thymosin-beta(4) and Disease-Models--Animal

There is an urgent need for new treatments for chronic kidney disease (CKD). Thymosin-β4 is a peptide that reduces inflammation and fibrosis and has the potential to restore endothelial and epithelial cell injury, biological processes involved in the pathophysiology of CKD. Therefore, thymosin-β4 could be a novel therapeutic direction for CKD.. Here, we review the current evidence on the actions of thymosin-β4 in the kidney in health and disease. Using transgenic mice, two recent studies have demonstrated that endogenous thymosin-β4 is dispensable for healthy kidneys. In contrast, lack of endogenous thymosin-β4 exacerbates mouse models of glomerular disease and angiotensin-II-induced renal injury. Administration of exogenous thymosin-β4, or its metabolite, Ac-SDKP, has shown therapeutic benefits in a range of experimental models of kidney disease.. The studies conducted so far reveal a protective role for thymosin-β4 in the kidney and have shown promising results for the therapeutic potential of exogenous thymosin-β4 in CKD. Further studies should explore the mechanisms by which thymosin-β4 modulates kidney function in different types of CKD. Ac-SDKP treatment has beneficial effects in many experimental models of kidney disease, thus supporting its potential use as a new treatment strategy.

Topics: Animals; Disease Models, Animal; Fibrosis; Humans; Inflammation; Kidney; Mice; Mice, Transgenic; Renal Insufficiency, Chronic; Thymosin

Thymosin β4 (Tβ4) is a 5K peptide which influences cellular migration by inhibiting organization of the actin-cytoskeleton. Treatment of acute stroke presently involves use of rt-PA and/or endovascular treatment with thrombectomy, both of which have time limitations. Therefore, development of a treatment beyond these times is necessary as most stroke patients present beyond these time limits. A drug which could be administered within 24 h from symptom onset would provide substantial benefit.. This review summarizes the data and results of two in-vivo studies testing Tβ4 in an embolic stroke model of young and aged rats. In addition, we describe in-vitro investigations of the neurorestorative and neuroprotective properties of Tβ4 in a variety of neuroprogenitor and oligoprogenitor cell models.. Tβ4 acts as a neurorestorative agent when employed in a young male rat model of embolic stroke while in an aged model it acts a neuroprotectant. However evaluation of Tβ4 as a treatment of stroke requires further preclinical evaluation in females and in males and females with comorbidities such as, hypertension and diabetes in models of embolic stroke to further define the mechanism of action and potential as a treatment of stroke in humans.

Topics: Animals; Disease Models, Animal; Female; Humans; Male; Nerve Regeneration; Neuroprotection; Rats; Stroke; Thymosin

Treatment with thymosin beta 4 (Tβ4) reduces infarct volume and preserves cardiac function in preclinical models of cardiac ischemic injury. These effects stem in part from decreased infarct size, but additional benefits are likely due to specific antifibrotic and proangiogenic activities. Injected or transgenic Tβ4 increase blood vessel growth in large and small animal models, consistent with Tβ4 converting hibernating myocardium to an actively contractile state following ischemia. Tβ4 and its degradation products have antifibrotic effects in in vitro assays and in animal models of fibrosis not related to cardiac injury. This large number of pleiotropic effects results from Tβ4's many interactions with cellular signaling pathways, particularly indirect regulation of cellular motility and movement via the SRF-MRTF-G-actin transcriptional pathway. Variation in effects and effect sizes in animal models may potentially be due to variable distribution of Tβ4. Preclinical studies of PK/PD relationships and a reliable pharmacodynamic biomarker would facilitate clinical development of Tβ4.

Topics: Actins; Animals; Cardiotonic Agents; Disease Models, Animal; Heart Diseases; Humans; Myocardial Infarction; Myocardium; Signal Transduction; Thymosin

Chronic ocular surface diseases such as dry eye, blepharitis, and neurotrophic keratopathies represent a significant and a growing therapeutic challenge. The basis of this expanding prevalence is multifactorial and may due to issues such as an aging population, an increasing use of video display terminals, and increases in frequency of refractive surgeries. The growing incidence of diseases such as diabetes may also be a contributing factor. Current treatments for ocular surface disease include artificial tears, lubricants, tear duct plugs, steroids, antibiotics, cyclosporine, scleral lenses, and serum tears. Treatment choices depend on the type and severity of the disease, but in general positive outcomes are limited because many of these treatments do not fully address the underlying disease process or promote mechanisms that facilitate long-term wound repair. From minor corneal injuries to more severe inflammatory-mediated pathologies, clinicians need agents that promote corneal healing and reduce the inflammatory response to prevent visual disturbances and improve quality of life. A focus on treatments that reduce the inflammatory responses while accelerating corneal epithelial growth would represent a major step forward from current treatment options. Increasing evidence suggests that thymosin beta 4 (Tβ4), a naturally occurring polypeptide, can elicit this spectrum of therapeutic responses: a rapid corneal reepithelialization and a reduction in corneal inflammation. This chapter serves as a review of standard therapies as well as recent advancements in the development of newer therapies that includes the use of Tβ4 that is proving to be an exciting new agent for the management of ocular surface disease.

Topics: Animals; Anti-Inflammatory Agents; Corneal Diseases; Disease Models, Animal; Dry Eye Syndromes; Humans; Thymosin; Wound Healing

Substantial evidence demonstrates a link of increased plasminogen activator inhibitor-1 (PAI-1) and glomerulosclerosis and kidney fibrosis, providing a novel therapeutic option for prevention and treatment of chronic kidney diseases. Several mechanisms contributing to increased PAI-1 will be addressed, including classic key profibrotic factors such as the renin-angiotensin-system (RAS) and transforming growth factor-beta (TGF-b???and novel molecules identified by proteomic analysis, such as thymosin- b4. The fibrotic sequelae caused by increased PAI-1 in kidney depend not only on its classic inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA), but also its influence on cell migration.

