investigated age related kidney tumor progression and compared three different rapamycin treatment schedules in cohorts of A/J Tsc2+/- mice. In addition, we used nude mice bearing Tsc2-/- subcutaneous tumors to evaluate the therapeutic utility of sunitinib, bevacizumab, vincristine, and asparaginase.. TSC related kidney disease severity is 5-10 fold higher in A/J Tsc2+/- mice compared with C57BL/6 Tsc2+/- mice. Similar to kidney angiomyolipomas associated with TSC, the severity of kidney cystadenomas increases with age in A/J Tsc2+/- mice. When rapamycin dosing schedules were compared in A/J Tsc2+/- cohorts, we observed a 66% reduction in kidney tumor burden in mice treated daily for 4 weeks, an 82% reduction in mice treated daily for 4 weeks followed by weekly for 8 weeks, and an 81% reduction in mice treated weekly for 12 weeks. In the Tsc2-/- subcutaneous tumor mouse model, vincristine is not effective, but angiogenesis inhibitors (sunitinib and bevacizumab) and asparaginase are effective as single agents. However, these drugs are not as effective as rapamycin in that they increased median survival only by 24-27%, while rapamycin increased median survival by 173%.. Our results indicate that the A/J Tsc2+/- mouse model is an improved, higher through-put mouse model for future TSC preclinical studies. The rapamycin dosing comparison study indicates that the duration of rapamycin treatment is more important than dose intensity. We also found that angiogenesis inhibitors and asparaginase reduce tumor growth in a TSC2 tumor mouse model and although these drugs are not as effective as rapamycin, these drug classes may have some therapeutic potential in the treatment of TSC related tumors.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Phytogenic; Asparaginase; Bevacizumab; Cystadenoma; Disease Models, Animal; Drug Administration Schedule; Female; Indoles; Kidney Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Pyrroles; Sirolimus; Sunitinib; Survival Rate; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Vincristine

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T ((n)T(reg)) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP(139-151)-induced relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-gamma, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4(+)FoxP3(+) cells were unaffected, we observed disappearance of CD4(+)CD45RB(high) effector T (T(eff)) cells and selective expansion of T(reg) cells bearing the CD4(+)CD45RB(low)FoxP3(+)CD25(+)CD103(+) extended phenotype. Finally, the dual action of rapamycin on both T(eff) and T(reg) cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Disease Models, Animal; Drug Administration Routes; Encephalomyelitis, Autoimmune, Experimental; Female; Immune Tolerance; Immunosuppressive Agents; Interferon-gamma; Interleukin-17; Mice; Multiple Sclerosis, Relapsing-Remitting; Myelin Proteolipid Protein; Peptide Fragments; Phenotype; Secondary Prevention; Sirolimus; T-Lymphocytes, Regulatory; Treatment Outcome

Complete blanching of port wine stain (PWS) birthmarks after laser therapy is rarely achieved for most patients. We postulate that the low therapeutic efficacy or treatment failure is caused by regeneration and revascularization of photocoagulated blood vessels due to angiogenesis associated with the skin's normal wound healing response. Rapamycin (RPM), an antiangiogenic agent, has been demonstrated to inhibit growth of pathological blood vessels. Our objectives were to (1) investigate whether topical RPM can inhibit reperfusion of photocoagulated blood vessels in an animal model and (2) determine the effective RPM concentration required to achieve this objective.. For both laser-only and combined laser and RPM treated animals, blood vessels in the dorsal window chambers implanted on golden Syrian hamsters were photocoagulated with laser pulses. Structural and flow dynamics of blood vessels were documented with color digital photography and laser speckle imaging to evaluate photocoagulation and reperfusion. For the combined treatment group, topical RPM was applied to the epidermal side of the window daily for 14 days after laser exposure.. In the laser-only group, 23 out of 24 photocoagulated blood vessels reperfused within 5-14 days. In the combined treatment group with different RPM formulae and concentrations, the overall reperfusion rate of 36% was much lower as compared to the laser-only group. We also found that the reperfusion rate was not linearly proportional to the RPM concentration.. With topical RPM application, the frequency of vessel reperfusion was considerably reduced, which implies that combined light and topical antiangiogenic therapy might be a promising approach to improve the treatment efficacy of PWS birthmarks.

Topics: Administration, Cutaneous; Animals; Blood Vessels; Combined Modality Therapy; Cricetinae; Disease Models, Animal; Laser Therapy; Lasers, Dye; Male; Port-Wine Stain; Random Allocation; Risk Factors; Sirolimus; Time Factors; Treatment Outcome

Drug-eluting stents (DESs) reduce neointima area and in-stent restenosis but delay re-endothelialization. Recently, we demonstrated that pharmacological expansion and functional enhancement of endothelial progenitor cells (EPCs) can be achieved by treatment with a glycogen synthase kinase-3beta inhibitor (GSKi)-even for feeble cells derived from coronary artery disease patients. GSKi treatment enhanced EPC adhesion via up-regulated expression of the alpha-4 integrin, ameliorated re-endothelialization, and reduced neointima formation in denuded murine arteries. Hence, we hypothesized that GSKi-coated stents (GSs) will enhance EPC adhesion and attenuate delayed vascular healing associated with rapamycin, a key DES agent.. In vitro human EPCs adhered to GS with affinities that were 2x, 14x, and 13x greater than vehicle (VSs)-, rapamycin (RSs)-, and rapamycin plus GSKi (RGSs)-coated stents, respectively. Stents were inserted in rabbit carotid arteries, and at 14 days, neointima area was 45 and 49% lower in GSs compared with bare metal stents (BMSs) and VSs. Moreover, RSs had a 47% larger neointima area than GSs, but RGSs reduced neointima area to a level comparable to GSs. Seven days after stenting, GSs displayed re-endothelialization that was 40, 33, and 42% greater than BMSs, VSs, and RSs, respectively. Moreover, RGSs had 41% more re-endothelialization than RSs. At 14 days, the 7-day re-endothelialization patterns persisted.. GSKi efficiently ameliorates the vascular response to stent implantation and has an important redeeming effect on the deleterious endothelial effects of rapamycin-coated stents.

Topics: Animals; Carotid Arteries; Carotid Artery Diseases; Cell Adhesion; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Prosthesis Design; Protein Kinase Inhibitors; Rabbits; Sirolimus; Stem Cells; Time Factors

Accumulation of amyloid-beta (Abeta) and Tau is an invariant feature of Alzheimer disease (AD). The upstream role of Abeta accumulation in the disease pathogenesis is widely accepted, and there is strong evidence showing that Abeta accumulation causes cognitive impairments. However, the molecular mechanisms linking Abeta to cognitive decline remain to be elucidated. Here we show that the buildup of Abeta increases the mammalian target of rapamycin (mTOR) signaling, whereas decreasing mTOR signaling reduces Abeta levels, thereby highlighting an interrelation between mTOR signaling and Abeta. The mTOR pathway plays a central role in controlling protein homeostasis and hence, neuronal functions; indeed mTOR signaling regulates different forms of learning and memory. Using an animal model of AD, we show that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates Abeta and Tau pathology by increasing autophagy. Indeed, we further show that autophagy induction is necessary for the rapamycin-mediated reduction in Abeta levels. The results presented here provide a molecular basis for the Abeta-induced cognitive deficits and, moreover, show that rapamycin, an FDA approved drug, improves learning and memory and reduces Abeta and Tau pathology.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Memory; Mice; Mice, Transgenic; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; tau Proteins; TOR Serine-Threonine Kinases

Acute graft-versus-host disease (aGVHD) is a serious complication of liver transplantation (LTx); it occurs in 1% to 2% of liver allograft recipients. The condition has a poor prognosis and poses major diagnostic and therapeutic challenges. A rat model of aGVHD after LTx has been developed, and a relative decrease in regulatory T (Treg) cells has been shown to be associated with this model. Interest has been expressed in the effects of different immunosuppressive agents on CD4+CD25+Foxp3+ Treg cell homeostasis. Rats with aGVHD after LTx were treated with tacrolimus (FK506), rapamycin (RAPA), or no immunosuppressive drug. Those that received RAPA survived longer (91.4 + or - 8.1 days) than those in the FK506 group (62.3 + or - 13.4 days) or the control group (22.9 + or - 1.2 days). Flow cytometry analysis showed that Treg cells, as a percentage of peripheral blood mononuclear cells (PBMCs), were more abundant in the RAPA group (6.8% + or - 0.8%) than in the FK506 group (1.7% + or - 0.4%) or the control group (2.0% + or - 0.4%). Immunohistochemistry demonstrated more Foxp3+ staining of intestinal cells in the RAPA group than in the FK506 group or the control group. In conclusion, the reduced mortality induced by RAPA in a rat model of aGVHD after LTx was associated with higher percentages of CD4+CD25+Foxp3+ Treg cells in PBMCs in blood and tissues than those occurring after the administration of FK506.

Topics: Animals; Biopsy; CD4 Antigens; Disease Models, Animal; Female; Forkhead Transcription Factors; Graft vs Host Disease; Homeostasis; Immune System; Immunosuppressive Agents; Interleukin-2 Receptor alpha Subunit; Liver Transplantation; Male; Rats; Rats, Inbred Lew; Sirolimus; Skin; T-Lymphocytes, Regulatory; Tacrolimus

Our group has shown that platelet inhibition with clopidogrel, an antagonist of the P2Y12 adenosine diphosphate receptor on platelets, reduced the formation of transplant arteriosclerosis. The aim of this study was to investigate whether a combination of cyclosporin or everolimus with clopidogrel has a beneficial effect on the development of transplant arteriosclerosis. Fully MHC mismatched C57Bl/6 (H2(b)) donor aortas were transplanted into CBA.J (H2(k)) recipients and mice received either clopidogrel alone (1 mg/kg/day) or in combination with cyclosporin (2 mg/kg/day) or everolimus (0.05 mg/kg/day). Grafts were analysed by histology and morphometry on day 30 after transplantation. In mice treated with clopidogrel alone, transplant arteriosclerosis was significantly reduced [intima proliferation 56 +/- 11% vs. 81 +/- 7% (control)/n = 7]. Daily application of everolimus reduced the development of transplant arteriosclerosis compared with untreated controls [intima proliferation of 29 +/- 9% vs. 81 +/- 7% (control)/n = 7]. Strikingly, combination of clopidogrel and everolimus almost abolished the formation of transplant arteriosclerosis [intima proliferation: 11 +/- 8% vs. 81 +/- 7% (control)/n = 7]. By contrast, combination of cyclosporin and clopidogrel compared with clopidogrel alone showed no additive effect. These results demonstrate that combination of platelet- and mammalian target of Rapamycin-inhibition can dramatically reduce the development of transplant arteriosclerosis.

Topics: Animals; Aorta, Abdominal; Arteriosclerosis; Clopidogrel; Disease Models, Animal; Drug Therapy, Combination; Everolimus; Graft Rejection; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Platelet Aggregation; Platelet Aggregation Inhibitors; Sirolimus; Ticlopidine; Transplantation, Homologous; Treatment Outcome

Selective drugs targeting dysregulated oncogenic pathways are promising cancer therapies. Because the mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in human follicular thyroid cancer (FTC), we hypothesized that its inhibition could block cancer development and progression. We, therefore, analyzed the effect of a treatment with a specific mTORC1 inhibitor (RAD001) in a faithful mouse model of FTC with constitutive mTORC1 activation (TRbeta(PV/PV)Pten(+/-) mice). The treatment did not prevent capsular and vascular invasion of the thyroid and the occurrence of lung metastasis. However, it substantially decelerated thyroid tumor growth, thereby prolonging TRbeta(PV/PV)Pten(+/-) mouse life span. RAD001 efficiently inhibited mTORC1 activity, as shown by the reduced phosphorylation of its downstream targets involved in the activity of the translation machinery, such as ribosomal S6 kinase (p70(S6K)), eukaryotic translation initiation factor 4E binding protein (4E-BP1) and the eukaryotic translation initiation factors eIF-4B and eIF-4G. Whereas mTORC1 signaling inhibition did not alter cell apoptosis, it induced a significant decrease in cell proliferation that was associated with the reduced abundance and altered activity of key regulators of cell cycle progression. Altogether, our data indicate that mTORC1 signaling plays a major role in the integration of the mitogenic signal in FTC. Therefore, our preclinical study with a relevant mouse model of FTC demonstrates for the first time that RAD001 efficaciously stabilizes cancer growth although it does not prevent its fatal outcome. In conclusion, our work underscores that in the treatment of FTC patients, RAD001 can only be used in combination with drugs and therapies inducing tumor shrinkage and blocking metastasis.

Topics: Animals; Apoptosis; Cell Cycle; Disease Models, Animal; Disease Progression; Everolimus; Extracellular Signal-Regulated MAP Kinases; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Thyroid Hormone Receptors beta; Thyroid Neoplasms; TOR Serine-Threonine Kinases; Transcription Factors

Although the therapeutic potential of regulatory T lymphocytes (Tregs) in preventing allograft rejection has been well documented, accumulating evidence indicates that supplemental measures, such as concomitant use of immunosuppressive agents, are essential for effective application of Treg cell therapy in clinical transplantation. Thus, it is important to know the effect of immunosuppressive agents on Treg cell therapy.. We examined the impact of various immunosuppressive agents on the in vivo proliferation and therapeutic efficacy of in vitro-expanded Tregs using the murine graft-versus-host reaction and skin allograft model (BDF1 [H-2] to C57BL/6 [H-2]), respectively.. All six immunosuppressive agents tested inhibited the alloantigen-stimulated proliferation of Tregs as efficiently as they inhibited the proliferative response of conventional CD3 T cells. We further show that blockade of the CD40-CD40L interaction by treatment with a MR-1 antibody significantly increased the therapeutic efficacy of Tregs, a synergistic effect that seemed to be related to the strong regulatory activity of adoptively transferred Tregs together with effector T-cell hyporesponsiveness. Although concomitant use of rapamycin marginally augmented the therapeutic effectiveness of Tregs, mycophenolate mofetil and cyclosporine A at their full therapeutic doses exerted an antagonistic effect on Treg cell therapy.. These results demonstrate that inhibition of CD40-CD40L interaction or treatment with rapamycin could be successfully combined with in vitro-expanded Treg cell therapy, but the concomitant use of mycophenolate mofetil or cyclosporine A in this type of Treg cell therapy should be carefully considered.

Topics: Adoptive Transfer; Animals; Antibodies; CD40 Antigens; CD40 Ligand; Cell Proliferation; Cells, Cultured; Cyclosporine; Disease Models, Animal; Forkhead Transcription Factors; Graft Rejection; Graft vs Host Disease; Immunosuppressive Agents; Interleukin-2 Receptor alpha Subunit; Isoantigens; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mycophenolic Acid; Sirolimus; Skin Transplantation; T-Lymphocytes, Regulatory; Time Factors; Transplantation Tolerance; Transplantation, Homologous

The aim of this study is to compare anti-inflammatory and intimal hyperplasia inhibiting efficacy between the Firebird rapamycin drug-eluting stent and a bare metal stent in a porcine coronary injury model.. Twelve rapamycin drug-eluting stents (Firebird), and 12 bare metal stents (BMS) were deployed with the oversizing method into porcine coronary arteries. Coronary angiography, histopathological and immunocytochemistry analysis were carried out at week 4 after stenting.. The distribution of stented vessels, diameter of reference vessels, and post-procedural minimal lumen diameter were compared between the two groups. At week 4 of follow-up, quantitative coronary angiography (QCA) showed that the minimal lumen diameter and late lumen loss were greater, and percent stenosis was less in the Firebird stent group than in the bare metal stent group. In the histopathological analysis, compared to the BMS group, injury score in the Firebird stent group (1.87 +/- 0.16 vs. 1.32 +/- 0.13) and inflammation score (1.86 +/- 0.55 vs. 1.12 +/- 0.35) decreased, P < 0.05. There are significant differences for neointimal area (4.60 +/- 1.39 mm2 in the BMS group vs. 1.51 +/- 0.45 mm2 in the TCS group, P < 0.05). The lumen area in the Firebird stent group enlarged (3.24 +/- 0.93 mm2 in the BMS group vs. 4.34 +/- 0.93 mm2 in the Firebird stent group, P < 0.05). Immunohistochemistry revealed that the Firebird stent suppressed cell proliferation (Ki67) and expression of nuclear factor-kappaB (NF-kappaB) in the arterial wall.. The Firebird stent showed suppression of constrictive remodelling, inhibition of neointimal hyperplasia through antiproliferation, and anti-inflammation acts via attenuated NF-KB activation, which has proved to be a feasible method for preventing restenosis after coronary angioplasty in pigs.

Topics: Animals; Anti-Bacterial Agents; Coronary Angiography; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Disease Progression; Drug-Eluting Stents; Female; Follow-Up Studies; Inflammation; Prosthesis Design; Sirolimus; Swine; Treatment Outcome

Topics: Animals; Disease Models, Animal; Drug-Eluting Stents; Humans; Paclitaxel; Risk Factors; Sirolimus; Swine; Thrombosis

Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms [1], [2], [3], [4]. It was recently demonstrated that long-term treatment with rapamycin, an inhibitor of the mTOR pathway[5], or ablation of the mTOR target p70S6K[6] extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway would delay or prevent age-associated disease such as AD remained to be determined.. We used rapamycin administration and behavioral tools in a mouse model of AD as well as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Abeta(42), a major toxic species in AD[7], in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR pathway can reduce Abeta(42) levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy was increased in neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice, suggesting that the reduction in Abeta and the improvement in cognitive function are due in part to increased autophagy, possibly as a response to high levels of Abeta.. Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical settings, may be a potentially effective therapeutic agent for the treatment of AD.

Topics: Aging; Alzheimer Disease; Amyloid beta-Peptides; Animals; Autophagy; Cognition Disorders; Disease Models, Animal; Immunosuppressive Agents; Mice; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Topics: Animals; Apoptosis; Autophagy; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Hepatocytes; Humans; Huntington Disease; Immunosuppressive Agents; Lysosomes; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Nerve Tissue Proteins; Neurons; Peptides; Serotonin Plasma Membrane Transport Proteins; Serum; Sirolimus; Subcellular Fractions; Thapsigargin; Time Factors; Vinca Alkaloids

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Ischemia; Disease Models, Animal; Down-Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Microtubule-Associated Proteins; Neostriatum; Neurogenesis; Neurons; Neuroprotective Agents; Phagosomes; Rats; Rats, Sprague-Dawley; Sequestosome-1 Protein; Sirolimus; Up-Regulation

The outcome of stent implantation for pulmonary vein stenosis (PVS) in children remains poor. Several reports describe placing drug-eluting stents to treat PVS, but their effectiveness remains unknown. In this study, three bare-metal stents (BMSs) and three sirolimus-eluting stents (SESs) were implanted in 1-month-old pigs. The pigs were killed 8 weeks later to compare in-stent stenosis rates. The extent of neointimal thickness, as measured by injury score, was significantly less in the SES group than in the BMS group (injury score 1: BMS 0.351 + or - 0.033 vs SES 0.226 + or - 0.031 mm; P < 0.01; injury score 2: BMS 1.232 + or - 0.244 vs SES 0.609 + or - 0.208 mm; P < 0.01). The pathologic findings showed confluence of inflammatory cells around the stent wires in BMS-treated areas and granuloma formation. Granuloma formation was not seen with SES. The degree of in-stent stenosis was significantly reduced in the SES group, suggesting that the use of drug-eluting stents is an effective treatment for PVS. Because of the small sample size and the considerable variation in injury scores and balloon-to-vein ratios, future studies with larger samples are necessary.

Topics: Animals; Disease Models, Animal; Drug-Eluting Stents; Female; Immunosuppressive Agents; Pulmonary Veno-Occlusive Disease; Secondary Prevention; Sirolimus; Treatment Outcome; Tunica Intima

Rapamycin, a novel macrolide immunosuppressive drug, is increasingly used as an agent for posttransplant immunosuppression and treatment of autoimmune disease. The molecular mechanism related to rapamycin-mediated immunosuppression is that rapamycin binds to FK-506 binding protein 12, and the formed complex inhibits the function of the mammalian target of rapamycin (mTOR), which in turn reduces protein phosphorylation, cell cycle progression, and cytokine production. The aim of this study was to examine the effect of rapamycin against the development of fibrosis and autoimmunity in 2 different types of systemic sclerosis (SSc) model mice.. Tight skin (TSK/+) mice and bleomycin- induced SSc model mice were used to evaluate the effect of rapamycin on fibrosis and immunologic abnormalities. Furthermore, the antifibrotic effect of rapamycin was assessed using TSK/+ mouse fibroblasts.. Treatment with rapamycin reduced skin fibrosis of TSK/+ mice and skin and lung fibrosis of bleomycin-induced SSc model mice. The production of fibrogenic cytokines, such as interleukin-4 (IL-4), IL-6, IL-17, and transforming growth factor beta1, was attenuated by rapamycin. Hypergammaglobulinemia and anti-topoisomerase I antibody production were also reduced by rapamycin treatment in TSK/+ mice. In addition, mTOR expression levels were increased in TSK/+ mouse fibroblasts compared with those in wild-type mouse fibroblasts. Rapamycin treatment inhibited proliferation and collagen production of TSK/+ mouse fibroblasts in a dose-dependent manner.. This study is the first to show that rapamycin has a significant inhibitory effect on fibrosis in both TSK/+ and bleomycin-induced SSc model mice. These results suggest that rapamycin might be an attractive candidate for clinical trials in SSc patients.

