bafilomycin-a1 and Disease-Models--Animal

Coronavirus disease 2019 is a global pandemic caused by SARS-CoV-2. The emergence of its variant strains has posed a considerable challenge to clinical treatment. Therefore, drugs capable of inhibiting SARS-CoV-2 infection, regardless of virus variations, are in urgently need. Our results showed that the endosomal acidification inhibitor, Bafilomycin A1 (Baf-A1), had an inhibitory effect on the viral RNA synthesis of SARS-CoV-2, and its Beta and Delta variants at the concentration of 500 nM. Moreover, the human lung xenograft mouse model was used to investigate the anti-SARS-CoV-2 effect of Baf-A1. It was found that Baf-A1 significantly inhibited SARS-CoV-2 replication in the human lung xenografts by in situ hybridization and RT-PCR assays. Histopathological examination showed that Baf-A1 alleviated SARS-CoV-2-induced diffuse inflammatory infiltration of granulocytes and macrophages and alveolar endothelial cell death in human lung xenografts. In addition, immunohistochemistry analysis indicated that Baf-A1 decreased inflammatory exudation and infiltration in SARS-CoV-2-infected human lung xenografts. Therefore, Baf-A1 may be a candidate drug for SARS-CoV-2 treatment.

Topics: Alveolar Epithelial Cells; Animals; COVID-19; Disease Models, Animal; Heterografts; Humans; Mice; SARS-CoV-2

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Topics: Animals; Autophagy; Azepines; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Inflammasomes; Inflammation; Intervertebral Disc Degeneration; Macrolides; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Nuclear Proteins; Nucleus Pulposus; Rats; Signal Transduction; Transcription Factor RelA; Transcription Factors; Triazoles

Alcoholic liver disease is associated with high morbidity and mortality, and treatment options are limited to date. Augmenter of liver regeneration (ALR) may protect against hepatic injury from chemical poisons, including ethanol. Autophagy appears to positively influence survival in cases of liver dysfunction, although the mechanisms are poorly understood. Herein, we investigated effects of ALR-induced autophagy in vitro and in vivo in an ethanol-induced model of acute liver injury. Decreased serum levels of alanine aminotransferase and aspartate aminotransferase and reduced histologic lesions revealed that mice overexpressing ALR experienced less liver damage than wild-type. ALR-knockdown mice experienced more severe liver damage than wild-type. ALR-transfected HepG2 cells showed increased survival rates, improved maintenance of mitochondrial membrane potential, and increased ATP levels after ethanol treatment. The observed protection was associated with up-regulation of autophagy-markers, including light chain 3II, beclin-1, and autophagy-related gene 5, and down-regulation of p62 by ALR. Autophagy was inhibited in ALR-knockdown mice and HepG2 cells, and autophagy inhibitor bafilomycin A1 attenuated the protective effects of ALR. Results showed phosphorylated mammalian target of rapamycin (mTOR) was down-regulated when ALR was overexpressed and up-regulated when ALR was knocked down. These data show that ALR is protective against ethanol-induced acute liver injury by promoting autophagy, probably via repressing the mTOR pathway. These results have potential implications for the clinical treatment of alcoholic liver disease patients.

Topics: Acute Lung Injury; Animals; Autophagy; Disease Models, Animal; Ethanol; Hep G2 Cells; Humans; Liver Diseases, Alcoholic; Liver Regeneration; Macrolides; Male; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Mitochondria, Liver; TOR Serine-Threonine Kinases

Major depression is a multifactorial disease. Emerging evidence has suggested that autophagy is involved in the pathological process of depressive disorders. Bafilomycin A1 (Baf A1), is an inhibitor of vacuolar H+‑ATPase that is frequently used at high concentrations to block late‑phase autophagy. However, whether Baf A1 has antidepressant effects remains to be elucidated. The current study aimed to evaluate the antidepressant effects of Baf A1 in rats with chronic unpredictable mild stress (CUMS) and its potential mechanism. The CUMS animal model was established. The sucrose preference test, open‑field test (OFT) and forced swim test (FST) were applied to evaluate the depressive behavior. Synaptic plasticity‑associated proteins synaptophysin and postsynaptic density protein 95 were measured by western blotting and immunofluorescence. Apoptosis‑ and autophagy‑associated proteins in addition to pro‑inflammatory cytokines, including interleukin‑1β and tumor necrosis factor‑α, were detected by western blotting, reverse transcription‑quantitative polymerase chain reaction or ELISA. A 4‑week treatment period with Baf A1 markedly ameliorated CUMS‑induced behavioral abnormalities, including increasing sucrose intake, improving locomotor activity in the OFT, and decreasing immobility time in the FST. In addition, treatment with Baf A1 restored the dysregulation of synaptic plasticity and inhibited neuroinflammation in rats exposed to CUMS. Furthermore, Baf A1 decreased the levels of apoptosis‑ and autophagy‑associated proteins induced by CUMS. The present study demonstrated that Bafilomycin A1 resulted in antidepressant effects in rats, which may be mediated by the reversal of apoptosis, autophagy and neuroinflammation in the hippocampus.