Topics: Angiotensins; Animals; Chronic Disease; Disease Models, Animal; Fibrosis; Humans; Kidney Diseases; Mice; Oligopeptides; Organ Specificity; Plasminogen Activator Inhibitor 1; Renin-Angiotensin System; Thymosin; Transforming Growth Factor beta1

We investigated the therapeutic effect of recombinant thymosin β4 (rTβ4) on rabbit autoimmune dacryoadenitis, an animal model of SS dry eye, and explore its mechanisms.. Rabbits were treated topically with rTβ4 or PBS solution after disease onset for 28 days, and clinical scores were determined by assessing tear secretion, break-up time, fluorescein, hematoxylin and eosin staining, and periodic acid-Schiff. The expression of inflammatory mediators in the lacrimal glands were measured by real-time PCR. The expression of T helper 17 (Th17) cell-related transcription factors and cytokines were detected by real-time PCR and Western blotting. The molecular mechanism underlying the effects of rTβ4 on Th17 cell responses was investigated by Western blotting.. Topical administration of rTβ4 after disease onset efficiently ameliorated the ocular surface inflammation and relieved the clinical symptoms. Further analysis revealed that rTβ4 treatment significantly inhibited the expression of Th17-related genes (RORC, IL-17A, IL-17F, IL-1R1, IL-23R, and granulocyte-macrophage colony-stimulating factor) and IL-17 protein in lacrimal glands, and meanwhile decreased the inflammatory mediators expression. Mechanistically, we demonstrated that rTβ4 repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Activation of the STAT3 signal pathway by Colivelin partly reversed the suppressive effects of rTβ4 on IL-17 expression in vitro.. rTβ4 could alleviate ongoing autoimmune dacryoadenitis in rabbits, probably by suppressing Th17 response via partly affecting the STAT3 pathway. These data may provide a new insight into the therapeutic effect and mechanism of rTβ4 in dry eye associated with Sjögren's syndrome.

Topics: Animals; Dacryocystitis; Disease Models, Animal; Dry Eye Syndromes; Interleukin-17; Rabbits; Tears; Th17 Cells

Dry eye disease (DED) is a multifactorial ocular disorder that interferes with daily living and reduces quality of life. However, there is no most ideal therapeutic treatment to address all the deleterious defects of DED. The purpose of this study was to investigate the ability of recombinant human thymosin β4 (rhTβ4) to promote healing in a benzalkonium chloride (BAC)-induced mice DED model and the anti-inflammatory effects involved in that process. Eye drops consisting of 0.05% and 0.1% rhTβ4 were used for treatment of DED. Tear volume and corneal staining scores were measured after 7 days. Periodic acid-Schiff staining for gobleT cells in conjunctiva, immunohistochemical staining for CD4

Topics: Animals; Anti-Inflammatory Agents; Benzalkonium Compounds; Cytokines; Disease Models, Animal; Dry Eye Syndromes; Humans; Mice; NF-kappa B; Ophthalmic Solutions; Quality of Life; Thymosin

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in interorgan cross talk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin β4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in the plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular cross talk.

Topics: Animals; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Diabetes Mellitus, Type 2; Disease Models, Animal; Humans; Insulin Resistance; Male; Mice, Inbred C57BL; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Diseases; Neuronal Outgrowth; Osteoblasts; Physical Endurance; Proteomics; Signal Transduction; Tandem Mass Spectrometry; Thymosin

Demyelination is observed in animal models of intractable epilepsy (IE). Epileptogenesis damages the myelin sheath and dysregulates oligodendrocyte precursor cell (OPC) development. However, the molecular pathways regulating demyelination in epilepsy are unclear. Here, we predicted the molecular mechanisms regulating demyelination in a rat model of lithium-pilocarpine hydrochloride-induced epilepsy. We identified DGKA/Mboat2/Inpp5j and NOS/Keratin 28 as the main target molecules that regulate demyelination via glycerolipid and glycerophospholipid metabolism, phosphatidylinositol signaling, and estrogen signaling in demyelinated forebrain slice cultures (FSCs). In seizure-like FCSs, the actin cytoskeleton was regulated by Cnp and MBP via Pak4/Tmsb4x (also known as Tβ4) and Kif5c/Kntc1. Tβ4 possibly prevented OPC differentiation and maturation and inhibited MBP phosphorylation via the p38MAPK/ERK1/JNK1 pathway. The MAPK signaling pathway was more likely activated in seizure-like FCSs than in demyelinated FCSs. pMBP expression was decreased in the hippocampus of lithium-pilocarpine hydrochloride-induced acute epilepsy rats. The expression of remyelination-related factors was suppressed in the hippocampus and corpus callosum in lithium-pilocarpine hydrochloride-induced epilepsy rats. These findings suggest that the actin cytoskeleton, Tβ4, and MAPK signaling pathways regulate the decrease in pMBP in the hippocampus in a rat model of epilepsy. Our results indicate that regulating the actin cytoskeleton, Tβ4, and MAPK signaling pathways may facilitate the prevention of demyelination in IE.

Topics: Animals; Cell Differentiation; Demyelinating Diseases; Disease Models, Animal; Epilepsy; Hippocampus; Lithium; Male; MAP Kinase Signaling System; Myelin Sheath; p38 Mitogen-Activated Protein Kinases; Pilocarpine; Proteomics; Rats; Rats, Sprague-Dawley; Signal Transduction; Thymosin

Thymosin beta-4 (Tβ4) is an actin-sequestering peptide that plays important roles in regeneration and remodeling of injured tissues. However, its function in a naturally occurring pathogenic bacterial infection model has remained elusive. We adopted Tβ4-overexpressing transgenic (Tg) mice to investigate the role of Tβ4 in acute pulmonary infection and systemic sepsis caused by

Topics: Animals; Cytokines; Disease Models, Animal; Disease Resistance; Ectopic Gene Expression; Host-Pathogen Interactions; Humans; Immunohistochemistry; Immunophenotyping; Legionella pneumophila; Legionnaires' Disease; Ligands; Male; Mice; Mice, Transgenic; Pneumonia, Bacterial; Sepsis; Thymosin; Toll-Like Receptors

Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Pooled knockout mouse DGC cells were transplanted into a spleen of syngeneic immunocompetent mice to study clonal advantages in context of a complex process of liver metastasis. Tmsb4x (thymosin beta-4 X-linked), Hmox1, Ifitm3, Ldhb, and Itgb7 were identified as strong candidate genes that promote metastasis. In particular, Tmsb4x enhanced DGC metastasis and stomach organoid-generated tumor growth in in vivo transplantation models. Tmsb4x promoted tumor clonogenicity and anoikis resistance. In situ hybridization analysis showed that Tmsb4x is highly expressed in E-cadherin-negative mouse DGC models compared with mouse IGC and intestinal cancer models. E-cadherin deficiency also increased Tmsb4x expression in stomach organoids via Wnt signaling activation. Collectively, these results demonstrate that Tmsb4x promotes DGC metastasis. In addition, this experimental system will aid in the identification of novel target genes responsible for DGC metastasis.