Topics: Animals; Cell Proliferation; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Immunohistochemistry; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Transgenic; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Sirolimus; Skin; Statistics, Nonparametric; TOR Serine-Threonine Kinases

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in one in 2000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicate that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared with a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well-characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

Topics: alpha 1-Antitrypsin; Animals; Autophagy; Disease Models, Animal; Enzyme Activators; Humans; Liver Cirrhosis; Liver Diseases; Mice; Mutant Proteins; Sirolimus

Renal transplantation is the definitive treatment for many metabolic abnormalities of uremic patients, although it is only partially effective for renal osteodystrophy, which may interact with posttransplant renal osteopathy. Osteopenic-osteoporotic syndrome represents, together with fractures secondary to osteoporosis and osteonecrosis, the bone complication most related to renal transplantation. Several factors contribute to the pathogenesis of posttransplantation osteoporosis, particularly immunosuppressive treatment. In this study, we evaluated the prevalence of factors related to posttransplant renal osteopathy and the clinical impact of immunosuppressive protocols. We studied 24 renal transplant recipients with hypercalcemia. Glomerular filtration rate was >50 mL/min. Mean age, time on dialysis, and time from transplantation were 49.6, 5.4, and 6.9 years, respectively. We evaluated serum and urine calcium and phosphorus, calcitonin, parathormone, bone-specific alkaline phosphatase, osteocalcin, urine deoxypyridinoline, telopeptide of type 1 procollagen, 1,25-(OH)(2) and 25-OH vitamin D, parathyroid ultrasound, and computerized bone mineralometry. The combination of sirolimus and steroids resulted in the most disadvantageous outcomes regarding alkaline phosphatase and mineralometry. Calcineurin inhibitors did not significantly influence bone metabolism markers; mycophenolate mofetil evidenced no effect on bone. According to the literature, steroids account for the abnormalities found in our patients and in severe osteopenia. Several factors may contribute to the development of osteoporosis and fractures in transplantation patients, although they are overcome by the prominent effect of steroids. In patients at high risk of osteoporosis, steroid-free therapy should be considered. Everolimus is indicated for diseases with bone loss. Combined therapy with everolimus and mycophenolic acid without cyclosporine and steroids, seemed to be particularly indicated. Prophylactic treatments should be commenced early. No single marker was useful to diagnose posttransplant renal osteopathy. The definitive diagnosis should be made by bone biopsy during transplantation, and noninvasive procedures, such as densitometry and evaluation of biologic markers, may be useful during follow-up.

Topics: Adult; Alkaline Phosphatase; Animals; Bone Density; Bone Diseases; Calcium; Disease Models, Animal; Female; Fractures, Bone; Humans; Hypercalcemia; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Osteocalcin; Osteoporosis; Phosphorus; Procollagen; Rats; Sirolimus; Uremia

Soluble oligomeric aggregates of the amyloid-beta (A beta) peptide are believed to be the most neurotoxic A beta species affecting the brain in Alzheimer disease (AD), a terminal neurodegenerative disorder involving severe cognitive decline underscored by initial synaptic dysfunction and later extensive neuronal death in the CNS. Recent evidence indicates that A beta oligomers are recruited at the synapse, oppose expression of long-term potentiation (LTP), perturb intracellular calcium balance, disrupt dendritic spines, and induce memory deficits. However, the molecular mechanisms behind these outcomes are only partially understood; achieving such insight is necessary for the comprehension of A beta-mediated neuronal dysfunction. We have investigated the role of the phosphatase calcineurin (CaN) in these pathological processes of AD. CaN is especially abundant in the CNS, where it is involved in synaptic activity, LTP, and memory function. Here, we describe how oligomeric A beta treatment causes memory deficits and depresses LTP expression in a CaN-dependent fashion. Mice given a single intracerebroventricular injection of A beta oligomers exhibited increased CaN activity and decreased pCREB, a transcription factor involved in proper synaptic function, accompanied by decreased memory in a fear conditioning task. These effects were reversed by treatment with the CaN inhibitor FK506. We further found that expression of hippocampal LTP in acutely cultured rodent brain slices was opposed by A beta oligomers and that this effect was also reversed by FK506. Collectively, these results indicate that CaN activation may play a central role in mediating synaptic and memory disruption induced by acute oligomeric A beta treatment in mice.

Topics: Amyloidogenic Proteins; Animals; Behavior, Animal; Calcineurin; Conditioning, Psychological; CREB-Binding Protein; Disease Models, Animal; Drug Interactions; Fear; Female; Immunosuppressive Agents; In Vitro Techniques; Injections, Intra-Articular; Long-Term Potentiation; Male; Membrane Potentials; Memory Disorders; Mice; Mice, Inbred C57BL; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus

Inhibition of mTOR by rapamycin has been shown to suppress seizures in TSC/PTEN genetic models. Rapamycin, when applied immediately before or after a neurological insult, also prevents the development of spontaneous recurrent seizures (epileptogenesis) in an acquired model. In the present study, we examined the mTOR pathway in rats that had already developed chronic spontaneous seizures in a pilocarpine model. We found that mTOR is aberrantly activated in brain tissues from rats with chronic seizures. Furthermore, inhibition of mTOR by rapamycin treatment significantly reduces seizure activity. Finally, mTOR inhibition also significantly suppresses mossy fiber sprouting. Our findings suggest the possibility for a much broader window for intervention for some acquired epilepsies by targeting the mTOR pathway.

Topics: Animals; Convulsants; Disease Models, Animal; Epilepsy; Hippocampus; Male; Mossy Fibers, Hippocampal; Neuronal Plasticity; Pilocarpine; Rats; Rats, Sprague-Dawley; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To analyze the immune modulatory effect of low-dose systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) in a high-responder corneal allograft model.. A total of 80 C57BL/6 mice received corneal grafts from BALB/c donors. Recipients were treated with either CsA 3 mg/kg/day or Rapa 0.5 mg/kg/day monotherapy or received combined treatment. Immunomodulatory treatment was started on the day of surgery, and continued for 14 days. The frequency of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in secondary lymphoid organs was measured by flow cytometry. Development of IFN-gamma producing alloreactive T cells was estimated by Elispot. In addition, corneal samples were subjected to real-time RT-PCR analysis for cytokine transcription.. Monotherapy with Rapa significantly delayed allograft rejection (13.4 +/- 1.34 days, p = 0.03). However, the combination of both, low-dose Rapa and CsA prolonged corneal allograft survival at a significantly higher level (MST = 17.1 +/- 1.37 days, p = 0.0001) than in the control group (MST = 11.2 +/- 1.91 days). Rapa monotherapy increased the frequency of CD4(+)CD25(+)Foxp3(+)Treg in draining lymph nodes, whereas addition of CsA reduced Tregs. Monotherapy with Rapa as well as combined treatment prevented development of IFN-gamma producing alloreactive T cells in spleen. Combined treatment resulted in down-regulation of intragraft CD3, IL-2, IFN-gamma and IL-10 transcription (p = 0.028, p = 0.027, p = 0.028 and p = 0.027 respectively).. Combined treatment with low-dose CsA and Rapa resulted in superior graft survival, and effectively modulated mRNA expression of inflammation and infiltration markers.

Topics: Animals; Cornea; Cyclosporine; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Flow Cytometry; Graft Survival; Immunosuppressive Agents; Injections, Intraperitoneal; Keratoplasty, Penetrating; Lymphoid Tissue; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; T-Lymphocytes, Regulatory; Transplantation, Homologous

Recent studies have revealed that T lymphocytes play a role in neuropathic pain following nerve injury in rats through releasing several cytokines. Sirolimus is an immunosuppressive antibiotic inhibiting T cell activation. This study aimed to determine the effect of sirolimus on hyperalgesia and allodynia and on serum and spinal cord TNF-alpha, IL-1beta and IL-6 levels in rat neuropathic pain. Neuropathic pain was induced by loose ligation of the sciatic nerve and evaluated by tests measuring the mechanical hyperalgesia and allodynia. Sirolimus (0.75 and 1.5 mg/kg) was administered intraperitoneally once every 3 days for 2 weeks (7 doses totally). This dosing regimen revealed acceptable blood concentrations in neuropathic rats. Chronic constriction injury of the sciatic nerve resulted in hyperalgesia and allodynia. Serum levels of cytokines remained unchanged in neuropathic rats. However, TNF-alpha, but not IL-1beta or IL-6, protein level was increased in the spinal cord tissue as evaluated by Western blotting analysis. Treatment with sirolimus resulted in antihyperalgesic and antiallodynic effects and prevented the increased spinal cord TNF-alpha level. It seems that sirolimus could be a promising immunosuppressive agent in the treatment of neuropathic pain.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Assays; Hyperalgesia; Immunologic Factors; Male; Pain Threshold; Rats; Rats, Wistar; Sciatica; Sirolimus; Spinal Cord; Time Factors

Inhibitors of TORC1 have been shown to be active in patients with metastatic renal cell carcinoma (RCC). As the phosphatidylinositol 3-kinase (PI3K) pathway activates numerous other kinases, transcription factors, and proteins associated with cell growth and survival besides mammalian target of rapamycin (mTOR), disruption of this pathway upstream of mTOR may be more effective than inhibition of TORC1 alone.. To investigate this possibility, the dual PI3K/mTOR inhibitor NVP-BEZ235 was compared with rapamycin in RCC cell lines and xenografts generated from 786-O and A498 cells.. Treatment of RCC cell lines with NVP-BEZ235 in vitro resulted in the nuclear translocation of p27, greater reduction in tumor cell proliferation, and more complete suppression of Akt, Mnk-1, eIF4E, and 4EBP-1 phosphorylation and cyclin D1 and hypoxia-inducible factor 2alpha (HIF2alpha) expression than that achieved with rapamycin. The reduction of HIF2alpha levels correlated with reduced HIF activity as determined by luciferase assay. NVP-BEZ235 induced growth arrest in both the 786-O and A498 xenografts that was associated with inhibition of Akt and S6 phosphorylation as well as the induction of apoptosis and reduction in markers of tumor cell proliferation. In contrast, rapamycin induced only minimal growth retardation.. Dual inhibition of PI3K/mTOR with NVP-BEZ235 induced growth arrest in RCC cell lines both in vitro and in vivo more effectively than inhibition of TORC1 alone. These results provide the rationale for the clinical assessment of agents such as NVP-BEZ235 in patients with advanced RCC.

Topics: Animals; Carcinoma, Renal Cell; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Imidazoles; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Multiprotein Complexes; Neoplasm Transplantation; Phosphoinositide-3 Kinase Inhibitors; Proteins; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/- mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/- mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/- mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/- mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function.

Topics: Animals; Body Weight; Cysts; Disease Models, Animal; Disease Progression; Humans; Immunosuppressive Agents; Kidney; Kidney Function Tests; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Mutant Strains; Multiprotein Complexes; Organ Size; Polycystic Kidney, Autosomal Dominant; Proteins; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Topics: Animals; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Everolimus; Glomerular Filtration Rate; Humans; Mutation; Phosphatidylinositol 3-Kinases; Polycystic Kidney, Autosomal Dominant; Rats; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Type 2 diabetes increases breast cancer risk and mortality, and hyperinsulinemia is a major mediator of this effect. The mammalian target of rapamycin (mTOR) is activated by insulin and is a key regulator of mammary tumor progression. Pharmacological mTOR inhibition suppresses tumor growth in numerous mammary tumor models in the non-diabetic setting. However, the role of the mTOR pathway in type 2 diabetes-induced tumor growth remains elusive. Herein, we investigated whether the mTOR pathway is implicated in insulin-induced mammary tumor progression in a transgenic mouse model of type 2 diabetes (MKR mice) and evaluated the impact of mTOR inhibition on the diabetic state. Mammary tumor progression was studied in the double transgenic MMTV-Polyoma Virus middle T antigen (PyVmT)/MKR mice and by orthotopic inoculation of PyVmT- and Neu/ErbB2-driven mammary tumor cells (Met-1 and MCNeuA cells respectively). mTOR inhibition by rapamycin markedly suppressed tumor growth in both wild-type and MKR mice. In diabetic animals, however, the promoting action of insulin on tumor growth was completely blunted by rapamycin, despite a worsening of the carbohydrate and lipid metabolism. Taken together, pharmacological mTOR blockade is sufficient to abrogate mammary tumor progression in the setting of hyperinsulinemia, and thus mTOR inhibitors may be an attractive therapeutic modality for breast cancer patients with type 2 diabetes. Careful monitoring of the metabolic state, however, is important as dose adaptations of glucose- and/or lipid-lowering therapy might be necessary.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blood Glucose; Body Weight; Breast Neoplasms; Diabetes Mellitus, Type 2; Disease Models, Animal; Eating; Female; Insulin; Mice; Mice, Transgenic; Sirolimus; TOR Serine-Threonine Kinases; Triglycerides

The purpose of this study was to investigate the oncolytic potential of the recombinant, granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus (VV) JX-594 in experimental malignant glioma (MGs) in vitro and in immunocompetent rodent models. We have found that JX-594 killed all MG cell lines tested in vitro. Intratumoral (i.t.) administration of JX-594 significantly inhibited tumor growth and prolonged survival in rats-bearing RG2 intracranial (i.c.) tumors and mice-bearing GL261 brain tumors. Combination therapy with JX-594 and rapamycin significantly increased viral replication and further prolonged survival in both immunocompetent i.c. MG models with several animals considered "cured" (three out of seven rats >120 days, terminated experiment). JX-594 infected and killed brain tumor-initiating cells (BTICs) from patient samples grown ex vivo, and did so more efficiently than other oncolytic viruses MYXV, Reovirus type-3, and VSV(ΔM51). Additional safety/toxicity studies in nontumor-bearing rodents treated with a supratherapeutic dose of JX-594 demonstrated GM-CSF-dependent inflammation and necrosis. These results suggest that i.c. administered JX-594 triggers a predictable GM-CSF-mediated inflammation in murine models. Before proceeding to clinical trials, JX-594 should be evaluated in the brains of nonhuman primates and optimized for the viral doses, delivery routes as well as the combination agents (e.g., mTOR inhibitors).

Topics: Animals; Antibiotics, Antineoplastic; Brain Neoplasms; Combined Modality Therapy; Disease Models, Animal; Female; Genetic Vectors; Glioma; Granulocyte-Macrophage Colony-Stimulating Factor; Immunoenzyme Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oncolytic Virotherapy; Rats; Rats, Inbred F344; Sirolimus; Survival Rate; Transgenes; Tumor Cells, Cultured; Vaccines, Synthetic; Vaccinia virus; Virus Replication

Pancreatic neuroendocrine tumors (PNETs), although rare, often metastasize, such that surgery, the only potentially curative therapy, is not possible. There is no effective systemic therapy for patients with advanced PNETs. Therefore, new strategies are needed. Toward that end, we investigated the potential benefit of dual therapeutic targeting of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) kinases, using a preclinical mouse model of PNET.. Rapamycin and erlotinib, inhibitors of mTOR and EGFR, respectively, were used to treat RIP-Tag2 transgenic mice bearing advanced multifocal PNET. Tumor growth and survival were monitored, and tumors were surveyed for potential biomarkers of response to the therapeutics.. Rapamycin monotherapy was notably efficacious, prolonging survival concomitant with tumor stasis (stable disease). However, the tumors developed resistance, as evidenced by eventual relapse to progressive tumor growth. Erlotinib monotherapy slowed tumor growth and elicited a marginal survival benefit. In combination, there was an unprecedented survival benefit in the face of this aggressive multifocal cancer and, in contrast to either monotherapy, the development of adaptive resistance was not apparent. Additionally, the antiapoptotic protein survivin was implicated as a biomarker of sensitivity and beneficial responses to the dual targeted therapy.. Preclinical trials in a mouse model of endogenous PNET suggest that combined targeting of the mTOR and EGFR signaling pathways could have potential clinical benefit in treating PNET. These results have encouraged development of an ongoing phase II clinical trial aimed to evaluate the efficacy of this treatment regimen in human neuroendocrine tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Survival; Clinical Trials, Phase II as Topic; Disease Models, Animal; ErbB Receptors; Erlotinib Hydrochloride; Humans; Immunohistochemistry; Immunosuppressive Agents; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Neuroendocrine Tumors; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Quinazolines; Repressor Proteins; RNA Interference; Signal Transduction; Sirolimus; Survival Analysis; Survivin; TOR Serine-Threonine Kinases; Treatment Outcome

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase II as Topic; Disease Models, Animal; ErbB Receptors; Erlotinib Hydrochloride; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Neuroendocrine Tumors; Pancreatic Neoplasms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Quinazolines; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases; Treatment Outcome

Targeting the mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming cisplatin resistance in ovarian cancer patients. Because mTOR inhibition affects cell proliferation, we aimed to study whether 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET could be useful for monitoring early response to treatment with mTOR inhibitors in an animal model of cisplatin-resistant ovarian tumor.. BALB/c nude mice bearing subcutaneous human SKOV3 ovarian cancer xenografts were treated with either the mTOR inhibitor everolimus (5 mg/kg) or vehicle, and (18)F-FLT PET was performed at baseline, day 2, and day 7 of treatment. (18)F-FLT uptake was evaluated by calculation of mean standardized uptake value (SUVmean) corrected for partial-volume effect. Ex vivo immunohistochemistry studies were performed on separate cohorts of mice treated as above and sacrificed at the same time points as for the PET studies. The ex vivo analysis included bromodeoxyuridine incorporation as a marker of cell proliferation, and phosphorylation of ribosomal protein S6 as a downstream marker of mTOR activation.. During the treatment period, no significant change in tumor (18)F-FLT uptake was observed in the vehicle group, whereas in everolimus-treated mice, (18)F-FLT SUVmean decreased by 33% (P = 0.003) at day 2 and 66% (P < 0.001) at day 7, compared with baseline. Notably, the reduction of (18)F-FLT uptake observed at day 2 in the everolimus group preceded changes in tumor volume, and a significant difference in (18)F-FLT uptake was observed between vehicle and drug-treated tumors at both day 2 (P = 0.0008) and day 7 (P = 0.01). In ex vivo studies, everolimus treatment resulted in a 98% reduction in phosphorylated ribosomal protein S6 immunostaining at day 2 (P = 0.02) and 91% reduction at day 7 (P = 0.003), compared with the vehicle group. Bromodeoxyuridine incorporation was reduced by 65% at day 2 (not significant) and by 41% at day 7 (P = 0.02) in drug versus vehicle groups.. Reduction in (18)F-FLT uptake correlates well with the level of mTOR inhibition by everolimus in the SKOV3 ovarian tumor model. These data suggest that early treatment monitoring by (18)F-FLT PET may be of use in future preclinical or clinical trials evaluating treatment of cisplatin-resistant ovarian tumors by mTOR inhibitors.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cisplatin; Dideoxynucleosides; Disease Models, Animal; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Mice; Mice, Nude; Ovarian Neoplasms; Positron-Emission Tomography; Radiopharmaceuticals; Reproducibility of Results; Sensitivity and Specificity; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

The two complexes of the mammalian target of rapamycin (mTOR), mTORC1 and mTORC2, have central functions in the integration of both extracellular and intracellular signals that are also critical players in the induction of post-ischemic long-term potentiation (i-LTP), a pathological form of plasticity inducible in striatal medium spiny neurons (MSNs) after a brief episode of in vitro ischemia. To evaluate the involvement of mTOR complexes during ischemia we analyzed the time course of i-LTP by intracellular recordings of MSNs from corticostriatal slices incubated with 1μM mTOR inhibitor rapamycin. Although rapamycin did not affect the amplitude and duration of ischemia-induced membrane depolarization it fully prevented i-LTP, leaving unaffected the capability to undergo activity-dependent LTP following high-frequency stimulation of corticostriatal fibers. The present results argue for a role of mTOR complex in i-LTP and suggest that rapamycin, by selectively blocking i-LTP, represents a promising therapeutic tool to limit cellular damage after ischemic brain insult.

Topics: Animals; Biophysics; Brain Ischemia; Corpus Striatum; Disease Models, Animal; Electric Stimulation; Excitatory Postsynaptic Potentials; Glucose; Hypoxia; Intracellular Signaling Peptides and Proteins; Long-Term Potentiation; Male; Neurons; Patch-Clamp Techniques; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

To evaluate the effects of sirolimus (SRL) on renal injury in rats with bile duct ligation.. A total of 21 male Sprague-Dawley rats weighing 220-260 g were used. Group 1 (Sham-control, n=7) rats were undergone laparotomy alone and bile duct was just dissected from the surrounding tissue. Group 2 rats (BDL/Untreated, n=7) were subjected to bile duct ligation and no drug was applied. Group 3 rats (BDL/SRL, n =7) received a daily dose of sirolimus (0.5 mg·day(-1) x kg(-1) dissolved 1 ml in saline) by orogastric tube for 14 days after BDL. At the end of the two-week period, biochemical and histological evaluation were processed.. AST, ALT, AP and TB levels values were decreased in group 3 when compared to group 2. There was no significant difference in serum levels of BUN and creatinine among all the experimental groups. Histological evaluation of the liver of BDL/Untreated group rats demonstrated marked portal fibrosis and signs of major bile duct obstruction with prominent portal and lobular inflammation. In BDL/SRL group, moderate damage was seen. Tubular injury scores were higher in the BDL subgroups; however, group 3 rats showed considerably fewer lesions in the tubules and interstitium compared to the group 2 rats. In group 2 animals, in the epithelial cells of proximal tubules presented vacuoles and hydropic changes, atrophy and inflammatory cell infiltrate in the medullar interstitium.. Sirolimus decreased tubulointerstitial lesions in kidney induced by bile duct ligation in rats. The improve effects of sirolimus on renal morphology can be due to improved liver function or due to direct action on the kidney.