Topics: Animals; Antidepressive Agents; Apoptosis; Autophagy; Depression; Disease Models, Animal; Hippocampus; Humans; Inflammation; Macrolides; Neuronal Plasticity; Rats; Stress, Psychological; Swimming; Vacuolar Proton-Translocating ATPases

Dengue virus (DENV) infection in neuronal cells was speculated to trigger neuropathy. Herein, we determined the blockade of DENV infection by targeting endocytic pathways in vitro and in vivo. In DENV-infected mouse brains, we previously showed that viral proteins are expressed in neuronal cells around the hippocampus with accompanying neurotoxicity. DENV caused infection, including entry, double-stranded (ds)RNA replication, protein expression, and virus release, followed by cytotoxicity in the mouse neuronal Neuro-2a cell line. Pharmacologically blocking clathrin-mediated endocytosis of the DENV retarded viral replication. Targeting vacuolar-type H

Topics: Animals; Antifungal Agents; Cell Line; Cell Survival; Clathrin; Dengue; Dengue Virus; Disease Models, Animal; Endocytosis; Humans; In Vitro Techniques; Macrolides; Mice; Neurons; Virus Internalization; Virus Replication

As lysosomal hydrolysis has long been suggested to be responsible for myelin clearance after peripheral nerve injury, in this study, we investigated the possible role of autophagolysosome formation in myelin phagocytosis by Schwann cells and its final contribution to nerve regeneration. We found that the canonical formation of autophagolysosomes was induced in demyelinating Schwann cells after injury, and the inhibition of autophagy via Schwann cell-specific knockout of the atg7 gene or pharmacological intervention of lysosomal function caused a significant delay in myelin clearance. However, Schwann cell dedifferentiation, as demonstrated by extracellular signal-regulated kinase activation and c-Jun induction, and redifferentiation were not significantly affected, and thus the entire repair program progressed normally in atg7 knockout mice. Finally, autophagic Schwann cells were also found during segmental demyelination in a mouse model of inflammatory peripheral neuropathy. Together, our findings suggest that autophagy is the self-myelin destruction mechanism of Schwann cells, but mechanistically, it is a process distinct from Schwann cell plasticity for nerve repair.

Topics: Animals; Autophagy; Autophagy-Related Protein 7; Demyelinating Diseases; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; In Vitro Techniques; Lysosomes; Macrolides; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Myelin Sheath; Organ Culture Techniques; Schwann Cells; Sciatica; Time Factors; Wallerian Degeneration

Obesity-induced insulin resistance and diabetes are significantly associated with infiltrates of inflammatory cells in adipose tissue. Previous studies recognized the involvement of autophagy in the regulation of metabolism in multiple tissues, including β-cells, hepatocytes, myocytes, and adipocytes. However, despite the importance of macrophages in obesity-induced insulin resistance, the role of macrophage autophagy in regulating insulin sensitivity is seldom addressed. In the present study, we show that macrophage autophagy is important for the regulation of systemic insulin sensitivity. We found that macrophage autophagy is downregulated by both acute and chronic inflammatory stimuli, and blockade of autophagy significantly increased accumulation of reactive oxygen species (ROS) in macrophages. Macrophage-specific Atg7 knockout mice displayed a shift in the proportion to pro-inflammatory M1 macrophages and impairment of insulin sensitivity and glucose homeostasis under high-fat diet conditions. Furthermore, inhibition of ROS in macrophages with antioxidant recovered adipocyte insulin sensitivity. Our results provide evidence of the underlying mechanism of how macrophage autophagy regulates inflammation and insulin sensitivity. We anticipate our findings will serve as a basis for development of therapeutics for inflammatory diseases, including diabetes.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue; Animals; Autophagy; Autophagy-Related Protein 7; Diet, High-Fat; Disease Models, Animal; Enzyme Inhibitors; Glucose; Inflammation; Insulin; Insulin Resistance; Macrolides; Macrophages; Male; Mice; Mice, Knockout; Obesity; RAW 264.7 Cells; Reactive Oxygen Species

Topics: Animals; Animals, Newborn; Autophagy; Cell Survival; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Macrolides; Mice; Mice, Transgenic; Motor Neurons; Muscular Atrophy, Spinal; Nerve Growth Factors; Nerve Tissue Proteins; Sequestosome-1 Protein; Spinal Cord; Survival of Motor Neuron 1 Protein; Survival of Motor Neuron 2 Protein

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression.. We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay.. In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity.. Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Animals; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Female; Gene Expression Regulation; Glioma; Humans; Macrolides; Male; Mice; Mice, Transgenic; Middle Aged; Neurons; Parkinson Disease; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR gamma; Resveratrol; RNA Polymerase II; Stilbenes; Substantia Nigra; TATA-Box Binding Protein; Transcription Factors