Topics: Animals; Biomarkers, Tumor; CRISPR-Cas Systems; Disease Models, Animal; Gene Expression; Gene Knockout Techniques; Humans; Mice; Neoplasm Metastasis; Real-Time Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Thymosin

Thymosin β4 (Tβ4) is the most abundant member of the β-thymosins and plays an important role in the control of actin polymerization in eukaryotic cells. While its effects in multiple organs and diseases are being widely investigated, the safety profile has been established in animals and humans, currently, little is known about its influence on Alzheimer's disease (AD) and the possible mechanisms. Thus, we aimed to evaluate the effects and mechanisms of Tβ4 on glial polarization and cognitive performance in APP/PS1 transgenic mice.. Behavior tests were conducted to assess the learning and memory, anxiety and depression in APP/PS1 mice. Thioflavin S staining, Nissl staining, immunohistochemistry/immunofluorescence, ELISA, qRT-PCR, and immunoblotting were performed to explore Aβ accumulation, phenotypic polarization of glial cells, neuronal loss and function, and TLR4/NF-κB axis in APP/PS1 mice.. We demonstrated that Tβ4 protein level elevated in all APP/PS1 mice. Over-expression of Tβ4 alone alleviated AD-like phenotypes of APP/PS1 mice, showed less brain Aβ accumulation and more Insulin-degrading enzyme (IDE), reversed phenotypic polarization of microglia and astrocyte to a healthy state, improved neuronal function and cognitive behavior performance, and accidentally displayed antidepressant-like effect. Besides, Tβ4 could downregulate both TLR4/MyD88/NF-κB p65 and p52-dependent inflammatory pathways in the APP/PS1 mice. While combination drug of TLR4 antagonist TAK242 or NF-κB p65 inhibitor PDTC exerted no further effects.. These results suggest that Tβ4 may exert its function by regulating both classical and non-canonical NF-κB signaling and is restoring its function as a potential therapeutic target against AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Astrocytes; Brain; Cognitive Dysfunction; Disease Models, Animal; Female; Male; Memory; Mice; Mice, Transgenic; Microglia; Neuroglia; Neurons; NF-kappa B; Phenotype; Presenilin-1; Signal Transduction; Thymosin

Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function.. Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1.. The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

Topics: Animals; Cell Differentiation; Cell Line; Cytoskeletal Proteins; Disease Models, Animal; Endothelial Cells; Gene Expression Profiling; Gene Expression Regulation; Hindlimb; Human Embryonic Stem Cells; Human Umbilical Vein Endothelial Cells; Humans; Ischemia; Mice, Knockout; Neovascularization, Physiologic; Nerve Tissue Proteins; Peptides; Protein Binding; RNA-Seq; RNA, Long Noncoding; Signal Transduction; Thymosin; Transcriptome

Topics: Animals; Cell Differentiation; Cell Movement; Cell Transplantation; Disease Models, Animal; Gene Expression Regulation; Hindlimb; Humans; Ischemia; Male; MAP Kinase Signaling System; Mesenchymal Stem Cells; Mice; Mice, Nude; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Thymosin; TOR Serine-Threonine Kinases; Wound Healing

Endothelial progenitor cells (EPCs) are bone-marrow derived cells that are critical in the maintenance of endothelial wall integrity and protection of ischemic myocardium through the formation of new blood vessels (vasculogenesis) or proliferation of pre-existing vasculature (angiogenesis). Diabetes mellitus (DM) and the metabolic syndrome are commonly associated with ischemic heart disease through its pathological effects on the endothelium and consequent endothelial dysfunction. Thymosin-β4 (Tβ4) which expressed in the embryonic heart is critical in epicardial and coronary artery formation. In this study, we explored the effects of Tβ4 treatment on diabetic EPCs in vitro and intramyocardial injection of Tβ4-treated and non-Tβ4 treated EPCs following acute myocardial infarction (MI) of diabetic rats in vivo. It was found that 10 ng/mL Tβ4 increased migration, tubule formation, and angiogenic factor secretion of diabetic EPCs in vitro. In vivo, although implantation of Tβ4 treated diabetic EPCs significantly increased capillary density and attracted more c-Kit positive progenitor cells into the infarcted hearts as compared with implantation of non-Tβ4 treated diabetic EPCs, the significantly improved left ventricular ejection fraction was only found in the rats which received non-Tβ4 treated EPCs. The data suggests that a low dose Tβ4 increases diabetic EPC migration, tubule formation, and angiogenic factor secretion. However, it did not improve the effects of EPCs on left ventricular pump function in diabetic rats with MI.

Topics: Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Echocardiography; Endothelial Progenitor Cells; Humans; Male; Microfilament Proteins; Myocardial Infarction; Obesity; Rats; Rats, Zucker; Thymosin

Thymosin‑β 4 (Tβ4) has been reported to exert a pro‑angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tβ4 on critical limb ischemia (CLI). The present study aimed therefore to investigate the mechanisms and pro‑angiogenic effects of Tβ4 in CLI mice. Tβ4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF‑κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis‑related factors angiopoietin‑2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α‑smooth muscle actin (α‑SMA) and the Notch/NF‑κB pathways‑related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF‑κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tβ4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF‑κB, and phosphorylated (p)‑p65 in HUVEC. In addition, Tβ4 promoted the expression of CD31, α‑SMA Ang2, tie2, VEGFA, N1ICD and p‑p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tβ4, whereas Tβ4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tβ4 may promote angiogenesis in CLI mice via regulation of Notch/NF‑κB pathways.

Topics: Animals; Cell Line; Disease Models, Animal; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Ischemia; Male; Mice; Mice, Inbred C57BL; Muscles; Neovascularization, Pathologic; NF-kappa B; Receptors, Notch; Signal Transduction; Thymosin; Vascular Endothelial Growth Factor A

The aim of the present study was to investigate the protective effects of thymosin β4 (Tβ4) on neuronal apoptosis in rat middle cerebral artery occlusion ischemia/reperfusion (MCAO I/R) injury, and determine the mechanisms involved in this process. Forty‑eight adult male Sprague‑Dawley rats were randomly divided into three groups (n=16 per group): A sham control group, an ischemia/reperfusion group (I/R group), and a Tβ4 group. The focal cerebral I/R model was established by blocking the right MCA for 2 h, followed by reperfusion for 24 h. The Zea‑Longa method was used to assess neurological deficits. Cerebral infarct volume was assessed using 2,3,5‑triphenyltetrazolium chloride staining, and pathological changes were observed via hematoxylin and eosin staining. The terminal dexynucleotidyl transferase (TdT)‑mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis. The expression of glucose‑regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase‑12 (CASP12) protein was assessed using immunohistochemistry and western blotting 24 h after reperfusion. Infarct volume and neuronal damage in the I/R and Tβ4 groups were significantly greater than those observed in the sham group. The Zea‑Longa score, neuronal apoptosis, and expression of GRP78, CHOP, and CASP12 in the I/R and Tβ4 groups were significantly higher than those reported in the sham group. However, the Longa score and neuronal apoptosis were lower in the Tβ4 group compared to the I/R group. The expression of GRP78 was significantly increased, whereas that of CHOP and CASP12 was significantly decreased in the Tβ4 group compared to the I/R group. The present data revealed that Tβ4 can inhibit neuronal apoptosis by upregulating GRP78 and downregulating CHOP and CASP12, thereby reducing cerebral I/R injury.