Topics: Acute Kidney Injury; Animals; Bile Ducts; Disease Models, Animal; Kidney; Ligation; Male; Nephritis; Random Allocation; Rats; Rats, Sprague-Dawley; Sirolimus

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Copper; Disease Models, Animal; Doxorubicin; Female; Liposomes; Mice; Nanoparticles; Sirolimus; Ultrasonic Therapy; Xenograft Model Antitumor Assays

Topics: Animals; Anticonvulsants; Consensus; Disease Models, Animal; Electroencephalography; Humans; Infant; Sirolimus; Spasms, Infantile; United States

The incidence of anal cancer is increasing especially among HIV-infected persons in the HAART era. Treatment of this cancer is based upon traditional chemoradiotherapeutic approaches, which are associated with high morbidity and of limited effectiveness for patients with high-grade disease. The mammalian target of rapamycin (mTOR) pathway has been implicated in several human cancers, and is being investigated as a potential therapeutic target. In archival human anal cancers, we observed mTOR pathway activation. To assess response of anal cancer to mTOR inhibition, we utilized two newly developed mouse models, one in which anal cancers are induced to arise in HPV16 transgenic mice and the second a human anal cancer xenograft model. Using the transgenic mouse model, we assessed the preventative effect of rapamycin on neoplastic disease. We saw significant changes in the overall incidence of tumors, and tumor growth rate was also reduced. Using both the transgenic mouse and human anal xenograft mouse models, we studied the therapeutic effect of rapamycin on preexisting anal cancer. Rapamycin was found to significantly slow, if not stop, the growth of both mouse and human anal cancers. As has been seen in other cancers, rapamycin treatment led to an activation of the MAPK pathway. These results provide us cause to pursue further the evaluation of rapamycin as a therapeutic agent in the control of anal cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antibiotics, Antineoplastic; Anus Neoplasms; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Disease Models, Animal; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Male; Mice; Mice, Nude; Mice, SCID; Mice, Transgenic; Oncogene Proteins, Viral; Papilloma; Papillomaviridae; Papillomavirus E7 Proteins; Repressor Proteins; Sirolimus

Topics: Animals; Disease Models, Animal; Epilepsy; Humans; Mice; Mice, Knockout; Models, Biological; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin is an immunosuppressive drug with potent antifibrotic activity. We evaluated the effect of rapamycin on murine adriamycin nephropathy, a model of progressive glomerulosclerosis and tubulointerstitial fibrosis.. Adriamycin nephropathy was induced in Balb/c mice by a single intravenous injection of adriamycin. The mice were treated orally with either saline or rapamycin, beginning at the time of adriamycin injection or rapamycin starting 1 week after adriamycin injection. The mice were sacrificed 6 weeks after adriamycin injection.. Saline-treated mice developed massive proteinuria and impaired renal function. Kidney sections from saline-treated mice showed marked focal segmental glomerulosclerosis, tubular dilation with protein cast deposition, interstitial fibrosis, and numerous infiltrating macrophages and T lymphocytes. The intrarenal expression of Collagen I and RANTES was also increased. In contrast, both groups of rapamycin-treated mice had markedly reduced proteinuria and preserved renal function, with only mild histological abnormalities. The intrarenal expression of Collagen I and RANTES was reduced, concomitant with a significant reduction in interstitial inflammatory cell infiltration.. Rapamycin is effective in attenuating the glomerular and tubulointerstitial abnormalities in adriamycin nephropathy. The beneficial effects of rapamycin are mediated, at least in part, through reduced RANTES expression and inflammatory cell infiltration.

Topics: Albuminuria; Animals; Antibiotics, Antineoplastic; Body Weight; Chemokine CCL5; Collagen Type I; Disease Models, Animal; Doxorubicin; Fibrosis; Gene Expression; Glomerulosclerosis, Focal Segmental; Immunosuppressive Agents; Kidney; Male; Mice; Mice, Inbred BALB C; Severity of Illness Index; Sirolimus; Survival Rate

Atherosclerosis, restenosis, and posttransplant graft atherosclerosis are characterized by endothelial damage, infiltration of inflammatory cells, and proliferation of smooth muscle cells. The CXCR3-activating chemokines interferon-gamma inducible protein 10 (IP10) and MIG (monokine induced by interferon-gamma) have been implicated in vascular repair and remodeling. The underlying molecular mechanisms, however, remain elusive. Here, we show that wire-mediated arterial injury induced local and systemic expression of IP10 and MIG, resulting in enhanced recruitment of CXCR3(+) leukocytes and hematopoietic progenitor cells. This was accompanied by profound activation of mammalian target of rapamycin complex (mTORC)1, increased reactive oxygen species production, apoptosis, and intimal hyperplasia. Genetic and pharmacological inactivation of CXCR3 signaling not only suppressed recruitment of inflammatory cells but also abolished mTORC1 activation, reduced reactive oxygen species generation, and blocked apoptosis of vascular cells, resulting in significant reduction of intimal hyperplasia in vivo. In vitro, stimulation of T cells with IP10 directly activated mTORC1 and induced generation of reactive oxygen species and apoptosis in an mTORC1-dependent manner. These results strongly indicate that CXCR3-dependent activation of mTORC1 directly links stimulation of the Th1 immune system with the proliferative response of intimal cells in vascular remodeling.

Topics: Animals; Apoptosis; Cardiovascular Agents; Carrier Proteins; Cell Proliferation; Chemokine CXCL10; Chemokine CXCL9; Chemotaxis; Disease Models, Animal; Everolimus; Femoral Artery; Hematopoietic Stem Cells; Humans; Hyperplasia; Inflammation; Jurkat Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; Receptors, CXCR3; Signal Transduction; Sirolimus; Th1 Cells; Time Factors; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti-angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti-angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin-like growth factor-I receptor (IGF-IR)-dependent and IGF-IR-independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF-IR, we sought to investigate whether Hsp90 blockade could improve growth-inhibitory and anti-angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth-inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin-induced activation of Akt and of the downstream effector nuclear factor kappa-B (NF-kappaB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet-derived growth factor-receptor-beta (PDGF-Rbeta) on VSMCs, and diminished vascular endothelial growth factor-receptor 2 (VEGFR-2) expression on ECs, which further improves the anti-angiogenic capacity of this regimen.. Blocking Hsp90 disrupts rapamycin-induced activation of alternative signaling pathways in HCCs and substantially improves the growth-inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Carrier Proteins; Cell Death; Cell Movement; Disease Models, Animal; DNA Fragmentation; DNA Primers; HSP90 Heat-Shock Proteins; Humans; Liver Neoplasms; Mice; Phosphotransferases (Alcohol Group Acceptor); RNA, Neoplasm; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Germline mutations in LKB1 cause Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder with a predisposition to gastrointestinal polyposis and cancer. Hyperactivation of mTOR-signaling has been associated with PJS. We previously reported that rapamycin treatment of Lkb1(+/-) mice after the onset of polyposis reduced the polyp burden. Here we evaluated the preventive efficacy of rapamycin on Peutz-Jeghers polyposis. We found that rapamycin treatment of Lkb1(+/-) mice initiated before the onset of polyposis in Lkb1(+/-) mice led to a dramatic reduction in both polyp burden and polyp size and this reduction was associated with decreased phosphorylation levels of S6 and 4EBP1. Together, these findings support the use of rapamycin as an option for chemoprevention and treatment of PJS.

Topics: AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Carrier Proteins; Disease Models, Animal; Germ-Line Mutation; Intestinal Polyps; Mice; Mice, Knockout; Peutz-Jeghers Syndrome; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Non-Fc-binding anti-CD3-specific antibodies represent a promising therapy for preserving C-peptide production in subjects with recent-onset type 1 diabetes. However, the mechanisms by which anti-CD3 exerts its beneficial effect are still poorly understood, and it is questionable whether this therapeutic approach will prove durable with regard to its ability to impart metabolic preservation without additional actions designed to maintain immunological tolerance. We used the NOD mouse model to test whether rapamycin, a compound well-known for its immunomodulatory activity in mice and humans, could increase the therapeutic effectiveness of anti-CD3 treatment in type 1 diabetes.. Rapamycin was administered to diabetic NOD mice simultaneously with anti-CD3 or to NOD mice cured by anti-CD3 therapy. The ability of this combined therapy to revert type 1 diabetes and maintain a state of long-term tolerance was monitored and compared with that of anti-CD3 therapy alone.. Rapamycin inhibited the ability of anti-CD3 to revert disease without affecting the frequency/phenotype of T-cells. Rapamycin also reinstated diabetes in mice whose disease was previously reversed by anti-CD3. Withdrawal of rapamycin in these latter animals promptly restored a normoglycemic state.. Our findings indicate that, when combined with anti-CD3, rapamycin exerts a detrimental effect on the disease outcome in NOD mice for as long as it is administered. These results suggest strong caution with regard to combining these treatments in type 1 diabetic patients.

Topics: Animals; Antibodies; Blood Glucose; CD3 Complex; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Immune Tolerance; Immunosuppressive Agents; Mice; Mice, Inbred NOD; Sirolimus

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

Topics: Analysis of Variance; Animals; Astrocytes; Cells, Cultured; Chromones; Disease Models, Animal; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Excitatory Amino Acid Transporter 2; Flavonoids; Glial Fibrillary Acidic Protein; Immunosuppressive Agents; Male; Monomeric GTP-Binding Proteins; Morpholines; Neuropeptides; Protein Kinases; Ras Homolog Enriched in Brain Protein; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Up-Regulation; Vimentin

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

Topics: Animals; Carrier Proteins; Collagen; Disease Models, Animal; Disease Progression; Doxycycline; Enzyme Inhibitors; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression Regulation; Humans; Lung; Mice; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proliferating Cell Nuclear Antigen; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Quinazolines; Respiratory Mechanics; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor alpha; Uteroglobin

To investigate the effect of human amniotic membrane transplantation (AMT) on T-cell immune response in murine corneas with herpetic stromal keratitis (HSK).. Herpes simplex virus (HSV)-1-infected BALB/c mice with necrotizing HSK were treated with AMT. CD3(+) cell apoptosis was determined in treated corneas and in vitro by flow cytometric analysis using the annexin V/7-AAD system. The effect of interleukin (IL)-2, cyclosporine, rapamycin, or Fas on T-cell survival was measured. Activation phenotype was measured by (3)H-thymidine uptake and flow cytometry (CD25, CD69, major histocompatibility complex class II). Cytokine/chemokine secretion from amniotic membrane (AM)-treated corneas or draining lymph node cells was measured. The immune-modulating capacity of long-term AMT treatment and adoptive transfer of AM-treated splenocytes was tested.. After AMT, HSK and corneal inflammatory cell infiltration improved, and T-lymphocyte apoptosis occurred. T-cell apoptosis was also induced in vitro, independently of rIL-2, cyclosporine, rapamycin, or Fas. AMT-treated corneas and cultured lymphocytes had reduced IL-2, IL-10, IL-12, CRG-2, and CCL-2 content. Long-term AMT treatment decreased the proliferative response and type 1 helper T-cell cytokine level in draining lymph node cells. The improvement in HSK did not persist. Delayed-type hypersensitivity or HSV-1-specific cytotoxicity was not altered. The results suggest that murine HSK improves after AMT through reduced local T-helper cell immune responses by inducing apoptosis in T lymphocytes, independently of passive apoptosis or activation-induced cell death. AM also reduces local T-helper cytokine and chemokine levels but does not result in immune deviation. Immunologic memory against HSV-1 is not affected by AMT, and long-term protection or tolerance is not induced.

Topics: Adoptive Transfer; Amnion; Animals; Apoptosis; CD3 Complex; Cell Culture Techniques; Cell Survival; Coculture Techniques; Corneal Stroma; Cyclosporine; Cytokines; Cytotoxicity, Immunologic; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Herpesvirus 1, Human; Hypersensitivity, Delayed; Keratitis, Herpetic; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Mice, Knockout; Microscopy, Confocal; Sirolimus; Spleen; T-Lymphocytes

We compared local vessel healing and inflammatory responses associated with nonoverlapping sirolimus-eluting stents (SES) and paclitaxel-eluting stents (PES).. Sirolimus and paclitaxel may have different effects on vascular healing. In the present study, we analyzed the local histologic effects of drug-eluting stents (DES).. We placed 43 stents (22 PES and 21 SES) in 16 Yucatan minipigs. Stents were randomly assigned and placed in the left anterior descending, circumflex, or right coronary arteries (one stent per artery), covering a region previously injured by balloon angioplasty.. Histopathologic analysis showed that the distribution of injury scores was similar between the two stent groups, reflecting the homogeneity of coronary injury secondary to balloon overstretch. Electron microscopy showed complete endothelialization in most cases. Incomplete endothelialization was present in 12.5% of PES and almost 20% of SES at 30 days. In the PES group, moderate to severe inflammation was found in eight arteries, whereas only one vessel had moderate inflammation in the SES group. Severe inflammation was observed significantly more often in the PES than in the sirolimus group (P = 0.006). With the PES group, stent struts overlying side branches had a significantly higher frequency of poor endothelialization scores than did stent struts that did not overlay side branches (P = 0.006).. In this preclinical study in a pig model of in-stent restenosis, implantation of nonoverlapping DES was associated with local inflammatory reactions and decreased endothelial repair. Impaired endothelialization was visualized in the struts overlying side branches.

Topics: Angioplasty, Balloon, Coronary; Animals; Cardiovascular Agents; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Inflammation; Paclitaxel; Platelet Endothelial Cell Adhesion Molecule-1; Platelet-Derived Growth Factor; Sirolimus; Swine; Swine, Miniature; Time Factors; Vascular Endothelial Growth Factor A; Wound Healing

Short-term studies have demonstrated that rapamycin or everolimus treatment decreases cyst formation and improves renal function in animal models of polycystic kidney disease (PKD). Autosomal dominant polycystic kidney disease (ADPKD) patients would likely require life-long treatment with rapamycin.. Male Han:SPRD rats with PKD (Cy/+) were treated with rapamycin (0.2 mg/kg/day IP) or vehicle from 1 to 12 months of age. Mean trough levels of rapamycin (ng/mL) were 6.6 +/- 0.1 at 8 weeks of age. Twelve-month-old littermates (+/+) were used as normal controls.. Twelve-month-old male Cy/+ rats treated with the vehicle had a more than doubling of kidney volume, severe chronic renal failure, severe hypertension and increased heart weight compared to normal littermate controls (+/+). After rapamycin treatment, 12-month-old Cy/+ rats had markedly improved kidney volume, renal function, blood pressure and heart weight not statistically different from controls. Rapamycin reduced the cyst volume density (CVD) by 72%. Mammalian target of rapamycin (mTOR) activation in the heart, as evidenced by a marked increase in the phospho-S6 protein that was inhibited by rapamycin, was demonstrated in 12-month-old Cy/+ rats.. In conclusion, long-term rapamycin treatment in Cy/+ rats results in a normalization of kidney volume, renal function, blood pressure and heart weight. The novel finding that rapamycin decreases hypertension, heart enlargement and mTOR signalling in the heart in PKD rats is reported. The only side effect of rapamycin treatment was an 11% decrease in body weight.

Topics: Animals; Blood Pressure; Body Weight; Disease Models, Animal; Heart; Hypertension; Immunoblotting; Immunosuppressive Agents; Kidney Function Tests; Male; Organ Size; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors

K-Ras mutations are characteristic of human lung adenocarcinomas and occur almost exclusively in smokers. In preclinical models, K-Ras mutations are necessary for tobacco carcinogen-driven lung tumorigenesis and are sufficient to cause lung adenocarcinomas in transgenic mice. Because these mutations confer resistance to commonly used cytotoxic chemotherapies and targeted agents, effective therapies that target K-Ras are needed. Inhibitors of mTOR such as rapamycin can prevent K-Ras-driven lung tumorigenesis and alter the proportion of cytotoxic and Foxp3+ regulatory T cells, suggesting that lung-associated T cells might be important for tumorigenesis.. Lung tumorigenesis was studied in three murine models that depend on mutant K-Ras; a tobacco carcinogen-driven model, a syngeneic inoculation model, and a transgenic model. Splenic and lung-associated T cells were studied using flow cytometry and immunohistochemistry. Foxp3+ cells were depleted using rapamycin, an antibody, or genetic ablation.. Exposure of A/J mice to a tobacco carcinogen tripled lung-associated Foxp3+ cells prior to tumor development. At clinically relevant concentrations, rapamycin prevented this induction and reduced lung tumors by 90%. In A/J mice inoculated with lung adenocarcinoma cells resistant to rapamycin, antibody-mediated depletion of Foxp3+ cells reduced lung tumorigenesis by 80%. Likewise, mutant K-Ras transgenic mice lacking Foxp3+ cells developed 75% fewer lung tumors than littermates with Foxp3+ cells.. Foxp3+ regulatory T cells are required for K-Ras-mediated lung tumorigenesis in mice. These studies support clinical testing of rapamycin or other agents that target Treg in K-Ras driven human lung cancer.

Topics: Animals; Disease Models, Animal; Forkhead Transcription Factors; Genes, ras; Lung Neoplasms; Mice; Mice, Transgenic; Mutation; Nicotiana; Sirolimus; T-Lymphocytes, Regulatory

To test the effect of rapamycin (RAPA) on hepatic tumor growth and metastasis in Sprague-Dawley (SD) rat model and explore the possible mechanism.. SD rat hepatocellular carcinoma (HCC) model with metastatic potential was induced by diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR). 120 SD rats were randomized into four groups 16 weeks after DEN and NMOR treatment, and received 4-week intraperitoneal injection of RAPA (1.5 or 4.5 mg x kg(-1) x d(-1)), CsA (25 mg x kg(-1) x d(-1)) or equal volume of 0.9% saline, respectively. Tumor growth and metastasis were checked after the 4-week treatment. Serum vascular endothelial growth factor (VEGF) was determined by enzyme-linked immunosorbent assay (ELISA). Antiangiogenetic effects were assessed by CD34 immunostaining. The levels of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and VEGF proteins and mRNAs were detected by immunohistochemistry, western blot and reverse transcriptase-polymerase chain reaction (RT-PCR).. The mean liver weight (5.58% +/- 0.42% and 5.69% +/- 0.74%), the metastatic liver nodules (5.12 +/- 0.68 and 5.67 +/-1.12), the metastasis lung nodules (0.43 +/- 0.11 and 0.45 +/- 0.83), and the lung metastasis rate (17.2% and 14.8%) were lower in rats treated with RAPA 1.5 mg x kg(-1) x d(-1) or 4.5 mg x kg(-1) x d(-1) than those in rats treated with saline, which were 10.42% +/- 1.86%, 12.36 +/- 3.45, 1.81 +/- 0.3 and 50.0% respectively (P < 0.01 or P < 0.05). The intratumoral microvessel density (MVD), serum VEGF, and the levels of HIF-1 alpha and VEGF were lower in RAPA-treated rats than those in control rats. However, CsA-treated rats showed an opposite trend compared with the RAPA-treated rats.. RAPA can repress the expression of angiogenesis-promoting factors HIF-1 alpha and VEGF, and significantly inhibits the growth and metastasis of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cyclosporine; Disease Models, Animal; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Immunosuppressive Agents; Liver Neoplasms, Experimental; Male; Microvessels; Neoplasm Metastasis; Neovascularization, Pathologic; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Vascular Endothelial Growth Factors

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by hamartomatous growths in the brain, skin, kidneys, lungs, and heart, which lead to significant morbidity. TSC is caused by mutations in the TSC1 or TSC2 genes, whose products, hamartin and tuberin, form a tumor suppressor complex that regulates the PI3K/Akt/mTOR pathway. Early clinical trials show that TSC-related kidney tumors (angiomyolipomas) regress when treated with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (also known as sirolimus). Although side effects are tolerable, responses are incomplete, and tumor regrowth is common when rapamycin is stopped. Strategies for future clinical trials may include the investigation of longer treatment duration and combination therapy of other effective drug classes.. Here, we examine the efficacy of a prolonged maintenance dose of rapamycin in Tsc2+/- mice with TSC-related kidney tumors. Cohorts were treated with rapamycin alone or in combination with interferon-gamma (IFN-g). The schedule of rapamycin included one month of daily doses before and after five months of weekly doses. We observed a 94.5% reduction in kidney tumor burden in Tsc2+/- mice treated (part one) daily with rapamycin (8 mg/kg) at 6 months

Topics: Animals; Atorvastatin; Benzenesulfonates; Cystadenoma; Disease Models, Animal; Doxycycline; Drug Evaluation, Preclinical; Drug Therapy, Combination; Female; Heptanoic Acids; Immunosuppressive Agents; Interferon-gamma; Kidney Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Neoplasms, Experimental; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Pyridines; Pyrroles; Sirolimus; Sorafenib; Survival Analysis; Treatment Outcome; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Burden; Tumor Suppressor Proteins

Because the size of renal cysts in the native kidneys of patients with ADPKD who have been transplanted was found to be reduced when rapamycin was the immunosuppressant, we tested the involvement of the mTOR pathway in cyst enlargement. Here, male pcy mice, with mutation in one of the nephronophthisis genes, were treated with rapamycin at an early (6 to 12 weeks of age) or a later (20 to 30 weeks of age) disease stage by means of slow-release pellets containing placebo or rapamycin. Effectiveness of the rapamycin dose and delivery was shown by the inhibition of insulin-stimulated phosphorylation of p70S6K, a marker of mTOR activity, in skeletal muscle. Early treatment did not affect initial cyst development but when started late, there was a significant reduction in the rate of cyst enlargement, kidney fibrosis, and the progressive loss of renal function as measured by blood urea nitrogen. Kidneys of the mice treated through 30 weeks of age tended to be smaller and have less fibrosis compared with those of untreated or placebo-treated pcy/pcy mice at 20 weeks when treatment was initiated. Our study shows that rapamycin can prevent the late- but not the early-stage progression of renal pathology and deterioration of renal functional in this model of nephronophthisis, presumably by inhibiting mTOR activity.