To investigate whether ezetimibe ameliorates hepatic steatosis and induces autophagy in a rat model of obesity and type 2 diabetes.. Male age-matched lean control LETO and obese and diabetic OLETF rats were administered either PBS or ezetimibe (10 mg/kg per day) via stomach gavage for 20 wk. Changes in weight gain and energy intake were regularly monitored. Blood and liver tissue were harvested after overnight fasting at the end of study. Histological assessment was performed in liver tissue. The concentrations of glucose, insulin, triglycerides (TG), free fatty acids (FFA), and total cholesterol (TC) in blood and TG, FFA, and TG in liver tissue were measured. mRNA and protein abundance involved in autophagy was analyzed in the liver. To investigate the effect of ezetimibe on autophagy and reduction in hepatic fat accumulation, human Huh7 hepatocytes were incubated with ezetimibe (10 μmol/L) together with or without palmitic acid (PA, 0.5 mmol/L, 24 h). Transmission electron microscopy (TEM) was employed to demonstrate effect of ezetimibe on autophagy formation. Autophagic flux was measured with bafilomycin A1, an inhibitor of autophagy and following immunoblotting for autophagy-related protein expression.. In the OLETF rats that received ezetimibe (10 mg/kg per day), liver weight were significantly decreased by 20% compared to OLETF control rats without changes in food intake and body weight (P < 0.05). Lipid parameters including TG, FFA, and TC in liver tissue of ezetimibe-administrated OLETF rats were dramatically decreased at least by 30% compared to OLETF controls (P < 0.01). The serum glucose, insulin, HOMA-IR, and lipid profiles were also improved by ezetimibe (P < 0.05). In addition, autophagy-related mRNA expression including ATG5, ATG6, and ATG7 and the protein level of microtubule-associated protein light chain 3 (LC3) were significantly increased in the liver in rats that received ezetimibe (P < 0.05). Likewise, for hepatocytes cultured in vitro, ezetimibe treatment significantly decreased PA-induced fat accumulation and increased PA-reduced mRNA and protein expression involved in autophagy (P < 0.05). Ezetimibe-increased autophagosomes was observed in TEM analysis. Immunoblotting analysis of autophagy formation with an inhibitor of autophagy demonstrated that ezetimibe-increased autophagy resulted from increased autophagic flux.. The present study demonstrates that ezetimibe-mediated improvement in hepatic steatosis might involve the induction of autophagy.

Topics: Animals; Anticholesteremic Agents; Autophagy; Biomarkers; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Type 2; Disease Models, Animal; Ezetimibe; Fatty Liver; Gene Expression Regulation; Hepatocytes; Lipids; Liver; Macrolides; Male; Obesity; Palmitic Acid; Rats, Inbred OLETF; RNA, Messenger

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

Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aβ antibody, which uses a monovalent binding mode to the TfR, increases β-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Blood-Brain Barrier; Brain; Cell Line, Transformed; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Lysosomes; Macrolides; Mice; Mice, Transgenic; Models, Immunological; Presenilin-1; Protein Binding; Protein Transport; Receptors, Transferrin; Single-Chain Antibodies; Subcellular Fractions; Time Factors; Transcytosis

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

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

It has been shown that a specific inhibitor of vacuolar H(+)-ATPases, bafilomycin A1, inhibits bone resorption by isolated chicken osteoclasts by blocking the proton pump in the ruffled border membrane. We report here the effects of bafilomycin A1 on bone resorption in vivo. Using a cannulated osmotic minipump delivery system, we infused bafilomycin locally to the eruption pathway of permanent premolars of beagle dogs. We used pit formation by osteoclasts in vitro to estimate the concentrations and heat stability of bafilomycin to be used in vivo. In this model, osteoclasts were cultured on thin bone slices, in which they form pits indicative of resorption. After 2 weeks preincubation at 37 degrees C, bafilomycin concentrations of 10(-6) and 10(-7) M but not 10(-8) M completely inhibited the resorptive activity of cultured osteoclasts, and the two larger doses were chosen for use in vivo. Local delivery of 10(-6) M bafilomycin to the eruption pathway of the fourth permanent mandibular premolar during mideruption inhibited tooth eruption by blocking bone resorption as assayed by radiography, light microscopy, and scanning electron microscopy. Bafilomycin at 10(-7) M had similar but less intensive effects. Moreover, osteoclasts in the alveolar bone of crypts treated with 10(-7) M bafilomycin A1 stained very weakly for tartrate-resistant acid phosphatase. The effect of bafilomycin on bone resorption was shown to be very local, and no side effects of treatment with bafilomycin were observed in adjacent teeth or the behavior of dogs. We report here, for the first time, inhibition of tooth eruption caused by inhibited bone resorption using bafilomycin A1 in vivo.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Bicuspid; Bone Resorption; Cells, Cultured; Disease Models, Animal; Dogs; Infusion Pumps, Implantable; Macrolides; Microscopy, Electron, Scanning; Osmosis; Osteoclasts; Proton-Translocating ATPases; Rabbits; Radiography; Tibia; Tooth Eruption