Topics: Animals; Apoptosis; Brain Ischemia; Disease Models, Animal; Immunohistochemistry; Male; Neurons; Rats; Reperfusion Injury; Thymosin

The antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is released from thymosin-β4 (Tβ4) by the meprin-α and prolyl oligopeptidase (POP) enzymes and is hydrolyzed by angiotensin-converting enzyme (ACE). Ac-SDKP is present in urine; however, it is not clear whether de novo tubular release occurs or if glomerular filtration is the main source. We hypothesized that Ac-SDKP is released into the lumen of the nephrons and that it exerts an antifibrotic effect. We determined the presence of Tβ4, meprin-α, and POP in the kidneys of Sprague-Dawley rats. The stop-flow technique was used to evaluate Ac-SDKP formation in different nephron segments. Finally, we decreased Ac-SDKP formation by inhibiting the POP enzyme and evaluated the long-term effect in renal fibrosis. The Tβ4 precursor and the releasing enzymes meprin-α and POP were expressed in the kidneys. POP enzyme activity was almost double that in the renal medulla compared with the renal cortex. With the use of the stop-flow technique, we detected the highest Ac-SDKP concentrations in the distal nephron. The infusion of a POP inhibitor into the kidney decreased the amount of Ac-SDKP in distal nephron segments and in the proximal nephron to a minor extent. An ACE inhibitor increased the Ac-SDKP content in all nephron segments, but the increase was highest in the distal portion. The chronic infusion of a POP inhibitor increased kidney medullary fibrosis, which was prevented by Ac-SDKP. We conclude that Ac-SDKP is released by the nephron and is part of an important antifibrotic system in the kidney.

Topics: Animals; Disease Models, Animal; Fibrosis; Kidney Diseases; Kidney Medulla; Male; Metalloendopeptidases; Nephrons; Oligopeptides; Prolyl Oligopeptidases; Rats, Sprague-Dawley; Serine Endopeptidases; Signal Transduction; Thymosin

Endoscopic submucosal dissection (ESD) is widely used in the treatment of early esophageal cancer. However, the incidence of postoperative esophageal stricture is relatively high, especially after full circumferential ESD. Previous studies have shown that thymosin β4 (Tβ4) has anti-fibrotic activity and prevents scar formation. In this study, we investigated the safety and therapeutic effect of Tβ4 injection in preventing esophageal stricture after circumferential ESD in a porcine model.. A total of 8 Bama pigs underwent esophageal circumferential ESD under anesthesia (n = 4 for experimental and control group). Local injection of Tβ4 gel was administered in the experimental group. Follow-up endoscopy was conducted, and balloon dilation (EBD) was performed to prevent the occurrence of esophageal stricture.. Esophageal stricture developed after circumferential ESD in all pigs. Local Tβ4 gel injection has shortened resolution of the stricture (p = 0.012) and was associated with a lesser number of EBD sessions (p = 0.002). The severity of esophageal stricture was milder in the experimental group (p = 0.046 vs. control group). No adverse events occurred in the study.. Local Tβ4 gel injection appeared to be safe and effective for the prevention of esophageal stricture after circumferential ESD in a porcine model.

Topics: Animals; Disease Models, Animal; Endoscopic Mucosal Resection; Esophageal Stenosis; Humans; Male; Postoperative Complications; Swine; Thymosin

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Endothelial Cells; Ganglia, Spinal; Interleukin-1 Receptor-Associated Kinases; Male; Mice; Mice, Transgenic; MicroRNAs; Neuronal Outgrowth; NF-kappa B; Sciatic Nerve; Signal Transduction; Thymosin; TNF Receptor-Associated Factor 6

Dipeptidyl peptidase type 4 (DPP-4) inhibitors were reported to have beneficial effects in experimental models of chronic kidney disease. The underlying mechanisms are not completely understood. However, these effects could be mediated via the glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP1R) pathway. Here we investigated the renal effects of the DPP-4 inhibitor linagliptin in Glp1r-/- knock out and wild-type mice with 5/6 nephrectomy (5/6Nx). Mice were allocated to groups: sham+wild type+placebo; 5/6Nx+ wild type+placebo; 5/6Nx+wild type+linagliptin; sham+knock out+placebo; 5/6Nx+knock out+ placebo; 5/6Nx+knock out+linagliptin. 5/6Nx caused the development of renal interstitial fibrosis, significantly increased plasma cystatin C and creatinine levels and suppressed renal gelatinase/collagenase, matrix metalloproteinase-1 and -13 activities; effects counteracted by linagliptin treatment in wildtype and Glp1r-/- mice. Two hundred ninety-eight proteomics signals were differentially regulated in kidneys among the groups, with 150 signals specific to linagliptin treatment as shown by mass spectrometry. Treatment significantly upregulated three peptides derived from collagen alpha-1(I), thymosin β4 and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and significantly downregulated one peptide derived from Y box binding protein-1 (YB-1). The proteomics results were further confirmed using western blot and immunofluorescence microscopy. Also, 5/6Nx led to significant up-regulation of renal transforming growth factor-β1 and pSMAD3 expression in wild type mice and linagliptin significantly counteracted this up-regulation in wild type and Glp1r-/- mice. Thus, the renoprotective effects of linagliptin cannot solely be attributed to the GLP-1/GLP1R pathway, highlighting the importance of other signaling pathways (collagen I homeostasis, HNRNPA1, YB-1, thymosin β4 and TGF-β1) influenced by DPP-4 inhibition.