Topics: Animals; Carrier Proteins; Cysts; Disease Models, Animal; Disease Progression; Fibrosis; Kidney Diseases, Cystic; Kidney Function Tests; Male; Mice; Mice, Inbred Strains; Phosphotransferases (Alcohol Group Acceptor); Sirolimus; TOR Serine-Threonine Kinases

Head and neck squamous cell carcinomas (HNSCC), the majority of which occur in the oral cavity, remain a significant cause of morbidity and mortality worldwide. A major limitation in HNSCC research has been the paucity of animal models to test the validity of current genetic paradigms of tumorigenesis and to explore the effectiveness of new treatment modalities and chemopreventive strategies. Here, we have developed an inducible oral-specific animal tumor model system, which consists in the expression of a tamoxifen-inducible Cre recombinase (CreER(tam)) under the control of the cytokeratin 14 (K14) promoter (K14-CreER(tam)) and mice in which the endogenous K-ras locus is targeted (LSL-K-ras(G12D)), thereby causing the expression of endogenous levels of oncogenic K-ras(G12D) following removal of a stop element. Surprisingly, whereas K14-CreER(tam) can also target the skin, K14-CreER(tam)/LSL-K-ras(G12D) mice developed papillomas exclusively in the oral mucosa within 1 month after tamoxifen treatment. These lesions were highly proliferative but never progressed to carcinoma. However, when crossed with p53 conditional knockout (p53(flox/flox)) mice, mice developed SCCs exclusively on the tongue as early as 2 weeks after tamoxifen induction, concomitant with a remarkable activation of the mammalian target of rapamycin (mTOR) signaling pathway. The availability of this ras and p53 two-hit animal model system recapitulating HNSCC progression may provide a suitable platform for exploring novel molecular targeted approaches for the treatment of this devastating disease. Indeed, we show here that mTOR inhibition by the use of rapamycin is sufficient to halt tumor progression in this genetically defined oral cancer model system, thereby prolonging animal survival.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Division; Disease Models, Animal; Genes, p53; Genes, ras; Head and Neck Neoplasms; Humans; Integrases; Mice; Mouth Mucosa; Mouth Neoplasms; Papilloma; Sirolimus; Tamoxifen

Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiological renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild-type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1(-/-) mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate upregulation of S6K2 expression in the kidneys of S6K1(-/-) mice. Phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1, another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1(-/-) and S6K1(+/+) littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1(-/-) mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild-type mice but failed to prevent the approximately 10% hypertrophy seen in S6K1(-/-) mice in response to uninephrectomy (UNX) although it did inhibit the basal rpS6 phosphorylation. Thus the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blood Glucose; Carrier Proteins; Cell Cycle Proteins; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Enzyme Inhibitors; Eukaryotic Initiation Factors; Gene Expression Regulation, Enzymologic; Hypertrophy; Kidney; Male; Mice; Mice, Knockout; Nephrectomy; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Topics: Analysis of Variance; Animals; Bromodeoxyuridine; Cell Death; Disease Models, Animal; Electroencephalography; Epilepsy, Temporal Lobe; Fluoresceins; Gene Expression Regulation; Immunosuppressive Agents; In Situ Nick-End Labeling; Kainic Acid; Male; Mossy Fibers, Hippocampal; Organic Chemicals; Protein Kinases; Rats; Rats, Sprague-Dawley; Seizures; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Video Recording

Liver injury induced by concanavalin A (Con A) is often used as a model to study the pathophysiology of immune mediated liver injury. Rapamycin (Rapa) is an effective immunosuppressant widely used for preventing immune activation and transplant rejection. However, the effect of Rapa on liver injury caused by Con A has not been carefully examined. In the present study, we examined the effect of Rapa on liver injury caused by Con A.. Mice received intraperitoneal Rapa injection before Con A intravenous administration. The liver injury was examined by measuring serum transaminase and pathology, and the level of cytokines was detected by enzyme linked immunosorbent assay (ELISA).. In the present study, we examined the effect of Rapa on liver injury after Con A injection in mice. We found that the treatment of mice with Rapa protected the liver from Con A-induced injury. Pretreatment with Rapa dramatically ameliorated Con A-induced mortality. This protection was associated with reduced transaminase levels in the blood and further confirmed by liver histology. ELISA showed that Rapa suppressed pro-inflammatory cytokines IFN-gamma and TNF-alpha production as compared with the untreated controls. Furthermore, intrahepatic lymphocyte proliferation was significantly inhibited.. These findings suggested that Rapa has the therapeutic potential for treatment of immune-mediated liver injury in the clinic.

Topics: Animals; Cell Proliferation; Chemical and Drug Induced Liver Injury; Concanavalin A; Cytoprotection; Disease Models, Animal; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Immunosuppressive Agents; Inflammation Mediators; Injections, Intraperitoneal; Interferon-gamma; Interleukin-4; Liver; Liver Diseases; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred C57BL; Sirolimus; Time Factors; Transaminases; Tumor Necrosis Factor-alpha

Calcineurin (CaN) is a neuronally enriched, calcium-dependent phosphatase, which plays an important role in a number of neuronal processes including development of learning and memory, and modulation of receptor's function and neuronal excitability as well as induction of apoptosis. It has been established in kindling model that the status epilepticus (SE)-induced increase in CaN activity is involved in the development of seizures through down-regulation of gamma-aminobutyric acid A receptor (GABA(A)R) activation. However, the mechanism by which CaN mediates GABA(A) receptor dephosphorylation in SE is not fully understood. Here, using a model of kainic acid (KA)-induced SE and CaN inhibitor FK506, we observed the behaviors induced by KA and levels of CaN activity and CaN expression in hippocampus by immunobloting. The results showed that the SE-induced CaN activity was time-dependent, with a peak at 2h and a return to basal level at 24h, whereas a significant increase in CaN expression was seen at 24h after SE. It is proposed that the rapid elevation in CaN activity after KA-induced SE is not likely due to an increase in CaN expression but rather an increase in CaN activation state or kinetics. In addition, we also demonstrated that pre-treatment with FK506 remarkably suppressed the SE-induced CaN activity and its expression, and reversed the SE-induced dephosphorylation of GABA(A)R 2/3 subunits. Taken together, our data suggest that down-regulation in inhibition of GABA(A)R 2/3 by CaN activity contributes to an elevation in neuronal excitability of hippocampus, which may be involved in development of chronic processes of seizures.

Topics: Animals; Behavior, Animal; Calcineurin; Disease Models, Animal; Gene Expression Regulation; Immunosuppressive Agents; Kainic Acid; Male; Phosphorylation; Rats; Rats, Wistar; Receptors, GABA-A; Sirolimus; Status Epilepticus; Tacrolimus; Time Factors

Rapamycin can inhibit tumor growth and angiogenesis in various human cancers. Cyclooxygenase 2 (COX-2) is involved in the angiogenic process. We hypothesized that the antiangiogenic effect of rapamycin may be mediated by suppression of COX-2.. Ewing sarcoma (ES) cells were implanted in athymic mice. Selected animals were treated with rapamycin for 5 weeks. Tumor vascularity was assessed by lectin perfusion angiography and immunohistochemistry. Phosphorylation of mammalian target of rapamycin pathway proteins was determined by Western blot analysis. Staining of COX-2 protein was determined by immunohistochemistry, and expression of COX-2 messenger RNA levels was assessed with quantitative real-time (RT) polymerase chain reaction.. Mean tumor weights were significantly reduced in the treated group (5.43 g +/- 1.43 SEM vs 0.49 g +/- 0.15 SEM, P < .003). There was abundant vasculature in the control group and blunted vascularity in the treated xenografts. The phosphorylation of p70s6k and Akt was not inhibited in the rapamycin-treated tumors. Cyclooxygenase 2 was suppressed in the treated xenografts at both the protein and messenger RNA levels.. Low-dose rapamycin inhibits tumor growth and angiogenesis in human ES without inhibiting the phosphorylation of p70s6k and Akt. Cyclooxygenase 2 levels are inhibited by low-dose treatment of ES with rapamycin. Cyclooxygenase 2 suppression may mediate the antiangiogenic effect of rapamycin in Ewing sarcoma.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cyclooxygenase 2; Disease Models, Animal; Female; Humans; Mice; Mice, Nude; Neovascularization, Pathologic; Sarcoma, Ewing; Signal Transduction; Sirolimus

Rapamycin represents a recognized drug-based therapeutic approach to treat cardiovascular disease. However, at least in the female heart, rapamycin may suppress the recruitment of putative signalling events conferring cardioprotection. The present study tested the hypothesis that rapamycin-sensitive signalling events contributed to the cardioprotective phenotype of the female rat heart after an ischemic insult. Rapamycin (1.5 mg/kg) was administered to adult female Sprague-Dawley rats 24 h after complete coronary artery ligation and continued for 6 days. Rapamycin abrogated p70S6K phosphorylation in the left ventricle of sham rats and the noninfarcted left ventricle (NILV) of 1-week postmyocardial-infarcted (MI) rats. Scar weight (MI 0.028 +/- 0.006, MI+rapamycin 0.064 +/- 0.004 g) and surface area (MI 0.37 +/- 0.08, MI+rapamycin 0.74 +/- 0.03 cm2) were significantly larger in rapamycin-treated post-MI rats. In the NILV of post-MI female rats, rapamycin inhibited the upregulation of eNOS. Furthermore, the increased expression of collagen and TGF-beta3 mRNAs in the NILV were attenuated in rapamycin-treated post-MI rats, whereas scar healing was unaffected. The present study has demonstrated that rapamycin-sensitive signalling events were implicated in scar formation and reactive fibrosis. Rapamycin-mediated suppression of eNOS and TGF-beta3 mRNA in post-MI female rats may have directly contributed to the larger infarct and attenuation of the reactive fibrotic response, respectively.

Topics: Animals; Blotting, Western; Cicatrix; Collagen; Disease Models, Animal; Female; Fibrosis; Heart Ventricles; Myocardial Infarction; Myocardium; Nitric Oxide Synthase Type III; Phosphorylation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Transforming Growth Factor beta3; Ventricular Remodeling

Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235.. Using ENU to enhance Tsc2+- kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation.. Both mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model.

Topics: Animals; Antineoplastic Agents; Carrier Proteins; Disease Models, Animal; Ethylnitrosourea; Everolimus; Imidazoles; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiprotein Complexes; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Proteins; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inactivating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamycin (mTOR), leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin (sirolimus), led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.

Topics: Animals; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Peutz-Jeghers syndrome (PJS) is a familial cancer disorder due to inherited loss of function mutations in the LKB1/ STK11 serine/threonine kinase. PJS patients develop gastrointestinal hamartomas with 100% penetrance often in the second decade of life, and demonstrate an increased predisposition toward the development of a number of additional malignancies. Among mitogenic signaling pathways, the mammalian-target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in tissues and tumors derived from LKB1-deficient mice. Consistent with a central role for mTORC1 in these tumors, rapamycin as a single agent results in a dramatic suppression of preexisting GI polyps in LKB1+/- mice. However, the key targets of mTORC1 in LKB1-deficient tumors remain unknown. We demonstrate here that these polyps, and LKB1- and AMPK-deficient mouse embryonic fibroblasts, show dramatic up-regulation of the HIF-1alpha transcription factor and its downstream transcriptional targets in an rapamycin-suppressible manner. The HIF-1alpha targets hexokinase II and Glut1 are up-regulated in these polyps, and using FDG-PET, we demonstrate that LKB1+/- mice show increased glucose utilization in focal regions of their GI tract corresponding to these gastrointestinal hamartomas. Importantly, we demonstrate that polyps from human Peutz-Jeghers patients similarly exhibit up-regulated mTORC1 signaling, HIF-1alpha, and GLUT1 levels. Furthermore, like HIF-1alpha and its target genes, the FDG-PET signal in the GI tract of these mice is abolished by rapamycin treatment. These findings suggest a number of therapeutic modalities for the treatment and detection of hamartomas in PJS patients, and potential for the screening and treatment of the 30% of sporadic human lung cancers bearing LKB1 mutations.

Topics: AMP-Activated Protein Kinases; Animals; Cell Proliferation; Disease Models, Animal; Down-Regulation; Fibroblasts; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Peutz-Jeghers Syndrome; Positron-Emission Tomography; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Burden; Up-Regulation

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by the presence of IgG autoantibodies against Dsg3. Our aim was to investigate the molecular events implicated in the development and localization of apoptosis and acantholysis in PV. We used a passive transfer mouse model together with immunohistochemical (IHC) techniques and the TUNEL assay, with quantification analysis in the basal layer of the epidermis. The activated signalling molecules analysed and apoptotic cells detected showed an identical localization. Herein, we found for the first time in vivo an increased expression of activated HER receptor isoforms in the basal layer in PV lesions. Besides, we observed the almost total lack of activated Akt compared with a higher level of activated mTOR within the basal cells of the epidermis. Our observations strongly support that the restriction of acantholysis to the basal layer may be due, at least in part, to the selective and increased presence of activated HER receptor isoforms in these cells. After phosphorylation of HER receptor isoforms, intracellular signalling pathways are activated in the basal layer. In addition, the imbalance in Akt/mTOR that takes place in the basal cells may provide intracellular signals necessary for the development of apoptosis and acantholysis.

Topics: Acantholysis; Animals; Apoptosis; Betacellulin; Carrier Proteins; Disease Models, Animal; Enzyme Activation; Epidermal Growth Factor; Epidermis; ErbB Receptors; Erlotinib Hydrochloride; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Intradermal Tests; Isoenzymes; Mice; Mice, Inbred C57BL; Pemphigus; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Quinazolines; Sirolimus; src-Family Kinases; TOR Serine-Threonine Kinases; Transforming Growth Factor alpha

Dentate granule cell axon (mossy fiber) sprouting is a common abnormality in patients with temporal lobe epilepsy. Mossy fiber sprouting creates an aberrant positive-feedback network among granule cells that does not normally exist. Its role in epileptogenesis is unclear and controversial. If it were possible to block mossy fiber sprouting from developing after epileptogenic treatments, its potential role in the pathogenesis of epilepsy could be tested. Previous attempts to block mossy fiber sprouting have been unsuccessful. The present study targeted the mammalian target of rapamycin (mTOR) signaling pathway, which regulates cell growth and is blocked by rapamycin. Rapamycin was focally, continuously, and unilaterally infused into the dorsal hippocampus for prolonged periods beginning within hours after rats sustained pilocarpine-induced status epilepticus. Infusion for 1 month reduced aberrant Timm staining (a marker of mossy fibers) in the granule cell layer and molecular layer. Infusion for 2 months inhibited mossy fiber sprouting more. However, after rapamycin infusion ceased, aberrant Timm staining developed and approached untreated levels. When onset of infusion began after mossy fiber sprouting had developed for 2 months, rapamycin did not reverse aberrant Timm staining. These findings suggest that inhibition of the mTOR signaling pathway suppressed development of mossy fiber sprouting. However, suppression required continual treatment, and rapamycin treatment did not reverse already established axon reorganization.

Topics: Animals; Anticonvulsants; Atropine Derivatives; Axons; Dentate Gyrus; Disease Models, Animal; Epilepsy, Temporal Lobe; Immunohistochemistry; Infusions, Parenteral; Injections, Intraperitoneal; Male; Mossy Fibers, Hippocampal; Muscarinic Agonists; Neural Inhibition; Neurons; Parasympatholytics; Pilocarpine; Protein Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Staining and Labeling; Status Epilepticus; Time Factors; TOR Serine-Threonine Kinases

Although both sirolimus (CYPHER) and paclitaxel (TAXUS) drug-eluting stents have demonstrated efficacy and safety in clinical trials, human autopsy data have raised concerns about long-term healing and the potential for local inflammatory reactions.. Overlapping stents (CYPHER drug-eluting stents, Bx SONIC bare metal stents, TAXUS drug-eluting stents, and Liberté bare metal stents) were implanted in noninjured coronary arteries of 58 domestic swine. Histopathological evaluation of proximal, overlapped, and distal stented segments was determined with emphasis on inflammation at 30, 90, and 180 days. Circumferential granulomatous inflammation in all stented segments was defined as inflammation consisting of macrophages, multinucleated giant cells, lymphocytes, and granulocytes, including many eosinophils, adjacent to almost all struts. Circumferential granulomatous inflammation was more prevalent in CYPHER (9 of 23, 39%) compared with TAXUS (1 of 21, 5%; P=0.01) and control bare metal stents (0 of 44) in the combined 90- and 180-day cohorts. Only CYPHER specimens showed marked adventitial inflammation (P=0.0025) and fibrosis (P=0.0055) accompanied by extensive remodeling. Fibrin deposition within neointima and medial smooth muscle cell death were greater (both P<0.001) in TAXUS than CYPHER at 30 days, with more fibrin in TAXUS than CYPHER through 90 days (P<0.05).. Although these data cannot be directly extrapolated to humans, the high prevalence in this porcine model of diffuse granulomatous inflammation seen with CYPHER stents, persisting at 180 days and associated with extensive remodeling of the artery, and persistent para-strut fibrin deposition with TAXUS stents emphasize the need for further investigation of biocompatibility with these and other novel combination drug/polymer drug-eluting stents.

Topics: Animals; Arteritis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Eosinophils; Female; Fibrin; Fibrosis; Granuloma, Foreign-Body; Paclitaxel; Sirolimus; Swine; Tunica Intima

We have generated an EL1-luc/TAg transgenic mouse model that develops spontaneous and bioluminescent acinar cell carcinomas. We applied this model to noninvasively monitor tumor development and drug response.. EL1-luc/TAg transgenic mice of 11 weeks of age were treated with rapamycin (5 mg/kg, i.p.) or vehicle for 6 to 12 weeks. Tumor development was monitored through bioluminescence imaging and necropsy at the study end point.. EL1-luc/TAg transgenic mice showed pancreas-specific bioluminescence signal before tumor progression and produced increasing light emission from the onset of the pancreatic acinar cell carcinomas. The latency of tumor development ranged from 10 to >20 weeks of age in these mice. Progression of the primary acinar cell carcinoma was accompanied by emergence of metastatic lesions in the abdominal organs, including liver and gastrointestinal fat tissues. Rapamycin treatment suppressed tumor development.. The EL1-luc/TAg mouse provides a noninvasive approach for monitoring spontaneous acinar cell carcinoma development and comprises a convenient tool for the evaluation of novel therapeutics against pancreatic cancers. Tumor growth suppression through inhibition of the mammalian target of rapamycin pathway further validates this model as clinically relevant.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Acinar Cell; Disease Models, Animal; Drug Monitoring; Longitudinal Studies; Luminescent Measurements; Mice; Mice, Transgenic; Pancreas; Pancreatic Neoplasms; Sirolimus

We have presently evaluated the effects of the immunomodulatory drug rapamycin on the course of protracted relapsing experimental allergic encephalomyelitis (PR-EAE) in Dark Agouti (DA) rats, which serves as a preclinical model of multiple sclerosis. The data show that the oral administration of rapamycin at 3 mg/kg for 28 consecutive days significantly ameliorated the course of PR-EAE in DA rats. The rats that received the medication had significantly lower clinical cumulative scores and shorter duration of the disease than did the control rats treated with the vehicle. The milder course of the disease was associated with a reduction of the histopathological signs associated to EAE: increased percentage of splenic CD4+CD25 + Foxp3+ Tregs and concomitant reduction of splenic CD8+T cells. These data suggest that rapamycin has pharmacological potential worthy of consideration in the treatment of MS patients.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Immunosuppressive Agents; Multiple Sclerosis; Rats; Rats, Inbred Strains; Sirolimus; T-Lymphocytes, Regulatory

The aim of this article was to study the effect of dual drug-eluting stent (DES) on both restenosis and thrombosis in a porcine coronary artery model. This study reports on the use of two drugs coated on the stent to simultaneously minimize both restenosis and thrombosis. The DES was prepared by spray coating a bare metal stent with a biodegradable polymer loaded with sirolimus and triflusal, to treat against restenosis and thrombosis, respectively. The two-layered dual drug-coated stent was characterized in vitro for surface properties before and after expansion, as well as for possible delamination by cross-sectioning the stent in vitro. In vivo animal studies (in a pig model) were then performed for acute thrombosis, inflammation, and restenosis. The results show a significant reduction in restenosis with a stent coated with both drugs compared with the controls (a bare metal stent, a sirolimus-coated, and a pure polymer-coated stent). The reduction in restenosis with a sirolimus/triflusal-eluting stent is associated with an inhibition of inflammation and thrombus formation, suggesting that such dual DES have a role to play for the treatment of coronary artery diseases.