Topics: Animals; Collagen Type I; Collagen Type I, alpha 1 Chain; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Down-Regulation; Glucagon-Like Peptide-1 Receptor; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; Kidney; Linagliptin; Male; Mice; Mice, Knockout; Nephrectomy; Renal Insufficiency, Chronic; RNA-Seq; Signal Transduction; Thymosin; Transcription Factors; Transforming Growth Factor beta1; Up-Regulation

We wanted to determine the impact of different doses of a pegylated and liposomal formulation of the cardiotoxic drug doxorubicin on cardiac function, fibrosis and survival in mice. The drug causes myocardial damage by producing reactive oxygen species, mitochondrial damage and lipid peroxidation. Thymosin beta 4 is a peptide with cardioprotective, anti-oxidant and anti-fibrotic properties and we further investigated whether the peptide could attenuate this drug-induced injury by measuring cardiac function and fibrosis.. Mice receiving high doses of doxorubicin died early during follow-up. Lowering the dose improved survival but did not markedly impair cardiac function on echocardiography and caused only limited fibrosis on histology. Thymosin beta 4 had only a mild protective effect on early cardiac function and did not significantly influence myocardial fibrosis. In conclusion, the use of pegylated and liposomal doxorubicin was not appropriate for inducing experimental cardiomyopathy. Thymosin beta 4 therapy was not beneficial in this setting.

Topics: Animals; Cardiomyopathies; Cardiotoxicity; Disease Models, Animal; Doxorubicin; Electrocardiography; Male; Mice; Polyethylene Glycols; Thymosin

To study the effects of exogenous thymosinβ4 (Tβ4) treatment in acetaminophen (APAP)-induced hepatotoxicity.. Liver injury was induced in mice by a single intraperitoneal injection of APAP (500 mg/kg). Exogenous Tβ4 was intraperitoneally administrated at 0 h, 2 h and 4 h after APAP injection. Chloroquine (CQ) (60 mg/kg) was intraperitoneally injected 2 h before APAP administration to inhibit autophagy. Six hours after APAP injection liver injury was evaluated by histological examinations, biochemical measurements and enzyme linked immunosorbent assay (ELISAs). Western blots were performed to detect proteins expression.. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased 6 h after APAP administration, but were significantly reduced by co-administration of Tβ4. Histological examinations demonstrated that Tβ4 reduced necrosis and inflammation induced by APAP. Immunofluorescence showed that Tβ4 suppressed APAP-induced translocation of high mobility group box-1 protein (HMGB1) from the nucleus to cytosol and intercellular space. Hepatic glutathione (GSH) depletion, malondialdehyde (MDA) formation and decreased superoxide dismutase (SOD) activities induced by APAP were all attenuated by Tβ4. APAP-induced increases in hepatic nuclear factor-κB (NF-κB) p65 protein expression and inflammatory cytokines production including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were reduced by Tβ4 treatment. Increased LC3 and p62 proteins in the liver tissues of APAP-treated mice were decreased by Tβ4 treatment, which indicated the enhancement of autophagy flux by Tβ4. Furthermore, inhibiting autophagy by CQ abrogated the protective effects of Tβ4 against APAP hepatotoxicity.. Exogenous Tβ4 treatment exerts protective effects against APAP-induced hepatotoxicity in mice. The underneath molecular mechanisms may involve autophagy enhancement and inhibition of oxidative stress by Tβ4.

Topics: Acetaminophen; Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Autophagy; Chemical and Drug Induced Liver Injury; Disease Models, Animal; HMGB1 Protein; Humans; Interleukin-1beta; Liver; Male; Mice; Mice, Inbred BALB C; Necrosis; Thymosin; Tumor Necrosis Factor-alpha

The transcription factor Islet-1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet-1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta-4 (TB4), a 43-aminoacid peptide, was associated with increased Islet-1 immunostaining, suggesting the induction of an Islet-1-positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1-nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet-1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet-1

Topics: Animals; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Heart Ventricles; LIM-Homeodomain Proteins; Mice; Mice, Transgenic; Myocardial Infarction; Sinoatrial Node; Stem Cells; Thymosin; Transcription Factors

Thymosin beta 4 (Tβ4) has demonstrated neuroprotective potential in models of neurlogical injury. The neuroprotective potential of Tβ4 has been associated with increased miR-200a and miR-200b within the brain following stroke. Here we tested the hypothesis that Tβ4 treatment could also alter miRNA profiles within the plasma following severe traumatic brain injury (TBI).. We used the rat lateral fluid percusion injury model of severe TBI to test this hypothesis. Highly sensitive and quantitative droplet digital polymerase chain reaction (ddPCR) was used to measure the plasma concentrations of miR-200 family members. In addition, we conducted RNAseq analysis of plasma miRNA to further identify changes associated with TBI and treatment with Tβ4.. ddPCR demonstrated that miR-200a-3p andmiR-200b-3p were both significantly increased in plasma following treatment with Tβ4 after severe TBI. RNAseq analysis suggested that miR-300-3p and miR-598-3p increased while miR-450-3p and miR-194-5p significantly decreased following TBI. In contrast, miR-194-5p significantly increased in Tβ4 treated rats following TBI. In addition, we identified nine plasma miRNAs whose expression significantly changed following treatment with Tβ4.. Tβ4 treatment significantly increased plasma levels of miR-200a-3p and miR-200b-3p, while RNAseq analysis identified miR-194-5p as a candidate miRNA that may be critical for neuroprotection.

Topics: Animals; Brain; Brain Injuries, Traumatic; Disease Models, Animal; Male; MicroRNAs; Percussion; Rats; Rats, Wistar; Severity of Illness Index; Thymosin; Transcriptome

Translations of new therapeutic options for cardiovascular disease from animal studies into a clinical setting have been hampered, in part by an improper reflection of a relevant patient population in animal models. In this study, we investigated the impact of thymosin β4 (Tβ4), which promotes collateralization and capillarization, during hypercholesterolemia, a known risk factor of coronary artery disease. Initial in vitro results highlighted an improved endothelial cell function upon Tβ4 treatment under control conditions and during hypercholesterolemic stress (scratch area [pixels]: oxidized low-density lipoprotein [oxLDL], 191,924 ± 7,717; and oxLDL + Tβ4, 105,621 ± 11,245). To mimic the common risk factor of hypercholesterolemia in vivo, pigs on regular (NC) or high-fat (HC) diet underwent chronic myocardial ischemia followed by recombinant adeno-associated virus (rAAV)-mediated transduction of Tβ4 or LacZ as a control. We show that Tβ4 overexpression improves capillarization and collateralization (collaterals: NC + rAAV.LacZ, 2.1 ± 0.5; NC + rAAV.Tβ4, 6.7 ± 0.5; HC + rAAV.LacZ, 3.0 ± 0.3; and HC + rAAV.Tβ4, 6.0 ± 0.4), ultimately leading to an improved myocardial function in both diet groups (ejection fraction [EF] at day 56 [%]: NC + rAAV.LacZ, 26 ± 1.1; NC + rAAV.Tβ4, 45 ± 1.5; HC + rAAV.LacZ, 26 ± 2.5; and HC + rAAV.Tβ4, 41 ± 2.6). These results demonstrate the potency of Tβ4 in a patient-relevant large animal model of chronic myocardial ischemia.

Topics: Animals; Dependovirus; Disease Models, Animal; Endothelial Cells; Hypercholesterolemia; Lipoproteins, LDL; Myocardial Ischemia; Myocardium; Neovascularization, Physiologic; Swine; Thymosin

Thymosin β4 (Tβ4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tβ4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTβ4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTβ4-Intein was constructed, in which rhTβ4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTβ4 could be fully acetylated when the rhTβ4-Intein was expressed in the engineering strain. After purification, the rhTβ4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTβ4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTβ4 can reach 200 mg per litre. The N-acetylated rhTβ4 could be stable at 2-8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTβ4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.

Topics: Acetylation; Animals; Disease Models, Animal; Dry Eye Syndromes; Escherichia coli; Female; Humans; Rats; Rats, Wistar; Recombinant Fusion Proteins; Thymosin

This study evaluated the clinical activity of RGN-259 (thymosin β4) in comparison with cyclosporine A (CsA), diquafosol (DQS), and lifitegrast (LFA) in a murine model of dry eye. The model was NOD.B10-H2

Topics: Animals; Conjunctiva; Cornea; Cyclosporine; Disease Models, Animal; Dry Eye Syndromes; Female; Goblet Cells; Humans; Inflammation Mediators; Lacrimal Apparatus; Male; Mice; Mice, Inbred NOD; Mucins; Ophthalmic Solutions; Phenylalanine; Polyphosphates; Prescription Drugs; Scopolamine; Sulfones; Tears; Thymosin; Treatment Outcome; Uracil Nucleotides

Corneal alkali burns are a severe disease and commonly encountered in the emergent clinic. A rapid medical treatment for the burn is very important. Gly-thymosin β. Rabbit alkali burns were induced with NaOH-contained filter paper. Phosphate-buffered solutions at pH 7.0, Gly-Tβ. Gly-Tβ

Topics: Administration, Topical; Alkalies; Animals; Burns, Chemical; Corneal Injuries; Corneal Neovascularization; Disease Models, Animal; Epithelium, Corneal; Eye Burns; Hydrogels; Ophthalmic Solutions; Rabbits; Thymosin

Thymosin β4 (Tβ4) is closely associated with the cytoskeleton, inflammation, wound healing, angiogenesis, apoptosis, and myocardial regeneration, but the effects of Tβ4 treatment on chronic renal tubular interstitial fibrosis (CRTIF) are poorly known. This study aimed to examine the effects of Tβ4 on the renal apoptosis and the expression of transforming growth factor (TGF-β), E-cadherin, and α-smooth muscle actin (α-SMA) in CRTIF rat models.. Male SD rats were randomized into four groups (sham group, unilateral ureteral obstruction (UUO) group, UUO + low-dose Tβ4 group, and UUO + high-dose Tβ4 group). The pathological changes of kidney tissue and its function were assessed two weeks after UUO. In renal interstitial tissue,TGF-β, E-cadherin and α-SMA expression was detected by western blot. In tubular epithelial cells, E-cadherin and α-SMA expression was detected using Real-time qPCR and western blot. Cell apoptosis of rat renal interstitial tissue and tubular epithelial cells was evaluated by immunofluorescence and western blot.. Two weeks after UUO, no differences in blood urea nitrogen and creatinine were observed between the four groups (P > 0.05). Compared to the UUO group, Tβ4 treatment decreased the 24-h proteinuria (P < 0.001) and reduced the area of pathological change (P < 0.01); this effect was more apparent in the UUO + high-dose Tβ4 group. Compared to the UUO group, a significant decrease in TGF-β and α-SMA protein expression was observed in the high-dose Tβ4 group. The level of E-cadherin protein was lower in the UUO group than the Tβ4 groups, and high-dose Tβ4 treatment further increased E-cadherin expression and improved cell apoptosis in the renal interstitial tissue. Analysis of in vitro tubular epithelial cells showed that α-SMA mRNA and protein expression decreased, while E-cadherin mRNA and protein expression increased by Tβ4 treatment. Similarly, these changes were more significant in the UUO + high-dose Tβ4 group. Tβ4 treatment improved the apoptosis of In vitro tubular epithelial cells compared with pure TGF-β stimulation, and equally, the decrease of apoptosis was more apparent in the TGF-β + high-dose Tβ4 group.. Tβ4 treatment might alleviate the renal fibrosis and apoptosis of tubular epithelial cells through TGF-β pathway inhibition in UUO rats with CRTIF.

Topics: Actins; Animals; Apoptosis; Cadherins; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Fibrosis; Kidney; Kidney Tubules; Male; Proteinuria; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Thymosin; Transforming Growth Factor beta; Ureteral Obstruction

Rapid vascular remodelling of damaged dermal tissue is required to heal burn wounds. Thymosin β4 (Tβ4) is a growth factor that has been shown to promote angiogenesis and dermal wound repair. However, the underlying mechanisms based on Tβ4 function have not yet been fully investigated. In the present study, we investigated how Tβ4 improves dermal burn wound healing via actin cytoskeletal remodelling and the action of heat-shock proteins (HSPs), which are a vital set of chaperone proteins that respond to heat shock. Our in vitro results achieved with the use of human umbilical vein endothelial cells (HUVECs) revealed a possible signal between Tβ4 and HSP70. Moreover, we confirmed that remodelling of filamentous actin (F-actin) was regulated by Tβ4-induced HSP70 in HUVECs. Based on these in vitro results, we confirmed the healing effects of Tβ4 in an adapted dermal burn wound in vivo model. Tβ4 improved wound-healing markers, such as wound closure and vascularization. Moreover, Tβ4 maintained the long-term expression of HSP70, which is associated with F-actin regulation during the wound-healing period. These results suggest that an association between Tβ4 and HSP70 is responsible for the healing of burn wounds, and that this association may regulate F-actin remodelling. Copyright © 2015 John Wiley & Sons, Ltd.