Topics: Animals; Blood Vessel Prosthesis; Blood Vessel Prosthesis Implantation; Chromium Alloys; Coronary Restenosis; Coronary Vessels; Disease Models, Animal; Drug Therapy, Combination; Drug-Eluting Stents; Immunosuppressive Agents; Platelet Aggregation Inhibitors; Prosthesis Design; Salicylates; Sirolimus; Swine; Thrombosis; Treatment Outcome

In clinical renal transplantation, an increase in proteinuria after conversion from calcineurin inhibitors to rapamycin has been reported. In contrast, there are studies showing a nephro-protective effect of rapamycin in proteinuric diseases characterized by progressive interstitial inflammatory fibrosis.. Because of the contradictory reports concerning rapamycin on proteinuria, we examined proteinuria and podocyte damage markers on two renal disease models, with clearly different pathophysiological mechanisms: a glomerular toxico-immunological model induced by puromycin aminonucleoside, and a chronic hyperfiltration and inflammatory model by mass reduction, both treated with a fixed high rapamycin dose.. In puromycin groups, rapamycin provoked significant increases in proteinuria, together with a significant fall in podocin immunofluorescence, as well as clear additional damage to podocyte foot processes. Conversely, after mass reduction, rapamycin produced lower levels of proteinuria and amelioration of inflammatory and pro-fibrotic damage. In contrast to the puromycin model, higher glomerular podocin and nephrin expression and amelioration of glomerular ultrastructural damage were found.. We conclude that rapamycin has dual opposing effects on subjacent renal lesion, with proteinuria and podocyte damage aggravation in the glomerular model and a nephro-protective effect in the chronic inflammatory tubulointerstitial model. Rapamycin produces slight alterations in podocyte structure when acting on healthy podocytes, but it clearly worsens those podocytes damaged by other concomitant injury.

Topics: Animals; Disease Models, Animal; Immunosuppressive Agents; Male; Nephrosis, Lipoid; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Sirolimus

Sirolimus (SRL) is a potent and specific immunosuppressive drug used in organ transplantation, as basic therapy or in combination with calcineurin inhibitors. Although SRL is a nonnephrotoxic drug, many reports have related its use with the development of proteinuria, especially after conversion. Therefore, the aim of this study was to elucidate the interrelation between early and late SRL administration on the development of glomerular hypertrophy and proteinuria in a model of renal mass reduction (RMR).. Rats underwent 2/3 cryoablation of the left kidney and subsequent right nephrectomy (n=42) or sham operations (n=29). Two weeks before (early study) or 12 weeks after (late study) surgery, SRL or vehicle was administered three times weekly. Creatinine clearance and proteinuria were determined throughout the study, and a complete histologic analysis was performed at the end of the study.. Treatment with SRL had no effect on creatinine clearance, independently of the administration time. Four weeks after RMR, a significant increase in proteinuria was observed. Proteinuria was stabilized after early and late SRL administration, whereas vehicle-treated animals showed a further increase in proteinuria. Glomerular hypertrophy was strongly associated with proteinuria, and early SRL introduction prevented glomerular enlargement. The histologic analysis showed less structural damage in the two groups of animals treated with SRL than in the control group.. Although early SRL introduction blocked glomerular hypertrophy, SRL treatment revealed the potential to halt progression of proteinuria and histologic damage at any time of administration in a model of RMR.

Topics: Animals; Creatinine; Disease Models, Animal; Disease Progression; Hypertrophy; Immunosuppressive Agents; Kidney; Kidney Diseases; Kidney Glomerulus; Male; Protein Kinases; Proteinuria; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases

Focal malformations of cortical development are highly associated with intractable epilepsy in children and adults. Most patients with focal cortical malformations and epilepsy will require epilepsy surgery. Recent studies have provided new insights into the developmental pathogenesis of cortical malformations specifically relating to alterations in cell signaling though the mammalian target of rapamycin (mTOR) pathway. Focal cortical dysplasias, hemimegalencephaly, and tubers in tuberous sclerosis complex all exhibit evidence for hyperactive mTOR signaling, suggesting that these disorders form a spectrum of malformations or "TORopathies" characterized by disorganized cortical lamination, cytomegaly, and intractable seizures. Alterations in mTOR activity in focal brain malformations provide a potential pathogenic pathway to investigate for gene mutations and to exploit for animal models. Most importantly, however, if select focal cortical malformations result from enhanced mTOR signaling, new therapeutic antiepileptic compounds, such as rapamycin, can be designed and tested that specifically target mTOR signaling.

Topics: Adult; Animals; Anticonvulsants; Disease Models, Animal; Drug Design; Epilepsies, Partial; Gene Expression; Humans; Malformations of Cortical Development; Mice; Mutation; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Focal cortical dysplasia (FCD) and related malformations of cortical development (MCDs) represent an increasingly recognized cause of medically intractable epilepsy. However, the underlying mechanisms of epileptogenesis are poorly understood, and treatments for epilepsy due to various cortical malformations are often limited or ineffective. Animal models offer a number of advantages for investigating cellular and molecular mechanisms of epileptogenesis and developing novel, rational therapies for MCD-related epilepsy. This review highlights specific examples of how animal models have been useful in addressing several clinically relevant issues about epilepsy due to FCDs and related cortical malformations, including the pathologic and clinical features, etiologic factors, localization of the epileptogenic zone, neuronal and astrocytic contributions to epileptogenesis, and the development of antiepileptogenic therapies.

Topics: Animals; Anticonvulsants; Cerebral Cortex; Disease Models, Animal; Drug Design; Epilepsy; Humans; Malformations of Cortical Development; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Proteins; Rats; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis

We examined the effect of 28 days of overload on mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) signaling in young adult (Y; 6-month old) and aged (O; 30-month old) Fischer 344 x Brown Norway rats subjected to bilateral synergist ablation (SA) of two thirds of the gastrocnemius muscle or sham surgery (CON). Although plantaris (PLA) muscle hypertrophy was attenuated by aging, mTOR phosphorylation was 44% and 35% greater in Y SA and O SA compared with CON (p = .038). Ribosomal protein S6 phosphorylation was 114% and 24% higher in Y SA and O SA compared with CON (p = .009). Eukaryotic initiation factor 2Bepsilon phosphorylation was 33% and 9% higher in Y SA and O SA compared with CON (p = .04). Translational signaling in young adult and aged plantaris muscle is equally responsive to chronic overload.

Topics: Age Factors; Aging; Analysis of Variance; Animals; Chronic Disease; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Hypertrophy; Immunoblotting; Linear Models; Male; Mitogen-Activated Protein Kinases; Muscle, Skeletal; Organ Size; Phosphorylation; Probability; Protein Kinases; Random Allocation; Rats; Rats, Inbred BN; Rats, Inbred F344; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Action Potentials; Animals; Apoptosis; Autophagy; Brain Diseases; Disease Models, Animal; Epilepsy; Neuroprotective Agents; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

The levels of a small heat shock protein (Hsp)20 and its phosphorylation are increased on ischemic insults, and overexpression of Hsp20 protects the heart against ischemia/reperfusion injury. However, the mechanism underlying cardioprotection of Hsp20 and especially the role of its phosphorylation in regulating ischemia/reperfusion-induced autophagy, apoptosis, and necrosis remain to be clarified.. Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20(S16A)). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation.. Our results demonstrated that Hsp20(S16A) hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20(S16A) hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20(S16A) hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20(S16A) hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20(S16A) hearts.. Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

Topics: Alanine; Animals; Apoptosis; Autophagy; Base Sequence; Disease Models, Animal; Heart Failure; HSP20 Heat-Shock Proteins; Humans; Male; Mice; Mice, Transgenic; Molecular Sequence Data; Mutation; Myocardial Contraction; Myocardium; Necrosis; Phosphorylation; Recovery of Function; Reperfusion Injury; Serine; Sirolimus; Time Factors; Ventricular Function, Left; Ventricular Pressure

Early-stage bladder cancer occurs as two distinct forms: namely, low-grade superficial disease and high-grade carcinoma in situ (CIS), which is the major precursor of muscle-invasive bladder cancer. Although the low-grade form is readily treatable, few, if any, effective treatments are currently available for preventing progression of nonmuscle-invasive CIS to invasive bladder cancer. Based on our previous findings that the mammalian target of Rapamycin (mTOR) signaling pathway is activated in muscle-invasive bladder cancer, but not superficial disease, we reasoned that suppression of this pathway might block cancer progression. To test this idea, we performed in vivo preclinical studies using a genetically engineered mouse model that we now show recapitulates progression from nonmuscle-invasive CIS to muscle-invasive bladder tumors. We find that delivery of Rapamycin, an mTOR inhibitor, subsequent to the occurrence of CIS effectively prevents progression to invasive bladder cancer. Furthermore, we show that intravesical delivery of Rapamycin directly into the bladder lumen is highly effective for suppressing bladder tumorigenesis. Thus, our findings show the potential therapeutic benefit of inhibiting mTOR signaling for treatment of patients at high risk of developing invasive bladder cancer. More broadly, our findings support a more widespread use of intravesical delivery of therapeutic agents for treatment of high-risk bladder cancer patients, and provide a mouse model for effective preclinical testing of potential novel agents.

Topics: Administration, Intravesical; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Disease Progression; Female; Integrases; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Composite tissue allotransplantation may have different immunosuppressive requirements and manifest different complications compared with solid organ transplantation. We developed a non-human primate facial composite tissue allotransplantation model to investigate strategies to achieve prolonged graft survival and immunologic responses unique to these allografts.. Composite facial subunits consisting of skin, muscle, and bone were heterotopically transplanted to mixed lymphocyte reaction-mismatched Cynomolgus macaques. Tacrolimus monotherapy was administered via continuous intravenous infusion for 28 days then tapered to daily intramuscular doses.. Five of the six animals treated with tacrolimus monotherapy demonstrated rejection-free graft survival up to 177 days (mean, 113 days). All animals with prolonged graft survival developed posttransplant lymphoproliferative disorders (PTLD). Three animals converted to rapamycin after 28 days of rejection of their allografts, but did not develop PTLD. Genotypic analysis of PTLD tumors demonstrated donor origin in three of the five analyzed by short-tandem repeats. Sustained alloantibodies were detected in rejecting grafts and absent in nonrejecting grafts.. Tacrolimus monotherapy provided prolonged rejection-free survival of composite facial allografts in a non-human primate model but was associated with the development of a high frequency of donor-derived PTLD tumors. The transplantation of a large volume of vascularized bone marrow in composite tissue allografts may be a risk factor for PTLD development.

Topics: Animals; Bone Marrow Transplantation; Disease Models, Animal; Facial Transplantation; Graft Rejection; Graft Survival; Immunosuppressive Agents; Infusions, Intravenous; Isoantibodies; Lymphocyte Culture Test, Mixed; Lymphoproliferative Disorders; Macaca fascicularis; Magnetic Resonance Imaging; Male; Risk Factors; Sirolimus; Tacrolimus; Time Factors; Transplantation, Homologous

We evaluated the impact of reduced nephron mass on nephrotoxicity by cyclosporine A (CsA) and/or sirolimus (SRL).. Renal function was tested in salt-depleted rats bearing two kidneys (2K), one kidney, or half a kidney (1/2K) and treated for 7 or 28 days with CsA (5 mg/kg) and/or SRL (0.8 mg/kg). We also measured the expression of aquaporin-2, sodium/phosphate cotransporter (NaPi)-2, paracellin-1, and kidney injury molecule (KIM)-1 by real-time polymerase chain reaction.. At 7 days in 2K, serum creatinine clearance (CrCl) was decreased only in CsA/SRL-treated group (P<0.05) compared with controls; in 1/2K, CrCl was decreased in all groups, but most dramatically in CsA/SRL group (P<0.05). Extended 28-day therapy worsened CrCl in all 1/2K groups (P<0.01). Although the expression of aquaporin-2, NaPi-2, and paracellin-1 mRNAs tended to increase in kidneys with a reduced nephron mass, NaPi-2 mRNA levels decreased in 1/2K rats exposed to CsA/SRL for 28 days (P<0.05). In contrast, low KIM-1 mRNA expression in control 2K rats increased fourfold in untreated 1/2K (P<0.05), and 50- to 200-fold in CsA/SRL-treated 1/2K (P=0.01).. Nephrotoxicity is significantly worsened by reduced nephron mass, which correlates with increased expression of KIM-1 and inhibited expression of NaPi-2.

Topics: Animals; Aquaporin 2; Cell Adhesion Molecules; Creatinine; Cyclosporine; Disease Models, Animal; Follow-Up Studies; Gene Expression Regulation; Immunosuppressive Agents; Kidney Diseases; Male; Nephrons; Polymerase Chain Reaction; Rats; Risk Factors; RNA, Messenger; Sirolimus; Sodium-Phosphate Cotransporter Proteins

Memory T cells specific for donor antigens are currently recognized as a significant barrier for maintaining a successful transplant. Furthermore, it has been shown that commonly used immunosuppressive drugs do not alleviate this memory response. Here, we report that rapamycin allows significant proliferation of memory T cells and bortezomib can abrogate the proliferation of rapamycin-resistant memory T cells when preserving the survival of regulatory T cells.. Peripheral blood mononuclear cells freshly isolated from non-human primates were stimulated with anti-CD3/CD28 antibodies, and inhibitory and apoptotic effects of rapamycin and bortezomib on memory T-cell proliferation were investigated. The CD95 marker in CD3+ T cells was used for the separate enrichment of memory T cells and naïve T cells.. Rapamycin at the level even higher than therapeutic concentration could not suppress the proliferation of a significant proportion of memory T cells. However, the combined administration of bortezomib and rapamycin abrogated the proliferation of rapamycin-resistant memory T cells. Furthermore, bortezomib preserved the survival of preexisting CD4+ FoxP3+ regulatory T cells, while inducing apoptosis of CD4+ FoxP3- conventional T cells. The combined administration of low doses of rapamycin and bortezomib also exerted an additive effect on suppressing T-cell proliferation. Cytokine analysis demonstrated that bortezomib could not only suppress rapamycin-permissive interleukin (IL)-6 production, but also production of interferon (IFN)-gamma, IL-4, and IL-10.. This article provides in vitro data from which immunosuppressive regimens for the effective control of memory T cells in non-human preclinical experiments and in clinical trials are selected.

Topics: Animals; Boronic Acids; Bortezomib; Cell Proliferation; Cell Survival; Disease Models, Animal; Drug Resistance; Graft Rejection; Immunologic Memory; Immunosuppressive Agents; Lymphocyte Activation; Macaca mulatta; Male; Pancreas Transplantation; Protease Inhibitors; Pyrazines; Sirolimus; Swine; Swine, Miniature; T-Lymphocytes; T-Lymphocytes, Regulatory

We aimed to explore the effect of Mycophenolate mofetil (MMF) on loss of renal function and cyst progression compared to rapamycin in Han: SPRD rats. We also sought to assess whether the effect of combination therapy of MMF plus rapamycin was better than that of monotherapy.. Sixty heterozygous (Cy/+) and littermate control (+/+) male Han: SPRD rats were weaned at 4 weeks of age, then divided into four groups randomly to receive different treatments by intragastric administration for 2 months: vehicle-treated group as control, MMF-treated group (20mg/kg/day), rapamycin-treated group (2mg/kg/day), and MMF+Rapa- treated group (MMF 20mg/kg/day plus Rapamycin 2mg/kg/day). RESULLS: After 2 months of treatment, rapamycin caused a 22 % decrease in body weight in comparison to the control group, whereas MMF had no significant effect on weight gain. The steady increase of BUN in Cy/+ rats was reduced by 15% in MMF-treated Cy/+ rats. However, rapamycin and combination therapy reduced BUN by 42% and 43%, respectively. CCr was 0.93+/-0.11ml/min in vehicle-treated Cy/+ rats, 1.67+/-0.23 ml/min in MMF-treated Cy/+ rats (P<0.05), 1.72+/-0.44 ml/min and 1.83+/-0.21 ml/min in rapamycin- and MMF+Rapa-treated Cy/+ rats, respectively (.P<0.01). Cyst volume density was 57.1 % in vehicle-treated Cy/+ rats, 45.2% in MMF-treated Cy/+ rats (P<0.05), 32.9% and 37.7% in rapamycin- and MMF+Rapa-treated Cy/+ rats, respectively (P<0.01). MMF markedly ameliorated interstitial inflammation and fibrosis. Rapamycin showed a similar effect on interstitial inflammation and fibrosis, but to a lesser degree.. MMF is more tolerable than rapamycin and can retard deterioration of renal function in Han: SPRD rats, though its effect is weaker than that of rapamycin. Combination therapy does not exert more favorable effect than monotherapy.

Topics: Animals; Disease Models, Animal; Drug Therapy, Combination; Immunosuppressive Agents; Male; Mycophenolic Acid; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors

A bioengineered microporous polycarbonate-siloxane polyurethane graft has been developed for coronary artery bypass grafting. Biological agents can be impregnated into its absorbable collagen and hyaluronan microstructure and stable macrostructure to promote patency. The objective of this study was to examine the in vivo biological performance and biomechanical characteristics of this graft.. Three types of graft (3.6-mm internal diameter, 24-mm length) were manufactured: heparin alone (H) grafts, heparin and sirolimus (HS) grafts, and grafts without any drug impregnation (C). All H and HS grafts were impregnated with 54 U of heparin in the microstructure for early elution to prevent acute graft thrombosis and 56 U of heparin in the macrostructure to prevent late thrombosis. In addition to the heparin, the HS graft was impregnated with 2.1 mg of sirolimus in the macrostructure for prolonged elution to inhibit intimal hyperplasia. All grafts (3.6-mm internal diameter, 24-mm length) were implanted into the abdominal aortas of rabbits (n = 55). Expanded polytetrafluoroethylene grafts (4.0-mm internal diameter, 24-mm length; n = 7) were implanted as controls. At 1, 3, and 6 months after surgery, the grafts were removed for histologic, scanning electron microscopic, immunohistochemical, and biomechanical evaluations.. The patency rate was 100% in the H, HS, and C grafts at each time point. Although the expanded polytetrafluoroethylene grafts were patent at 1 and 3 months after surgery, 1 of 2 grafts (50%) were occluded at 6 months. None of the H or HS grafts had any stenosis or thrombus. Scanning electron microscopic examination proved that endothelial cells propagated smoothly from the anastomotic sites after 6 months in the H and HS grafts in comparison with the expanded polytetrafluoroethylene grafts, which had rare endothelialization. Neointima formation was inhibited in the HS graft compared with the H or C graft at 6 months (123 +/- 126 microm vs 206 +/- 158 microm or 202 +/- 67 microm; P < .05). In addition, the H, HS, and C grafts had greater cellular infiltration inside the graft than the expanded polytetrafluoroethylene grafts. All grafts except the expanded polytetrafluoroethylene graft had marked neocapillary formation 6 months after surgery. The graft compliance between 80 and 120 mm Hg was 6.0% +/- 2.5% and 6.2% +/- 0.9% at 6 months in the H and HS grafts, respectively. The graft macrostructure was unchanged according to the biomechanical evaluation in the H and HS grafts.. A unique drug-eluting graft had excellent patency throughout the 6 months after implantation. The heparin-sirolimus graft encouraged luminal endothelialization without excessive intimal hyperplasia. This graft performed significantly better than the expanded polytetrafluoroethylene graft. This graft has the potential to become an implantable graft for coronary artery bypass grafting.

Topics: Animals; Aorta, Abdominal; Biomedical Engineering; Blood Vessel Prosthesis; Blood Vessel Prosthesis Implantation; Disease Models, Animal; Drug-Eluting Stents; Graft Occlusion, Vascular; Graft Rejection; Graft Survival; Heparin; Probability; Prosthesis Design; Rabbits; Random Allocation; Risk Assessment; Sensitivity and Specificity; Sirolimus; Vascular Patency

Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5' end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factor-4E; Female; HeLa Cells; Humans; Lymphoma; Mice; Mice, Inbred C57BL; Peptide Chain Initiation, Translational; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polyribosomes; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; Thapsigargin; Triterpenes

The ciliopathies are a class of rare human genetic disease whose aetioligies lie in defective primary cilia. Typical ciliopathies include Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Jeune, Joubert, oro-facial-digital (OFD1) and Meckel (MKS) syndromes. All ciliopathies have the common denominator of renal disease, often including tubular cysts. In this study, we have modelled a range of ciliopathies in zebrafish and shown in all cases that knocking down these genes causes cystic lesions in the kidney. We have identified two drugs, rapamycin and roscovitine, which ameliorate the renal phenotype, both morphologically and functionally. This is the first study in which zebrafish has been used to identify potential therapeutic modalities for ciliopathic renal disease, and the results pave the way for further investigations in mammalian models.

Topics: Animals; Antineoplastic Agents; Bardet-Biedl Syndrome; Cilia; Disease Models, Animal; Immunosuppressive Agents; Kidney; Polycystic Kidney Diseases; Purines; Recovery of Function; Roscovitine; Sirolimus; Zebrafish

The aim was to study the influence of sirolimus (SRL) on body weight in a rat model and in kidney transplant patients. Wistar rats (15 weeks old) were either treated with vehicle (VEH; n = 8) or SRL (n = 7) 1.0 mg/kg three times per week for 12 weeks. Body mass and food intake were measured weekly. Adipocyte diameter was determined in hematoxylin-eosin stains. The body mass index (BMI) obtained from clinical kidney transplant trials comparing SRL-based with cyclosporine-based therapy was analyzed.. SRL produced a decrease of the weight gain curve. At the end of the study, mean body weight in the SRL group was lower than in the VEH group (356 vs. 507 g, P < 0.01) in spite of comparable food intake normalized for body weight was not different. Mean adipocyte diameter was 36 mum in VEH and 25 mum in SRL rats (P = 0.009). Mean SRL blood trough concentration was 38 ng/ml. Kidney transplant patients: Two years after transplantation, BMI was significantly lower in the SRL-based treatment arm compared to cyclosporine (24.17 +/- 2.99 vs. 25.97 +/- 5.01 kg/m(2), P = 0.031). SRL treatment leads to less body mass. Adipocyte cell diameter was reduced in SRL-treated animals. A possible explanation may be the effects of SRL on metabolic regulation and cell growth.