Topics: Actin Cytoskeleton; Animals; Biomarkers; Burns; Cell Survival; Dermis; Disease Models, Animal; HSP70 Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Mice, Inbred C57BL; Models, Biological; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Thymosin; Vascular Endothelial Growth Factor A; Wound Healing

To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β. AAV-Tβ. Recombinant AAVs efficiently delivered LacZ and Tβ. Tβ

Topics: Animals; Cell Proliferation; Colitis, Ulcerative; Colon; Crohn Disease; Dependovirus; Dextran Sulfate; Disease Models, Animal; DNA, Recombinant; Enterocytes; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Immunochemistry; Interleukin-10; Interleukin-1beta; Intestinal Mucosa; Male; Malondialdehyde; Mice; Mice, Inbred BALB C; Peroxidase; Superoxide Dismutase; Thymosin; Trinitrobenzenesulfonic Acid; Tumor Necrosis Factor-alpha

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). No effective remyelination therapies are in use. We hypothesized that thymosin beta4 (Tβ4) is an effective remyelination treatment by promoting differentiation of oligodendrocyte progenitor cells (OPCs), and that the epidermal growth factor receptor (EGFR) signaling pathway contributes to this process. Two demyelination animal models were employed in this study: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated daily for 30days, with Tβ4 or saline treatment initiated on the day of EAE onset; and 2) cuprizone diet model, a non-inflammatory demyelination model. The mice were treated daily for 4weeks with Tβ4 or saline after fed a cuprizone diet for 5weeks. Immunofluorescent staining and Western blot were performed to measure the differentiation of OPCs, myelin and axons, respectively. To obtain insight into mechanisms of action, the expression and activation of the EGFR pathway was measured. AG1478, an EGFR inhibitor, was employed in a loss-of-function study. Data revealed that animals in both demyelination models exhibited significant reduction of myelin basic protein (MBP(+)) levels and CNPase(+) oligodendrocytes. Treatment of EAE mice with Tβ4 significantly improved neurological outcome. Double immunofluorescent staining showed that Tβ4 significantly increased the number of newly generated oligodendrocytes identified by BrdU(+)/CNPase(+) cells and MBP(+) mature oligodendrocytes, and reduced axonal damage in the EAE mice compared with the saline treatment. The newly generated mature oligodendrocytes remyelinated axons, and the increased mature oligodendrocytes significantly correlated with functional improvement (r=0.73, p<0.05). Western blot analysis revealed that Tβ4 treatment increased expression and activation of the EGFR pathway. In the cuprizone demyelination model, Tβ4 treatment was confirmed that significantly increased OPC differentiation and remyelination, and increased the expression of EGFR and activated the EGFR pathway in the demyelinating corpus callosum. In cultured OPCs, blockage of the activation of the EGFR pathway with AG1478 abolished the Tβ4-increased OPC differentiation. Collectively, these findings indicate that: 1) Tβ4 increases proliferation of OPCs and the maturation of OPCs to myelinating oligodendrocytes which in concert, likely contribute to the beneficial effect of Tβ4 on EAE, 2) EGFR upregul

Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chelating Agents; Cuprizone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; ErbB Receptors; Female; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Proteolipid Protein; Oligodendroglia; Peptide Fragments; Proteoglycans; Quinazolines; Stem Cells; Thymosin; Time Factors; Tyrphostins

The therapeutic efficiency of recombinant thymosin β4 (rTβ4) synthesized by us was studied in vivo on spontaneous CBRB mouse model that is adequate to human chronic dermatitis. Three applications of the drug during a week significantly alleviated symptoms of the disease in female mice, and in complex with subsequent antibacterial and antifungal therapy led to a pronounced and lasting (2 months) therapeutic effect. The results attest to a possibility of using rTβ4 in combination with the known treatment protocols for chronic inflammatory diseases of the skin.

Topics: Animals; Chronic Disease; Dermatitis; Disease Models, Animal; Female; Mice; Recombinant Proteins; Thymosin

Peripheral neuropathy is a chronic complication of diabetes mellitus. To investigated the efficacy and safety of the extended treatment of diabetic peripheral neuropathy with thymosin β4 (Tβ4), male diabetic mice (db/db) at the age of 24 weeks were treated with Tβ4 or saline for 16 consecutive weeks. Treatment of diabetic mice with Tβ4 significantly improved motor (MCV) and sensory (SCV) conduction velocity in the sciatic nerve and the thermal and mechanical latency. However, Tβ4 treatment did not significantly alter blood glucose levels. Treatment with Tβ4 significantly increased intraepidermal nerve fiber density. Furthermore, Tβ4 counteracted the diabetes-induced axon diameter and myelin thickness reductions and the g-ratio increase in sciatic nerve. In vitro, compared with dorsal root ganglia (DRG) neurons derived from nondiabetic mice, DRG neurons derived from diabetic mice exhibited significantly decreased neurite outgrowth, whereas Tβ4 promoted neurite growth in these diabetic DRG neurons. Blockage of the Ang1/Tie2 signaling pathway with a neutralized antibody against Tie2 abolished Tβ4-increased neurite outgrowth. Our data demonstrate that extended Tβ4 treatment ameliorates diabetic-induced axonal degeneration and demyelination, which likely contribute to therapeutic effect of Tβ4 on diabetic neuropathy. The Ang1/Tie2 pathway may mediate Tβ4-induced axonal remodeling.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Ganglia, Spinal; Male; Mice; Nerve Fibers; Sciatic Nerve; Signal Transduction; Thymosin; Treatment Outcome

The molecular anatomy of healthy and atherosclerotic tissue is pursued here to identify ongoing molecular changes in atherosclerosis development. Subclinical atherosclerosis cannot be predicted and novel therapeutic targets are needed. Mass spectrometry imaging (MSI) is a novel unexplored ex vivo imaging approach in CVD able to provide in-tissue molecular maps. A rabbit model of early atherosclerosis was developed and high-spatial-resolution MALDI-MSI was applied to comparatively analyze histologically-based arterial regions of interest from control and early atherosclerotic aortas. Specific protocols were applied to identify lipids and proteins significantly altered in response to atherosclerosis. Observed protein alterations were confirmed by immunohistochemistry in rabbit tissue, and additionally in human aortas. Molecular features specifically defining different arterial regions were identified. Localized in the intima, increased expression of SFA and lysolipids and intimal spatial organization showing accumulation of PI, PG and SM point to endothelial dysfunction and triggered inflammatory response. TG, PA, SM and PE-Cer were identified specifically located in calcified regions. Thymosin β4 (TMSB4X) protein was upregulated in intima versus media layer and also in response to atherosclerosis. This overexpression and localization was confirmed in human aortas. In conclusion, molecular histology by MS Imaging identifies spatial organization of arterial tissue in response to atherosclerosis.