Topics: Adipocytes; Animals; Body Mass Index; Body Weight; Cyclosporine; Disease Models, Animal; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Protein Kinases; Radioimmunoassay; Rats; Rats, Wistar; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Everolimus is a novel proliferation signal inhibitor that has potent immunosuppressive activity. As previously shown, systemic administration of the drug could effectively enhance the mean survival time (MST) of corneal allografts. Commonly, the topical application of immunomodulatory agents is preferred over systemic use, in order to reduce the side effects.. To investigate the efficacy of topically applied everolimus to prevent corneal graft rejection in an experimental model.. A total of 45 female Lewis rats received 3.5-mm grafts of MHCI/II incompatible Dark Agouti donors. Recipients were randomly assigned to receive either: (1) 0.05% everolimus microemulsion, (2) 0.025% everolimus microemulsion or (3) a vehicle as the control. Treatment was started on the day of surgery and applied 5 times daily. Grafts were graded every day and a rejection score was generated based on cornea clarity and oedema.. Local administration of 0.05 or 0.025% everolimus was effective in prolonging the mean survival time of corneal grafts (MST = 21 +/- 6.57 days and 16.4 +/- 2.3 days, respectively) as compared to vehicle control group (MST = 13.3 +/- 1.7 days; p < 0.001 and p < 0.001). Real-time PCR demonstrated that topical administration of everolimus increased the mRNA levels of CD25, IL-10 and IFN-gamma, but this was significant only for IL-10 (p = 0.015).. These data indicate that topically applied everolimus is effective in prolonging corneal allograft survival in an experimental keratoplasty model.

Topics: Administration, Topical; Animals; Cytokines; Disease Models, Animal; Everolimus; Female; Graft Rejection; Graft Survival; Immunosuppressive Agents; Keratoplasty, Penetrating; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Transplantation, Homologous

We previously reported that melatonin prevents neuronal cell death in ischemic brain injury through the activation of Akt and the inhibition of apoptotic cell death. We investigated whether melatonin inhibits the apoptotic signal through the activation of a mammalian target of rapamycin (mTOR) and p70S6 kinase and its downstream target, S6 phosphorylation. It is known that mTOR is a downstream target of Akt and a central regulator of protein synthesis, cell growth, and cell cycle progression. Adult male rats were treated with melatonin (5mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24h after MCAO and infarct volumes were analyzed. We confirmed that melatonin significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Brain injury induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Melatonin prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our results suggest that melatonin prevents cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.

Topics: Analysis of Variance; Animals; Antioxidants; Cell Line, Transformed; Cell Survival; Disease Models, Animal; Functional Laterality; Gene Expression Regulation; Glutamates; Infarction, Middle Cerebral Artery; Male; Melatonin; Mice; Neurons; Neurotoxins; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus

Disruptions in the normal program of tissue repair can result in poor wound healing, which perturbs the integrity of barrier tissues such as the skin. Such defects in wound repair occur in transplant recipients treated with the immunosuppressant drug rapamycin (sirolimus). Intraepithelial lymphocytes, such as gammadelta T cells in the skin, mediate tissue repair through the production of cytokines and growth factors. The capacity of skin-resident T cells to function during rapamycin treatment was analyzed in a mouse model of wound repair. Rapamycin treatment renders skin gammadelta T cells unable to proliferate, migrate, and produce normal levels of growth factors. The observed impairment of skin gammadelta T cell function is directly related to the inhibitory action of rapamycin on mammalian target of rapamycin. Skin gammadelta T cells treated with rapamycin are refractory to IL-2 stimulation and attempt to survive in the absence of cytokine and growth factor signaling by undergoing autophagy. Normal wound closure can be restored in rapamycin-treated mice by addition of the skin gammadelta T cell-produced factor, insulin-like growth factor-1. These studies not only reveal that mammalian target of rapamycin is a master regulator of gammadelta T cell function but also provide a novel mechanism for the increased susceptibility to nonhealing wounds that occurs during rapamycin administration.

Topics: Animals; Cell Line; Disease Models, Animal; Epidermis; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Culture Techniques; Protein Kinases; Receptors, Antigen, T-Cell, gamma-delta; Sirolimus; Skin; T-Lymphocyte Subsets; Time Factors; TOR Serine-Threonine Kinases; Wound Healing

Vascular healing of intracoronary stents has been shown to be delayed in drug-eluting stents (DES) due to the cytotoxic compounds on the stent surface that prevent stent ingrowth and endothelialization. The lack of endothelialization explains the occurrence of late and very late stent thrombosis in DES.. In 11 house swines (body weight 38-45 kg), 3 stents were implanted randomly into the 3 large epicardial coronary arteries, namely a bare-metal stent (BMS), a sirolimus-eluting stent with slow-release (SES) and a SES with extended-release (SESXR). Stent length was 18 mm, and stent diameter 3 mm. All stents were of identical design. Animals were followed for 3 (n = 3), 7 (n = 4) and 14 (n = 4) days, respectively. One animal died before implantation due to hyperthermia. On the day of explantation, the animals were euthanized and endothelialization was tested by scanning electron microscopy after drying and sputtering the samples. Endothelial coverage was determined semiquantitatively by 2 observers.. Endothelialization was more rapid with BMS and SESXR than SES at 3 and 14 days. At 7 days there were no significant differences between the 2 SES.. Endothelialization of intracoronary stents is faster with BMS and SESXR at 3 days than with SES. These differences persist at 14 days, suggesting delayed vascular healing with the slow-release SES.

Topics: Animals; Coronary Restenosis; Coronary Vessels; Delayed-Action Preparations; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Immunosuppressive Agents; Metals; Sirolimus; Stents; Swine; Wound Healing

Huntington's disease (HD) is caused by a polyglutamine expansion mutation in the huntingtin protein that confers a toxic gain-of-function and causes the protein to become aggregate-prone. Aggregate-prone proteins are cleared by macroautophagy, and upregulating this process by rapamycin, which inhibits the mammalian target of rapamycin (mTOR), attenuates their toxicity in various HD models. Recently, we demonstrated that lithium induces mTOR-independent autophagy by inhibiting inositol monophosphatase (IMPase) and reducing inositol and IP3 levels. Here we show that glycogen synthase kinase-3beta (GSK-3beta), another enzyme inhibited by lithium, has opposite effects. In contrast to IMPase inhibition that enhances autophagy, GSK3beta inhibition attenuates autophagy and mutant huntingtin clearance by activating mTOR. In order to counteract the autophagy inhibitory effects of mTOR activation resulting from lithium treatment, we have used the mTOR inhibitor rapamycin in combination with lithium. This combination enhances macroautophagy by mTOR-independent (IMPase inhibition by lithium) and mTOR-dependent (mTOR inhibition by rapamycin) pathways. We provide proof-of-principle for this rational combination treatment approach in vivo by showing greater protection against neurodegeneration in an HD fly model with TOR inhibition and lithium, or in HD flies treated with rapamycin and lithium, compared with either pathway alone.

Topics: Animals; Autophagy; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Drosophila; Drosophila Proteins; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Huntington Disease; Inositol; Lithium Compounds; Male; Mice; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

The present study was conducted to determine the magnitude and duration of ribosomal protein translation in response to pressure overload and determine if additional, paracrine events associated with mechanical transduction, such as integrin activation using a bioactive peptide ligand, RGD or endothelin stimulation lead to ribosomal protein translation. Polysome analysis of ventricular tissue samples obtained from an in vivo model of right-ventricular pressure overload (RVPO) showed a significant shift in the proportion of a 5'-terminal oligopyrimidine (5'-TOP) mRNA, rpL32, associated with the polysomal fraction when compared with non-5'-TOP mRNAs, beta-actin and beta-myosin heavy chain (beta-MHC), in the early stages of the hypertrophic response (24-48 h). Furthermore, this increase in polysome-bound rpL32 mRNA was accompanied by the phosphorylation of mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and S6 ribosomal protein. In our in vitro studies, treatment of primary cultures of adult feline cardiomyocytes (cardiocytes) with 100 nM endothelin, 9 mM RGD, 100 nM insulin, or 100 nM TPA activated mTOR via distinct signaling pathways and resulted in an increased proportion of polysome-bound rpL32 mRNA. Pre-treatment of cardiocytes with the mTOR inhibitor rapamycin blocked the agonist-induced rpL32 mRNA mobilization to polysomes. These results show that mechanisms that regulate ribosomal biogenesis in the myocardium are dynamically sensitive to pressure overload. Furthermore, our in vitro studies indicate that distinct pathways are operational during the early course of hypertrophic growth and converge to activate mTOR resulting in the translational activation of 5'-TOP mRNA.

Topics: Actins; Analysis of Variance; Animals; Blotting, Western; Cats; Cells, Cultured; Disease Models, Animal; Endothelins; Hypertrophy, Right Ventricular; Insulin; Myocytes, Cardiac; Myosin Heavy Chains; Nonmuscle Myosin Type IIB; Oligopeptides; Phosphorylation; Polyribosomes; Protein Biosynthesis; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6; Ribosomal Protein S6 Kinases; RNA 5' Terminal Oligopyrimidine Sequence; RNA, Messenger; Signal Transduction; Sirolimus; Tetradecanoylphorbol Acetate; TOR Serine-Threonine Kinases

Despite excellent clinical results for sirolimus (rapamycin)-eluting stents, the exact mechanisms of antirestenotic activity and affected cellular targets are incompletely understood. Therefore, we determined the presence and tem- porospatial expression pattern of FKBP12, the primary intracellular receptor of rapamycin, in rat carotid arteries after balloon injury, as well as in human in-stent restenosis and primary stable coronary atheroma. FKBP12 expression was assessed by immunohistochemistry. Rat carotid arteries revealed maximal expression in 57.7 +/- 4.0% of neointimal cells at day 7. A large proportion of these FKBP12+ cells showed luminally confined co-expression with dendritic cell markers. Despite a considerably thicker neointima at day 28, presence of FKBP12 decreased (8.5 +/- 1.9%, p = 0.02) with a scattered pattern in luminal and deep neointima. Likewise, human in-stent restenosis atherectomy specimens (time after stent implantation 2-12 months) revealed a comparable extent of cellular rapamycin receptor expression (9.3 +/- 1.0%) that significantly differed from that found in primary stable atheroma (1.3 +/- 0.4%, p < 0.001). In conclusion, the rapamycin receptor is predominantly present during early neointima formation, while mature neointimal atheromas show a relatively low expression without confinement to luminal areas. Co-expression of FKBP12 and dendritic cell markers suggests that dendritic cells may be another important target for early and long-term rapamycin effects.

Topics: Aged; Animals; Atherectomy, Coronary; Cardiovascular Agents; Carotid Artery Injuries; Catheterization; Coronary Restenosis; Coronary Stenosis; Disease Models, Animal; Drug-Eluting Stents; Female; Humans; Male; Middle Aged; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus Binding Protein 1A; Time Factors; Tunica Intima

Remodelling of matrix and tubular basement membranes (TBM) is a characteristic of polycystic kidney disease. We hypothesized that matrix and TBM degradation by metalloproteinases (MMPs) could promote cyst formation. We therefore investigated the renal expression of MMPs in the Han:SPRD rat model of autosomal dominant polycystic kidney disease (ADPKD) and examined the effect of sirolimus treatment on MMPs.. 5-week-old male heterozygous (Cy/+) and wild-type normal (+/+) rats were treated with sirolimus (2 mg/kg/day) through drinking water for 3 months.. The mRNA and protein levels of MMP-2 and MMP-14 were markedly increased in the kidneys of heterozygous Cy/+ animals compared to wild-type +/+ as shown by RT-PCR and Western blot analyses for MMP-2 and MMP-14, and by zymography for MMP-2. Strong MMP-2 expression was detected by immunoperoxidase staining in cystic epithelial cells that also displayed an altered, thickened TBM. Tissue inhibitor of metalloproteinases-2 (TIMP-2) expression was not changed in Cy/+ kidneys. Sirolimus treatment leads to decreased protein expression of MMP-2 and MMP-14 in Cy/+, whereas MMP-2 and MMP-14 mRNA levels and TIMP-2 protein levels were not affected by sirolimus.. In summary, in kidneys of the Han:SPRD rat model of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with a marked improvement of MMP-2 and MMP-14 overexpression, and this correlated also with less matrix and TBM alterations and milder cystic disease.

Topics: Animals; Basement Membrane; Disease Models, Animal; Extracellular Matrix; Immunosuppressive Agents; Kidney; Male; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Oligonucleotide Array Sequence Analysis; Polycystic Kidney Diseases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; Tissue Inhibitor of Metalloproteinase-2

Studies in animals and patients indicate that rapamycin affects vasodilatation differently in outer and inner curvatures of blood vessels. We evaluated in this study whether rapamycin affects endothelial nitric oxide synthase (eNOS) responsiveness to shear stress under normo- and hypercholesteraemic conditions to explain these findings.. Shear stress levels were varied over a large range of values in carotid arteries of transgenic mice expressing human eNOS fused to enhanced green fluorescence protein. The mice were divided into control, low-dose rapamycin (3 microg/kg/day), and high-dose rapamycin (3 mg/kg/day) groups and into normocholesteraemic and hypercholesteraemic (ApoE-/- on high cholesterol diet for 3-4 weeks) groups. The effect of rapamycin treatment on eNOS was evaluated by quantification of eNOS expression and of intracellular protein levels by en face confocal microscopy. A sigmoid curve fit was used to described these data. The efficacy of treatment was confirmed by measurement of rapamycin serum levels (2.0 +/- 0.5 ng/mL), and of p27kip1 expression in vascular tissue (increased by 2.4 +/- 0.5-fold). In control carotid arteries, eNOS expression increased by 1.8 +/- 0.3-fold in response to rapamycin. In the treated vessels, rapamycin reduced maximal eNOS expression at high shear stress levels (>5 Pa) in a dose-dependent way and shifted the sigmoid curve to the right. Hypercholesteraemia had a tendency to increase the leftward shift and the reduction in maximal eNOS expression (P = 0.07).. Rapamycin is associated with high eNOS in low shear regions, i.e. in atherogenic regions, protecting these regions against atherosclerosis, and is associated with a reduction of eNOS at high shear stress affecting vasomotion in these regions.

Topics: Animals; Apolipoproteins E; Cardiovascular Agents; Carotid Arteries; Carotid Artery Diseases; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Green Fluorescent Proteins; Humans; Hypercholesterolemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Nitric Oxide Synthase Type III; Pulsatile Flow; Recombinant Fusion Proteins; Sirolimus; Stress, Mechanical

Treatment options in human mesangioproliferative glomerulonephritis/sclerosis, mostly IgA nephropathy, are limited. Progressive mesangioproliferative nephropathy represents a major cause of end-stage kidney disease. The present study explores the efficacy of low-dose mTOR inhibition by rapamycin in a chronic-progressive model of mesangioproliferative glomerulosclerosis (cGS). cGS was induced by high-dose anti-thy1 antibody injection into uninephrectomized rats. Rapamycin administration (2.5 mg.kg(-1).body wt(-1)) was started 10 days after antibody injection and continued until week 20. cGS was characterized by advancing proteinuria, increased blood pressure, marked tubulointerstitial and glomerular fibrosis, cell proliferation and round cell infiltration, and impaired renal function. Kruskal-Wallis and Mann-Whitney U-tests were used for statistical analysis. The course of chronic anti-thy1-induced glomerulosclerosis was significantly attenuated by low-dose rapamycin treatment. In week 20, this was demonstrated by improvements in proteinuria (-38%), systolic blood pressure (-16 mmHg), tubulointerstitial and glomerular histological matrix accumulation (-61 and -24%), transforming growth factor-beta1 overexpression (-41 and -47%), collagen I deposition (-53 and -65%), cell proliferation (-90 and -76%), and leukocyte number (macrophages -52 and -53%; lymphocytes -58 and 51%), respectively. Rapamycin improved renal function as well (blood creatinine -0.68 mg/dl, urea -66.7 mg/day, and creatinine clearance +0.13 ml.min(-1).100 g body wt(-1)). In conclusion, low-dose mTOR inhibition by rapamycin limits the progressive course of anti-thy1-induced renal disease toward chronic glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency. Renoprotection by rapamycin involved significant beneficial effects on multiple key pathways in the progression of chronic renal disease, i.e., proteinuria, extracellular matrix accumulation, renal cell proliferation, and inflammatory cell infiltration.

Topics: Animals; Blood Pressure; Body Weight; Cell Movement; Cell Proliferation; Collagen Type I; Creatinine; Disease Models, Animal; Extracellular Matrix Proteins; Fibrosis; Glomerular Filtration Rate; Glomerulonephritis, Membranoproliferative; Isoantibodies; Kidney; Kidney Function Tests; Leukocytes, Mononuclear; Lipids; Male; Protein Kinases; Rats; Rats, Wistar; Sirolimus; Tissue Inhibitor of Metalloproteinase-1; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Urea

Inactivating mutations in NF1 underlie the prevalent familial cancer syndrome neurofibromatosis type 1 [1]. The NF1-encoded protein is a Ras GTPase-activating protein (RasGAP) [2]. Accordingly, Ras is aberrantly activated in NF1-deficient tumors; however, it is unknown which effector pathways critically function in tumor development. Here we provide in vivo evidence that TORC1/mTOR activity is essential for tumorigenesis. Specifically, we show that the mTOR inhibitor rapamycin potently suppresses the growth of aggressive NF1-associated malignancies in a genetically engineered murine model. However, in these tumors rapamycin does not function via mechanisms generally assumed to mediate tumor suppression, including inhibition of HIF-1alpha and indirect suppression of AKT, but does suppress the mTOR target Cyclin D1 [3]. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for this commonly untreatable malignancy. Moreover, they indicate that mTOR inhibitors do not suppress all tumor types via the same mechanism, suggesting that current biomarkers that rely on HIF-1alpha suppression may not be informative for all cancers. Finally, our results reveal important differences between the effects of mTOR inhibition on the microvasculature in genetically engineered versus xenograft models and indicate that the former may be required for effective preclinical screening with this class of inhibitors.

Topics: Animals; Cell Line; Cyclin D; Cyclins; Disease Models, Animal; Gene Expression Regulation; Genes, Neurofibromatosis 1; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Mammalian target of rapamycin (mTOR) and its downstream target S6 kinase 1 (S6K1) mediate nutrient-induced insulin resistance by downregulating insulin receptor substrate proteins with subsequent reduced Akt phosphorylation. Therefore, mTOR/S6K1 inhibition could become a therapeutic strategy in insulin-resistant states, including type 2 diabetes. We tested this hypothesis in the Psammomys obesus (P. obesus) model of nutrition-dependent type 2 diabetes, using the mTOR inhibitor rapamycin.. Normoglycemic and diabetic P. obesus were treated with 0.2 mg x kg(-1) x day(-1) i.p. rapamycin or vehicle, and the effects on insulin signaling in muscle, liver and islets, and on different metabolic parameters were analyzed.. Unexpectedly, rapamycin worsened hyperglycemia in diabetic P. obesus without affecting glycemia in normoglycemic controls. There was a 10-fold increase of serum insulin in diabetic P. obesus compared with controls; rapamycin completely abolished this increase. This was accompanied by weight loss and a robust increase of serum lipids and ketone bodies. Rapamycin decreased muscle insulin sensitivity paralleled by increased glycogen synthase kinase 3beta activity. In diabetic animals, rapamycin reduced beta-cell mass by 50% through increased apoptosis. Rapamycin increased the stress-responsive c-Jun NH(2)-terminal kinase pathway in muscle and islets, which could account for its effect on insulin resistance and beta-cell apoptosis. Moreover, glucose-stimulated insulin secretion and biosynthesis were impaired in islets treated with rapamycin.. Rapamycin induces fulminant diabetes by increasing insulin resistance and reducing beta-cell function and mass. These findings emphasize the essential role of mTOR/S6K1 in orchestrating beta-cell adaptation to hyperglycemia in type 2 diabetes. It is likely that treatments based on mTOR inhibition will cause exacerbation of diabetes.

Topics: Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Gerbillinae; Insulin; Insulin Secretion; Insulin-Secreting Cells; Protein Kinases; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases

Patients with Birt-Hogg-Dubé (BHD) syndrome harbor germline mutations in the BHD tumor suppressor gene that are associated with an increased risk for kidney cancer. BHD encodes folliculin, a protein that may interact with the energy- and nutrient-sensing 5'-AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathways.. We used recombineering methods to generate mice with a conditional BHD allele and introduced the cadherin 16 (KSP)-Cre transgene to target BHD inactivation to the kidney. Kidney cell proliferation was measured by BrdU incorporation and phospho-histone H3 staining. Kidney weight data were analyzed with Wilcoxon's rank-sum, Student's t, and Welch's t tests. Hematoxylin and eosin staining and immunoblot analysis and immunohistochemistry of cell cycle and signaling proteins were performed on mouse kidney cells and tissues. BHD knockout mice and kidney cells isolated from BHD knockout and control mice were treated with the mTOR inhibitor rapamycin. Mouse survival was evaluated by Kaplan-Meier analyses. All statistical tests were two-sided.. BHD knockout mice developed enlarged polycystic kidneys and died from renal failure by 3 weeks of age. Targeted BHD knockout led to the activation of Raf-extracellular signal-regulated protein kinase (Erk)1/2 and Akt-mTOR pathways in the kidneys and increased expression of cell cycle proteins and cell proliferation. Rapamycin-treated BHD knockout mice had smaller kidneys than buffer-treated BHD knockout mice had (n = 4-6 mice per group, relative kidney/body weight ratios, mean = 4.64% vs 12.2%, difference = 7.6%, 95% confidence interval = 5.2% to 10.0%; P < .001) and longer median survival time (n = 4-5 mice per group, 41.5 vs 23 days; P = .0065 ).. Homozygous loss of BHD may initiate renal tumorigenesis in the mouse. The conditional BHD knockout mouse may be a useful research model for dissecting multistep kidney carcinogenesis, and rapamycin may be considered as a potential treatment for Birt-Hogg-Dubé syndrome.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Southern; Cell Proliferation; Disease Models, Animal; Estrone; Fluorescent Antibody Technique; Gene Silencing; Genotype; Germ-Line Mutation; Immunoblotting; Immunohistochemistry; Kaplan-Meier Estimate; Kidney; Kidney Neoplasms; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Polycystic Kidney Diseases; Protein Kinases; Proto-Oncogene Proteins; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Syndrome; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Skin manifestations of Tuberous Sclerosis Complex (TSC) cause significant morbidity. The molecular mechanism underlying TSC is understood and there is evidence that systemic treatment with rapamycin or other mTOR inhibitors may be a useful approach to targeted therapy for the kidney and brain manifestations. Here we investigate topical rapamycin in a mouse model for TSC-related tumors.. 0.4% and 0.8% rapamycin ointments were applied to nude mice bearing subcutaneous, TSC-related tumors. Topical treatments were compared with injected rapamycin and topical vehicle. Rapamycin levels in blood and tumors were measured to assess systemic drug levels in all cohorts.. Treatment with topical rapamycin improved survival and reduced tumor growth. Topical rapamycin treatment resulted in systemic drug levels within the known therapeutic range and was not as effective as injected rapamycin.. Topical rapamycin inhibits TSC-related tumor growth. These findings could lead to a novel treatment approach for facial angiofibromas and other TSC skin lesions.

Topics: Administration, Cutaneous; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Disease Models, Animal; Enzyme Inhibitors; Mice; Mice, Nude; Neoplasm Transplantation; Protein Kinases; Sirolimus; Skin Absorption; Soft Tissue Neoplasms; Survival Analysis; TOR Serine-Threonine Kinases; Tuberous Sclerosis

This study evaluated the inflammatory reaction at the site of overlapping drug-eluting stents (DES) in a porcine model of in-stent restenosis.. Twenty bare metal stents (BMS) (group I; n=10), 20 sirolimus-eluting stents (SES) (group II: n=10), 20 paclitaxel-eluting stent (PES) (group III: n=10), and 10 PES and 10 SES (group IV: n=10) were overlapped in the left anterior descending coronary arteries of 40 pigs. Follow-up coronary angiography and histopathology were performed at 4 weeks after stenting. For the overlapped segments, the minimal luminal diameter at 4 weeks was smaller in group I than in the other groups (1.78+/-0.13 mm, 2.79+/-0.09 mm, 2.90+/-0.04 mm, 2.80+/-0.07 mm, respectively; p<0.001), and the neointimal area (5.51+/-0.58 mm2, 2.38+/-0.53 mm2, 2.07+/-0.37 mm2, 2.39+/-0.58 mm2, respectively; p<0.001) and area stenosis (68.74+/-4.02%, 27.79+/-4.73%, 23.66+/-3.24%, 27.63+/-4.07%, respectively; p<0.001) were higher in group I than in the other groups; however, the inflammatory score was higher in group III than in the other groups (1.80+/-0.42, 2.10+/-0.32, 2.90+/-0.31, 2.50+/-0.52, respectively; p<0.001) and the endothelization score was lower in group III than in the other groups (2.80+/-0.42, 2.30+/-0.67, 1.30+/-0.48, 2.10+/-0.74, respectively; p<0.001).. Compared with BMS, DES inhibit neointimal hyperplasia, but inflammation and poor endothelization occur at the site of overlapping stents.

Topics: Animals; Cell Proliferation; Coronary Restenosis; Coronary Stenosis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Endothelium, Vascular; Female; Hyperplasia; Inflammation; Paclitaxel; Sirolimus; Stents; Swine; Tunica Intima

Mouse models of human cancers afford unique opportunities to evaluate novel therapies in preclinical trials. For this purpose, we analyzed three genetically engineered mouse (GEM) models of low-grade glioma resulting from either inactivation of the neurofibromatosis-1 (Nf1) tumor suppressor gene or constitutive activation of KRas in glial cells. Based on tumor proliferation, location, and penetrance, we selected one of these Nf1 GEM models for preclinical drug evaluation. After detection of an optic glioma by manganese-enhanced magnetic resonance imaging, we randomized mice to either treatment or control groups. We first validated the Nf1 optic glioma model using conventional single-agent chemotherapy (temozolomide) currently used for children with low-grade glioma and showed that treatment resulted in decreased proliferation and increased apoptosis of tumor cells in vivo as well as reduced tumor volume. Because neurofibromin negatively regulates mammalian target of rapamycin (mTOR) signaling, we showed that pharmacologic mTOR inhibition in vivo led to decreased tumor cell proliferation in a dose-dependent fashion associated with a decrease in tumor volume. Interestingly, no additive effect of combined rapamycin and temozolomide treatment was observed. Lastly, to determine the effect of these therapies on the normal brain, we showed that treatments that affect tumor cell proliferation or apoptosis did not have a significant effect on the proliferation of progenitor cells within brain germinal zones. Collectively, these findings suggest that this Nf1 optic glioma model may be a potential preclinical benchmark for identifying novel therapies that have a high likelihood of success in human clinical trials.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Cell Proliferation; Dacarbazine; Disease Models, Animal; Eye Neoplasms; Magnetic Resonance Imaging; Mice; Mice, Transgenic; Neurofibromin 1; Optic Nerve Glioma; Protein Kinases; Signal Transduction; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases

Tuberous sclerosis complex (TSC) represents one of the most common genetic causes of epilepsy. TSC gene inactivation leads to hyperactivation of the mammalian target of rapamycin signaling pathway, raising the intriguing possibility that mammalian target of rapamycin inhibitors might be effective in preventing or treating epilepsy in patients with TSC. Mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1(GFAP)CKO mice) develop glial proliferation, progressive epilepsy, and premature death. Here, we tested whether rapamycin could prevent or reverse epilepsy, as well as other cellular and molecular brain abnormalities in Tsc1(GFAP)CKO mice.. Tsc1(GFAP)CKO mice and littermate control animals were treated with rapamycin or vehicle starting at postnatal day 14 (early treatment) or 6 weeks of age (late treatment), corresponding to times before and after onset of neurological abnormalities in Tsc1(GFAP)CKO mice. Mice were monitored for seizures by serial video-electroencephalogram and for long-term survival. Brains were examined histologically for astrogliosis and neuronal organization. Expression of phospho-S6 and other molecular markers correlating with epileptogenesis was measured by Western blotting.. Early treatment with rapamycin prevented the development of epilepsy and premature death observed in vehicle-treated Tsc1(GFAP)CKO mice. Late treatment with rapamycin suppressed seizures and prolonged survival in Tsc1(GFAP)CKO mice that had already developed epilepsy. Correspondingly, rapamycin inhibited the abnormal activation of the mammalian target of rapamycin pathway, astrogliosis, and neuronal disorganization, and increased brain size in Tsc1(GFAP)CKO mice.. Rapamycin has strong efficacy for preventing seizures and prolonging survival in Tsc1(GFAP)CKO mice.

Topics: Animals; Disease Models, Animal; Epilepsy; Mice; Mice, Knockout; Sirolimus; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Rapamycin is a potent immunosuppressive drug currently used mainly for rejection prophylaxis in renal transplantation. The aim of this study was to determine the effect of rapamycin treatment on the development of nephritis in lupus-prone New Zealand Black/White F1 (NZB/W F1) mice. Twelve-week-old female NZB/W F1 mice were treated with rapamycin (3 mg/kg body weight) or saline once daily by oral gavage for 20 weeks. The severity of nephritis was assessed by clinical and biochemical parameters, renal histology, immunohistochemistry and gene expression studies. Rapamycin treatment markedly reduced proteinuria, improved renal function, decreased serum anti-double stranded DNA antibody levels and diminished splenomegaly. Kidney sections from saline-treated mice showed marked mesangial proliferation, tubular dilation with protein cast deposition and interstitial inflammatory cell infiltration. Rapamycin-treated mice had near normal renal histology, with marked reduction in glomerular immune deposition and the infiltration by T cells, B cells and macrophages. Rapamycin treatment was associated with down-regulation of intra-renal expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein. We conclude that rapamycin is highly effective in preventing the development of nephritis in NZB/W F1 mice. The beneficial effects of rapamycin are mediated through inhibition of lymphoproliferation and reduced MCP-1 expression.

Topics: Administration, Oral; Animals; Antibodies, Antinuclear; Cell Proliferation; Chemokine CCL2; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gene Expression; Immunohistochemistry; Immunosuppressive Agents; Kidney Glomerulus; Lupus Nephritis; Mice; Mice, Inbred NZB; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; T-Lymphocytes

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.

Topics: Animals; Autophagy; Caspase 3; Caspase Inhibitors; Cell Line; Cell Line, Tumor; Disease Models, Animal; Everolimus; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Protein Kinase Inhibitors; Protein Kinases; Radiation-Sensitizing Agents; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation; Xenograft Model Antitumor Assays

The immunosuppressive drug rapamycin (RAPA) prevents rejection in organ transplantation by inhibiting interleukin-2-stimulated T-cell division. Rapamycin has also been suggested to possess strong anti-angiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF). Angiogenesis and VEGF are thought to play a crucial role in fracture healing and as osteoporotic and traumatic fractures are common complications in immunosuppressed, organ transplantation patients, we conducted this study to analyze the effect of rapamycin treatment on bone repair.. We investigated the effect of rapamycin treatment on bone repair in a murine closed femur fracture model using radiological, histomorphometric, immunohistochemical, biomechanical and protein biochemical analyses.. X-ray analyses demonstrated that rapamycin treatment inhibits callus formation after two weeks of fracture healing. The radiologically observed lack of callus formation was confirmed by histomorphometric analyses, which revealed a significantly diminished callus size and a reduced amount of bone formation when compared with vehicle-treated controls. Biomechanical testing further demonstrated that rapamycin significantly reduces torsional stiffness of the callus. Interestingly, this effect was associated with decreased vessel formation; a diminished proliferation of osteoblasts, endothelial cells and periosteal cells; and a reduced VEGF expression in hypertrophic chondrocytes. After five weeks treatment, however, the negative impact of rapamycin on fracture healing was overcome.. Thus, rapamycin initially delays fracture healing, most probably by inhibiting cell proliferation and neovascularization in the callus. These undesirable effects should be considered when rapamycin is administered to patients sustaining bone fractures.

Topics: Angiogenesis Inhibitors; Animals; Biomechanical Phenomena; Blotting, Western; Bony Callus; Cell Proliferation; Chondrocytes; Disease Models, Animal; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Femoral Fractures; Fracture Healing; Immunohistochemistry; Immunosuppressive Agents; Mice; Neovascularization, Physiologic; Osteoblasts; Osteogenesis; Sirolimus; Time Factors

Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC.. Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting.. Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation.. Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aorta; Apoptosis; Carcinoma, Hepatocellular; Cells, Cultured; Disease Models, Animal; Doxorubicin; Endothelial Cells; Intracellular Signaling Peptides and Proteins; Liver Neoplasms, Experimental; Male; Neoplasm Transplantation; Oncogene Protein p21(ras); Phosphorylation; Polyethylene Glycols; Protein Serine-Threonine Kinases; Rats; Rats, Inbred ACI; Sirolimus; Transcription Factors

Hepatocellular carcinoma is a leading cause of global cancer mortality, with standard chemotherapy being minimally effective in prolonging survival. We investigated if combined targeting of vascular endothelial growth factor protein and expression might affect hepatocellular carcinoma growth and angiogenesis.. We treated patient-derived hepatocellular carcinoma xenografts with (i) bevacizumab; (ii) rapamycin; and (iii) bevacizumab plus rapamycin. Western blotting was employed to determine changes in the proteins. Apoptosis, vascular endothelial growth factor expression, microvessel density, and cell proliferation were analyzed by immunohistochemistry.. Hepatocellular carcinoma growth was inhibited by bevacizumab plus rapamycin treatment to a significantly greater degree than bevacizumab or rapamycin monotherapy. Reductions in tumor growth by bevacizumab plus rapamycin were associated with inhibition of downstream targets of the mammalian target-of-rapamycin pathway, reductions in vascular endothelial growth factor expression, and tumor microvessel density. Potentially additive effects of bevacizumab plus rapamycin included reductions in vascular endothelial growth factor expression, cyclin D1, and cyclin B1. In an intra-peritoneal model of hepatocellular carcinoma, bevacizumab plus rapamycin potently inhibited both intra-liver and intra-abdominal tumor growth, reduced ascites levels, and significantly prolonged mouse survival.. Bevacizumab and rapamycin, which are both clinically approved drugs, may represent a novel molecularly-targeted combination treatment for hepatocellular carcinoma.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Carcinoma, Hepatocellular; Disease Models, Animal; Drug Therapy, Combination; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, SCID; Multiprotein Complexes; Proteins; Signal Transduction; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Transcription Factors; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Tuberous sclerosis (TSC) is a hamartoma syndrome attributable to mutations in either TSC1 or TSC2 in which brain involvement causes epilepsy, mental retardation, and autism. We have reported recently (Meikle et al., 2007) a mouse neuronal model of TSC in which Tsc1 is ablated in most neurons during cortical development. We have tested rapamycin and RAD001 [40-O-(2-hydroxyethyl)-rapamycin], both mammalian target of rapamycin mTORC1 inhibitors, as potential therapeutic agents in this model. Median survival is improved from 33 d to more than 100 d; behavior, phenotype, and weight gain are all also markedly improved. There is brain penetration of both drugs, with accumulation over time with repetitive treatment, and effective reduction of levels of phospho-S6, a downstream target of mTORC1. In addition, there is restoration of phospho-Akt and phospho-glycogen synthase kinase 3 levels in the treated mice, consistent with restoration of Akt function. Neurofilament abnormalities, myelination, and cell enlargement are all improved by the treatment. However, dysplastic neuronal features persist, and there are only modest changes in dendritic spine density and length. Strikingly, mice treated with rapamycin or RAD001 for 23 d only (postnatal days 7-30) displayed a persistent improvement in phenotype, with median survival of 78 d. In summary, rapamycin/RAD001 are highly effective therapies for this neuronal model of TSC, with benefit apparently attributable to effects on mTORC1 and Akt signaling and, consequently, cell size and myelination. Although caution is appropriate, the results suggest the possibility that rapamycin/RAD001 may have benefit in the treatment of TSC brain disease, including infantile spasms.

Topics: Age Factors; Animals; Animals, Newborn; Brain; Cell Size; Dendrites; Disease Models, Animal; Everolimus; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neurons; Oncogene Protein v-akt; Signal Transduction; Sirolimus; Time Factors; Transcription Factors; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

The purpose of this study was to compare the effects of granulocyte-colony stimulating factor (G-CSF) on in-stent restenosis (ISR) in bare and sirolimus-eluting stents (SES) in a porcine myocardial infarction model.. Using G-CSF to mobilize stem cells has shown promise in infarcted heart. However, G-CSF may aggravate ISR and an aggressive strategy to prevent ISR is needed.. Bare stents and SES were implanted in coronary arteries (Group I, bare stents; Group II, bare stents with G-CSF; Group III, SES; Group IV, SES with G-CSF, n=10 in each group) 72 h after experimental myocardial infarction (MI). G-CSF (10 microg/kg/day) was injected for 7 days from 24 h after stent implantation.. In coronary angiographic and histomorphometric analysis, percent area stenosis was significantly increased in Group II compared with that in Group I at 28 days (P<0.05). The ratio of inflammatory cells in the neointima was higher in Group II (P<0.05). No significant differences were observed between Group III and IV. In Group II, phosphorylated signal transducers and activators of transcription (STAT)-3, STAT-3, and vascular endothelial growth factor (VEGF) showed increased neointimal expression. In porcine aortic smooth muscle cells (PASMC), G-CSF increased the growth rate, migration, STAT-3 phosphorylation, and VEGF, which were suppressed by rapamycin and AG490, a STAT-3 inhibitor.. STAT-3 and VEGF are important in the development of enhanced ISR by G-CSF in bare stents. SES could be a good strategy to prevent the G-CSF-stimulated proliferation and migration of smooth muscle cells, which could be responsible for neointimal hyperplasia.

Topics: Analysis of Variance; Angioplasty, Balloon, Coronary; Animals; Blood Chemical Analysis; Cell Proliferation; Coronary Angiography; Coronary Restenosis; Disease Models, Animal; Female; Granulocyte Colony-Stimulating Factor; Immunohistochemistry; Myocardial Infarction; Probability; Random Allocation; Sensitivity and Specificity; Sirolimus; Stents; Sus scrofa

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol-3-kinase and hypoxia-inducible factor-1alpha (HIF-1alpha). Since mTOR is an upstream regulator of HIF-1alpha, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho-mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E-BP-1 and HIF-1alpha activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra-peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal-skin-fold chamber model. Immunohistochemical analyses showed strong expression of phospho-mTOR in 60% of intestinal- and 64% of diffuse-type human gastric adenocarcinomas. In vitro, rapamycin-treatment effectively blocked S6K1, 4E-BP-1 and HIF-1alpha activation, and significantly impaired tumor cell migration. In vivo, rapamycin-treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31-positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal-skin-fold chamber model, rapamycin-treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy.

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intestinal Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stomach Neoplasms; Survival Rate; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Regulated gene expression may be required for the clinical development of certain gene therapies. Several approaches have been developed that allow pharmacologic control of transgene expression, including the dimerizer-regulated transcriptional system in which rapamycin or its analogs function as transcriptional inducers. These compounds can also act as direct antitumor agents via inhibition of mammalian target of rapamycin (mTOR). We describe the development of an optimized recombinant adeno-associated virus (AAV) expression cassette that allows dimerizer-regulated gene expression from a single vector in vitro and in vivo. After demonstrating multiple cycles of rapamycin-dependent transgene induction following a single administration of an AAV vector in vivo, application of this regulated AAV gene expression system to the pharmacologic control of antiangiogenic therapy was evaluated in preclinical tumor models. Dimerizer-regulated vectors were constructed encoding a soluble inhibitor of the vascular endothelial growth factor (VEGF) pathway. In two subcutaneous models of glioblastoma, regulated expression of the VEGF inhibitor via recombinant AAV-mediated gene transfer, in combination with rapamycin, was shown to decrease tumor growth rate significantly. The dual properties of rapamycin--as a transcriptional inducer and mTOR inhibitor--are exploited in combination with an AAV-encoded antiangiogenic agent to provide a novel approach for the treatment of malignant diseases.

Topics: Angiostatins; Animals; Blotting, Western; Cell Line; Cell Line, Tumor; Dependovirus; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glioblastoma; Humans; Immunosuppressive Agents; Mice; Mice, Nude; Neoplasms; Prealbumin; Promoter Regions, Genetic; Receptors, Vascular Endothelial Growth Factor; Sirolimus; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

Topics: Administration, Oral; Animals; Antibodies, Antinuclear; Apoptosis; Autoantigens; Cell Movement; Chromatin; Complement C3; Complement C3 Nephritic Factor; Cyclophosphamide; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Female; Fingolimod Hydrochloride; Glomerular Mesangium; Immunoglobulin G; Immunosuppressive Agents; Injections, Intraperitoneal; Kidney Glomerulus; Lupus Nephritis; Lymphocytes; Mice; Mice, Inbred NZB; Nucleosomes; Propylene Glycols; Proteinuria; Receptors, Lysosphingolipid; Sirolimus; Sphingosine

Chronic allograft nephropathy (CAN) remains the most common cause of late graft loss especially in sensitized patients. The aim of this study is to evaluate the therapeutic effect of everolimus on cellular and humoral mechanisms of chronic allograft damage in a rat model with sensitized recipients.. F344 kidneys were transplanted to LEW.RNU rats. The athymic recipients were reconstituted with 3.5 x 10(7) or 5 x 10(7) presensitized CD4+T-lymphocytes. In the treatment group, everolimus was introduced five weeks posttransplantation. Rats were monitored for peripheral blood lymphocytes, renal function, histological changes in the graft, and the development of donor-specific alloantibodies.. Rats developed cell dose-dependent renal failure. Increased urinary albumin excretion and glomerulopathy were frequently accompanied by the development of donor-specific major histocompatibility complex (MHC) alloantibodies. In the everolimus group, five of six animals survived for 20 weeks with stable serum creatinine and displayed neither acute cellular rejection nor CAN. Prolonged survival was accompanied with significantly reduced tubulointerstitial cell infiltrate in the graft. Increased urinary albumin excretion was present in all, acute tubular necrosis in five of six, and glomerular sclerosis in two grafts. MHC alloantibodies were found in four of six animals.. The used rat model offers the opportunity to study the influence of everolimus on the interaction of humoral and cellular mechanisms involved in chronic renal damage. Everolimus leads to a prolongation of allograft survival, reduced cell infiltrate in the graft, and prevents tubular atrophy and interstitial fibrosis. The development of alloantibodies and albuminuria was not prevented. These data suggest that although cellular rejection is clearly suppressed, humoral mechanisms of CAN cannot be completely controlled by everolimus treatment in the sensitized rat model.

Topics: Animals; Antibodies; Antibody Formation; Chronic Disease; Disease Models, Animal; Everolimus; Graft Rejection; Histocompatibility Antigens Class I; Immunohistochemistry; Kidney Diseases; Lymphocyte Count; Male; Rats; Sirolimus; Survival Rate; Transplantation, Homologous

Chronic hypoxia induces pulmonary arterial hypertension (PAH). Smooth muscle cell (SMC) proliferation and hypertrophy are important contributors to the remodeling that occurs in chronic hypoxic pulmonary vasculature. We hypothesized that rapamycin (RAPA), a potent cell cycle inhibitor, prevents pulmonary hypertension in chronic hypoxic mice.. Mice were held either at normoxia (N; 21% O2) or at hypobaric hypoxia (H; 0.5 atm; ~10% O2). RAPA-treated animals (3 mg/kg*d, i.p.) were compared to animals injected with vehicle alone. Proliferative activity within the pulmonary arteries was quantified by staining for Ki67 (positive nuclei/vessel) and media area was quantified by computer-aided planimetry after immune-labeling for alpha-smooth muscle actin (pixel/vessel). The ratio of right ventricle to left ventricle plus septum (RV/[LV+S]) was used to determine right ventricular hypertrophy.. Proliferative activity increased by 34% at day 4 in mice held under H (median: 0.38) compared to N (median: 0.28, p = 0.028) which was completely blocked by RAPA (median HO+RAPA: 0.23, p = 0.003). H-induced proliferation had leveled off within 3 weeks. At this time point media area had, however, increased by 53% from 91 (N) to 139 (H, p < 0.001) which was prevented by RAPA (H+RAPA: 102; p < 0.001). RV/[LV+S] ratio which had risen from 0.17 (N) to 0.26 (H, p < 0.001) was attenuated in the H+RAPA group (0.22, p = 0.041). For a therapeutic approach animals were exposed to H for 21 days followed by 21 days in H +/- RAPA. Forty two days of H resulted in a media area of 129 (N: 83) which was significantly attenuated in RAPA-treated mice (H+RAPA: 92). RV/[LV+S] ratios supported prevention of PH (N 0.13; H 0.27; H+RAPA 0.17). RAPA treatment of N mice did not influence any parameter examined.. Therapy with rapamycin may represent a new strategy for the treatment of pulmonary hypertension.

Topics: Animals; Disease Models, Animal; Female; Hypertrophy, Right Ventricular; Hypoxia; Lung; Male; Mice; Sirolimus

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3(+) regulatory T-cell (T(REG)) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for T(REG) homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on T(REG) homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4(+)CD25(+)FoxP3(+) T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25(+)FoxP3(+) T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4(+)FoxP3(+) T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4(+)FoxP3(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.

Topics: Animals; Cyclosporine; Disease Models, Animal; Forkhead Transcription Factors; Graft vs Host Disease; Homeostasis; Immune Tolerance; Immunosuppressive Agents; Interleukin-2; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; Thymus Gland

Renal fibrosis is a hallmark of end-stage renal diseases and of chronic allograft nephropathy (CAN). Rapamycin, besides its action through blockade of lymphocyte proliferation, also has antiproliferative, antiviral, and antitumor actions. Its use in clinical in patients with CAN has recently been advocated.. Our goal was to evaluate the effect of rapamycin in an established model of renal fibrosis, unilateral ureteral obstruction.. C57BL/6 mice were divided into two groups, treated or not with daily doses of rapamycin (0.2 mg/kg) beginning on day-1. The obstruction was performed as day 0. Blood and kidney tissues were collected at 1, 4, 7, and 14 days after the surgery to quantify bone morphogenic protein (BMP)-7 and transforming growth factor (TGF)-beta mRNA by real time PCR.. Daily treatment with rapamycin caused a significant reduction in serum creatinine at day 1 (0.57 +/- 0.03 vs 0.95 +/- 0.15 mg/dL, P = .002) and at day 14 (0.56 +/- 0.04 vs 0.73 +/- 0.07 mg/dL, P = .040). This profile was corroborated by histological morphometric analyses showing less fibrosis at day 14. However, rapamycin surprisingly induced an upregulation of TGF-beta at day 4 (3.05 +/- 0.46 vs 1.85 +/- 0.41, P = .006) and at day 7 (6.33 +/- 0.55 vs 4.97 +/- 0.38, P = .024) with a reduced expression by day 14 (4.03 +/- 1.07 vs 7.89 +/- 0.83, P < .001). Surprisingly, rapamycin also promoted an increment in BMP-7, completely reversing the ratio of TGF-beta to BMP-7, allowing a more protective phenotype.. Rapamycin slightly ameliorated the renal dysfunction and, at later time points, induced less fibrosis and less decrease in the TGF-beta to BMP-7 ratio.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Creatinine; Disease Models, Animal; Disease Progression; Fibrosis; Immunosuppressive Agents; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Male; Mice; Mice, Inbred C57BL; RNA, Messenger; Sirolimus; Transforming Growth Factor beta

The aim of this study was to assess the efficacy of FK778 to prevent acute and chronic allograft rejection compared with other immunosuppressive agents.. Heterotopic Brown-Norway (BN)-to-Lewis rat cardiac transplantations and heterotopic BN-to-Lewis tracheal transplantations were performed to study acute heart rejection and the development of chronic obliterative airway disease (OAD), respectively. Recipients were treated with FK778, tacrolimus, MMF, or sirolimus for 10 days (acute rejection study) or 28 days (chronic OAD study) at varying doses.. In untreated recipients, cardiac allograft survival was 6.2 +/- 0.4 days. FK778 (20 mg/kg), tacrolimus (2 or 8 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly prolonged graft survival to 17.0 +/- 2.8, 18.5 +/- 2.7, 25.0 +/- 2.5, 20.7 +/- 3.8, 14.5 +/- 2.2, and 23.2 +/- 1.5 days, respectively (P < .05). Tracheal grafts in untreated recipients showed intense infiltration and complete luminal obliteration by day 28. FK778 (20 mg/kg), tacrolimus (1 or 4 mg/kg), MMF (10 or 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly inhibited tracheal luminal obliteration (19.5% +/- 16.4%, 44.2% +/- 33.6%, 12.3% +/- 3.3%, 61.7% +/- 18.6%, 18.3% +/- 11.3%, 55.0% +/- 30.9%, and 8.5% +/- 3.5% (P < .05). All 4 high-dose groups showed similar efficacy.. When used in therapeutic doses, tacrolimus and sirolimus were more effective than FK778 to prolong cardiac allograft survival. However, with its antiproliferative effects on smooth muscle cells, its good tolerability, and its blockade of cytomegalovirus replication, FK778 proved effective to prevent chronic OAD development. Thus, FK778 may acquire an important role in maintenance therapy for the prevention of long-term fibroproliferative complications.

Topics: Acute Disease; Alkynes; Animals; Chronic Disease; Disease Models, Animal; Graft Rejection; Graft Survival; Heart Transplantation; Immunosuppressive Agents; Isoxazoles; Nitriles; Rats; Rats, Inbred BN; Rats, Inbred Lew; Sirolimus; Tacrolimus; Trachea; Transplantation, Homologous

The mammalian target of rapamycin (mTOR) is thought to play a critical role in regulating cell growth, cell cycle progression, and tumorigenesis. Because the AKT-mTOR pathway is frequently hyperactivated in ovarian cancer, we hypothesized that the mTOR inhibitor RAD001 (Everolimus) would inhibit ovarian tumorigenesis in transgenic mice that spontaneously develop ovarian carcinomas. We used TgMISIIR-TAg transgenic mice, which develop bilateral ovarian serous adenocarcinomas accompanied by ascites and peritoneal dissemination. Fifty-eight female TgMISIIR-TAg mice were treated with 5 mg/kg RAD001 or placebo twice weekly from 5 to 20 weeks of age. To monitor tumor development, mice were examined biweekly using magnetic resonance microimaging. In vivo effects of RAD001 on Akt-mTOR signaling, tumor cell proliferation, and blood vessel area were analyzed by immunohistochemistry and Western blot analysis. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by approximately 84%. In addition, ascites formation, together with peritoneal dissemination, was detected in only 21% of RAD001-treated mice compared with 74% in placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. Treatment with RAD001 diminished the expression of vascular endothelial growth factor in tumor-derived cell lines and inhibited angiogenesis in vivo. RAD001 also attenuated the expression of matrix metalloproteinase-2 and inhibited the invasiveness of tumor-derived cells. Taken together, these preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer.

Topics: Animals; Disease Models, Animal; Disease Progression; Everolimus; Female; Matrix Metalloproteinase 2; Mice; Mice, Transgenic; Neovascularization, Pathologic; Ovarian Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

A limited number of therapeutic strategies are currently available for patients with inflammatory bowel disease (IBD). In particular, the maintenance therapy after remission in Crohn's disease (CD) is not satisfactory and new approaches are needed. Interleukin-10 gene-deficient (IL-10-/-) mice, a well-characterized experimental model of CD, develop severe chronic colitis due to an aberrant Th1 immune response. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), a new immunosuppressive reagent, has been used successfully in animal models for heart, liver, lung and kidney transplantation. In the present study, we examined the efficacy of everolimus in the treatment of chronic colitis in an IL-10-/- mouse model. Everolimus was administered orally for a period of 4 weeks to IL-10-/- mice with clinical signs of colitis. The gross and histological appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. The 4-week administration of everolimus resulted in a significant decrease in the severity of colitis, together with a significant reduction in the number of CD4+ T cells in the colonic lamina propria as well as IFN-gamma production in colonic lymphocytes. Everolimus treatment of established colitis in IL-10-/- mice ameliorated the colitis, probably as a result of decreasing the number of CD4+ T cells in the colonic mucosa and an associated reduction in IFN-gamma production.

Topics: Animals; Body Weight; CD4-Positive T-Lymphocytes; Cells, Cultured; Crohn Disease; Cytokines; Disease Models, Animal; Everolimus; Female; Immunosuppressive Agents; Interferon-gamma; Interleukin-10; Intestinal Mucosa; Lymph Nodes; Lymphocyte Count; Male; Mice; Mice, Inbred C57BL; Sirolimus; Spleen; T-Lymphocyte Subsets; Treatment Outcome

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2 values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

Topics: Animals; Animals, Newborn; Binding, Competitive; Cell Proliferation; Coronary Vessels; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Hypersensitivity; Encephalomyelitis, Autoimmune, Experimental; Graft Rejection; Half-Life; Heart Transplantation; Humans; Hypersensitivity, Delayed; Immunosuppressive Agents; Inhibitory Concentration 50; Lymphocyte Culture Test, Mixed; Male; Myocytes, Smooth Muscle; Rats; Rats, Inbred BN; Rats, Inbred Lew; Rats, Sprague-Dawley; Sirolimus; T-Lymphocytes; Tacrolimus Binding Protein 1A

Is a common feature of progressive renal diseases regardless of the initiating insult To clarify the role of connective tissue growth factor (CTGF) in after (UUO) in renal interstitial fibrosis and effects of rapamycin (RAP) thereupon.. Eighteen Sprague-Dawley rats were randomly divided into 3 equal groups: unilateral ureteral obstruction (UUO) model group, undergoing ligation of the left ureter; RAP treatment group, undergoing ligation of the left ureter and intraperitoneal injection of RAP 0.04 mg.kg(-1).d(-1); and sham operation group. The right kidneys were taken out 7 and 14 days after the operation respectively to undergo renal pathological examination by Masson staining. Semi-quantitative RT-PCR was used to detect the mRNA expression of CTGF. Western blotting was performed to examine the protein expression of CTGF and fibronectin (FN).. In comparison with the sham operation group, renal interstitial fibrosis was significant more expression in the 2 UUO groups, especially the UUO model group (P < 0.01). Seven and 14 days after the operation the levels of CTGF mRNA expression of the UUO model and RAP treatment groups (both P < 0.01), and the level of CTGF mRNA expression of the RAP treatment group was significantly lower than that of the UUO model group (P < 0.01), however, there was no significant difference in the level of CTGF mRNA expression between the 2 UUO groups 14 days after the operation. Seven and 14 days after the operation the levels of CTGF protein expression of the UUO model and RAP treatment groups were both significantly higher than that of the sham operation group (both P < 0.01), and the levels of CTGF protein expression of the RAP treatment group were significantly lower than that of the UUO model group (P < 0.05 and P < 0.01). The levels of FN expression 7 and 14 days after the operation of the 2 UUO groups were both significantly higher than that of the sham operation group (both P < 0.01). and the level of FN expression 7 days after of the RAP treatment group was significantly lower than that of the UUO model group (P < 0.01), however, there was no significant difference in the level of FN expression between the 2 UUO groups 14 days after the operation.. The expression of CTGF mRNA and that of CTGF protein increase after UUO. Rapamycin play a protective role in the kidney by downregulating the CTGF expression and alleviating the renal interstitial fibrosis following UUO.

Topics: Animals; Blotting, Western; Connective Tissue Growth Factor; Disease Models, Animal; Fibrosis; Gene Expression; Immediate-Early Proteins; Immunosuppressive Agents; Injections, Intraperitoneal; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Ureteral Obstruction

FK506 Binding Protein 12 and its related isoform 12.6 (FKBP12/12.6) stabilize a closed state of intracellular Ca2+ release channels (ryanodine receptors [RyRs]), and in myocytes removal of FKBP12/12.6 from RyRs alters intracellular Ca2+ levels. The immunosuppressive drugs rapamycin and FK506 bind and displace FKBP12/12.6 from RyRs, and can also cause endothelial dysfunction and hypertension. We tested whether rapamycin and FK506 cause an intracellular Ca2+ leak in endothelial cells and whether this affects endothelial function and blood pressure regulation.. Rapamycin or FK506 concentration-dependently caused a Ca2+ leak in isolated endothelial cells, decreased aortic NO production and endothelium-dependent dilation, and increased systolic blood pressure in control mice. Rapamycin or FK506 at 10 micromol/L abolished aortic NO production and endothelium-dependent dilation. Similar results were obtained in isolated endothelial cells and aortas from FKBP12.6-/- mice after displacement of FKBP12 with 1 micromol/L rapamycin or FK506. In hypertensive FKBP12.6-/- mice, systolic blood pressures were further elevated after treatment with either rapamycin or FK506. Blockade of the Ca2+ leak with ryanodine normalized NO production and endothelium-dependent dilation.. Complete removal of FKBP12 and 12.6 from endothelial RyRs induces an intracellular Ca2+ leak which may contribute to the pathogenesis of endothelial dysfunction and hypertension caused by rapamycin or FK506.

Topics: Animals; Calcium Channels; Calcium Signaling; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Gene Deletion; Hypertension; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Nitric Oxide; Ryanodine Receptor Calcium Release Channel; Sensitivity and Specificity; Sirolimus; Tacrolimus; Tacrolimus Binding Protein 1A; Vasoconstriction; Vasodilation

Glucagon Like Peptide-1 (GLP-1), one of the most potent incretin hormones, has potential beneficial actions on the ischaemic and failing heart. This study sought to further identify the mechanisms of action of GLP-1 on the ischaemic heart using an in vitro isolated perfused rat heart model of ischaemic-reperfusion injury (measuring infarct size to area of risk (%)) subjected to 35 min regional ischaemia and 2 h reperfusion. To examine the effect of intact GLP-1 we used an inhibitor of GLP-1 breakdown, Valine pyrrolidide (VP). The downstream target of phosphatidylinositol 3-kinase includes the mTOR/p70s6 kinase pathway which was pharmacologically inhibited by rapamycin.. GLP-1 alone did not decrease myocardial infarction (54.4 +/- 3.1%). VP alone did not decrease myocardial infarction (52.5 +/- 4%). GLP-1 in the presence of VP produced significant reduction in myocardial infarction compared to control hearts (28.4 +/- 2.7% vs. 56.4 +/- 3.9% vs. P < 0.05). Inhibiting p70s6 Kinase with rapamycin completely abolished GLP-1 induced protection (57.1 +/- 4.9% vs. 28.4 +/- 2.7% P < 0.05). There was no detectable increase in the phosphorylated p70s6k after either 5 or 10 min of treatment with GLP-1/VP or with VP alone in comparison to control blots. In conclusion we show for the first time that the protective effects of GLP-1 are mediated by intact GLP-1 and can be inhibited by blocking the p70s6 kinase.

Topics: Animals; Blotting, Western; Disease Models, Animal; Enzyme Inhibitors; Glucagon-Like Peptide 1; In Vitro Techniques; Incretins; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Phosphatidylinositol 3-Kinases; Protein Kinases; Pyrroles; Random Allocation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Valine

Observational clinical studies have shown that patients with diabetes have less favorable results after percutaneous coronary intervention compared with the non-diabetic counterparts, but its mechanism remains unclear. The aim of this study was to examine the changes of neointimal hyperplasia after sirolimus-eluting stent (SES) implantation in a diabetic porcine model, and to evaluate the impact of aortic inflammation on this proliferative process.. Diabetic porcine model was created with an intravenous administration of a single dose of streptozotocin in 15 Chinese Guizhou minipigs (diabetic group); each of them received 2 SES (Firebird, Microport Co, China) implanted into 2 separated major epicardial coronary arteries. Fifteen non-diabetic minipigs with SES implantation served as controls (control group). At 6 months, the degree of neointimal hyperplasia was determined by repeat coronary angiography, intravascular ultrasound (IVUS) and histological examination. Tumor necrosis factor (TNF)-alpha protein level in the aortic intima was evaluated by Western blotting, and TNF-alpha, interleukin (IL)-1beta and IL-6 mRNA levels were assayed by reverse transcription and polymerase chain reaction.. The distribution of stented vessels, diameter of reference vessels, and post-procedural minimal lumen diameter were comparable between the two groups. At 6-month follow-up, the degree of in-stent restenosis (40.4 +/- 24.0% vs. 20.2 +/- 17.7%, p < 0.05), late lumen loss (0.33 +/- 0.19 mm vs. 0.10 +/- 0.09 mm, p < 0.001) by quantitative angiography, percentage of intimal hyperplasia in the stented area (26.7 +/- 19.2% vs. 7.3 +/- 6.1%, p < 0.001) by IVUS, and neointimal area (1.59 +/- 0.76 mm2 vs. 0.41 +/- 0.18 mm2, p < 0.05) by histological examination were significantly exacerbated in the diabetic group than those in the controls. Significant increases in TNF-alpha protein and TNF-alpha, IL-1beta and IL-6 mRNA levels were observed in aortic intima in the diabetic group.. Neointimal hyperplasia persisted at least up to 6 months after SES implantation in diabetic porcine, which may be partly related to an exaggerated inflammatory response within the blood vessel wall. Our results provide theoretical support for potential direct beneficial effects of anti-diabetic and anti-inflammation medications in reducing the risk of restenosis after stenting.

Topics: Animals; Catheters, Indwelling; Coronary Vessels; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Disease Models, Animal; Hyperplasia; Male; Sirolimus; Stents; Swine; Tunica Intima

Rapamycin inhibits cyst growth in polycystic kidney disease by targeting the mammalian target of rapamycin (mTOR). To determine if this is a class effect of the mTOR inhibitors, we examined the effect of everolimus, the analogue of rapamycin, on disease progression in the Han:SPRD rat model of polycystic kidney disease.. Four-week-old male heterozygous cystic (Cy/+) and wild-type normal (+/+) Han:SPRD rats were administered everolimus or vehicle (3 mg/kg/day) by gavage for 5 weeks. Kidney function and whole-blood trough levels of everolimus were monitored. After treatment kidney weight and cyst volume density were assessed. Tubule epithelial cell proliferation was assessed by BrdU staining.. Everolimus trough levels between 5 and 7 microg/l were sufficient to significantly reduce kidney and cyst volume density by approximately 50 and 40%, respectively. The steady decrease of kidney function in Cy/+ rats was reduced by 30% compared with vehicle-treated Cy/+ rats. Everolimus treatment markedly reduced the number of 5-bromo-2-deoxyuridine-labeled nuclei in cyst epithelia. Body weight gain and kidney function were impaired in everolimus-treated wild-type rats.. Moderate dosage of everolimus inhibits cystogenesis in Han:SPRD rats. The inhibitory effect of everolimus appears to represent a class effect of mTOR inhibitors.

Topics: Animals; Cysts; Disease Models, Animal; Everolimus; Kidney Function Tests; Male; Polycystic Kidney Diseases; Rats; Rats, Mutant Strains; Sirolimus

Inducible nitric oxide synthase (iNOS) produces nitric oxide and modulates many biologic processes critical in the development of rejection; however, its role in chronic rejection (CR) in small-bowel transplantation (SBT) is largely unknown.. FK506 prevented acute rejection (AR); however, recipients eventually lost their bowel grafts to CR. Combined FK506 and rapamycin treatment prevented CR, thus leading to long-term graft survival. We investigated iNOS expression in our rat orthotopic SBT CR model.. Histologically, mesentery vascular occlusion and fibrosis, which are hallmarks of CR, were apparent in bowel grafts in an FK506 single-treatment group. In contrast, patients with long-term surviving grafts receiving FK506 and rapamycin developed mild vascular occlusion and fibrosis. Unlike in AR, low iNOS expression, which is associated with decreased macrophage infiltration, was observed in CR grafts. However, iNOS expression and macrophage infiltration was higher in long-term-surviving grafts than CR grafts. Immunofluorescence staining revealed that the majority of macrophages expressed iNOS in long-term surviving grafts.. Sequential treatment combining FK506 and rapamycin prolonged survival of SBT animals with decreased vasculopathy and collagen deposition of the intestinal grafts. iNOS may play opposing roles in AR and CR in SBT.

Topics: Animals; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Graft Rejection; Graft Survival; Immunosuppressive Agents; Intestine, Small; Macrophages; Male; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Strains; Sirolimus; Tacrolimus