Topics: Animals; Aorta; Atherosclerosis; Disease Models, Animal; Humans; Lipids; Mass Spectrometry; Rabbits; Thymosin; Tunica Intima; Vascular Calcification

Dry eye syndrome is a common condition that affects up to 20% of the population aged 45 and older. There are no successful treatments to date. The goal of this research was to determine the efficacy of various doses and the optimal frequency of thymosin β4 (Tβ4) treatment in a murine severe dry eye model.. The study was performed using a controlled adverse environment chamber (CAE) in combination with scopolamine to induce moderate to severe dry eye in mice. The study included five mice per group and tested six different doses of Tβ4 twice per day for 12 days. Tβ4 at 0.1% was also administered 2 - 4 times per day for 12 days. Healing was measured by fluorescein staining.. Tβ4 significantly reduced the signs of dry eye relative to controls. The treatment effect was more pronounced than the positive controls, doxycycline and Restasis (cyclosporine 0.05%). Active doses of 0.1 and 0.5% were determined, and it was found that the frequency of dosing at 2 times per day was the most effective for healing.. Tβ4 has the potential to be an important new effective therapeutic for dry eye.

Topics: Administration, Topical; Animals; Cyclosporine; Disease Models, Animal; Dose-Response Relationship, Drug; Dry Eye Syndromes; Environment, Controlled; Female; Humans; Mice; Mice, Inbred C57BL; Ophthalmic Solutions; Thymosin; Wound Healing

Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis.. In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis.. We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin.. In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days.. We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Cells, Cultured; Disease Models, Animal; Fibroblasts; Humans; Lung; Male; Mice; Oligopeptides; Pneumonia; Protein Structure, Tertiary; Pulmonary Fibrosis; Thymosin; Transforming Growth Factor beta

Thymosin β4 (Tβ4) is a highly conserved peptide with immunomodulatory properties. In this research we investigated the effects of Tβ4 on the bleomycin-induced lung damage in CD-1 mice and the changes in the number of IL-17-producing cells as well as the IL-17 expression in the lung. Male CD-1 mice were treated with bleomycin (1mg/kg) in the absence or the presence of Tβ4 (6mg/kg delivered intra-peritoneally on the day of bleomycin treatment and for 2 additional doses). After sacrifice one week later, lung histology, measurement of collagen content of the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, evaluation of IL17-producing cells in the blood as well as RT-PCR and IHC in the lung tissue were performed. As expected, bleomycin-induced inflammation and lung damage were substantially reduced by Tβ4 treatment in CD-1 mice, as shown by the significant reduction of (i) leukocytes in BALF, (ii) histological evidence of the lung damage, and (iii) total collagen content in the lung. Importantly, the bleomycin-induced increase in the number of IL17-producing cells in the blood was significantly blocked by Tβ4. Accordingly, IHC and RT-PCR results demonstrated that Tβ4 substantially inhibited bleomycin-induced IL-17 over-expression in the lung tissue. This is the first report showing that a decreased amount of IL17-producing cells and inhibited IL-17 expression in the lung with Tβ4 treatment correlate with its anti-inflammatory and anti-fibrotic effects.

Topics: Animals; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Disease Models, Animal; Inflammation; Interleukin-17; Leukocytes; Lung; Lung Injury; Male; Mice; Pulmonary Fibrosis; Random Allocation; Thymosin

Thymosin β4 (Tβ4) has many physiological functions that are highly relevant to spinal cord injury (SCI), including neuronal survival, anti-inflammation, wound repair promotion, and angiogenesis. The present study investigated the therapeutic value of Tβ4 in SCI, with a focus on its neuroprotective, anti-inflammatory, and vasculoprotective properties. Tβ4 or a saline control was administered by intraperitoneal injection 30 min, 3 days, or 5 days after SCI with mild compression in rat. Locomotor recovery was tested with the Basso-Beattie-Bresnahan scale and a footprint analysis. All behavioral assessments were markedly improved with Tβ4 treatment. Histological examination at 7 days post injury showed that the numbers of surviving neurons and oligodendrocytes were significantly increased in Tβ4-treated animals compared to saline-treated controls. Levels of myelin basic protein, a marker of mature oligodendrocytes, in Tβ4-treated rats were 57.8% greater than those in saline-treated controls. The expression of ED1, a marker of activated microglia/macrophages, was reduced by 36.9% in the Tβ4-treated group compared to that of the saline-treated group. Tβ4 treatment after SCI was also associated with a significant decrease in pro-inflammatory cytokine gene expression and a significant increase in the mRNA levels of IL-10 compared to the control. Moreover, the size of lesion cavity delineated by astrocyte scar in the injured spinal cord was markedly reduced in Tβ4-treated animals compared to saline-treated controls. Given the known safety of Tβ4 in clinical trials and its beneficial effects on SCI recovery, the results of this study suggested that Tβ4 is a good candidate for SCI treatment in humans.

Topics: Animals; Cell Survival; Cytokines; Disease Models, Animal; Interleukin-10; Macrophages; Male; Microglia; Myelin Basic Protein; Neuroimmunomodulation; Neurons; Neuroprotective Agents; Oligodendroglia; Random Allocation; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Thymosin; Time Factors

Thymosin β4 (Tβ4) is a 5K actin binding peptide. Tβ4 improves neurological outcome in a rat model of embolic stroke and research is now focused on optimizing its dose for clinical trials. The purpose of this study was to perform a dose-response study of Tβ4 to determine the optimal dose of neurological improvement in a rat model of embolic stroke.. Male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were divided into 4 groups of 10 animals/group: control, 2, 12 and 18 mg/kg. Tβ4 was administered intraperitoneally 24h after MCAo and then every 3 days for 4 additional doses in a randomized controlled fashion. Neurological tests were performed after MCAo and before treatment and up to 8 weeks after treatment. The rats were sacrificed 56 days after MCAo and lesion volumes measured. Generalized estimating equation was used to compare the treatment effect on long term functional recovery at day 56. A quartic regression model was used for an optimal dose determination.. Tβ4 significantly improved neurological outcome at dose of 2 and 12 mg/kg at day 14 and extended to day 56 (p-values <0.05). The higher dose of 18 mg/kg did not show significant improvement. The estimated optimal dose of 3.75 mg/kg would provide optimal neurological improvement.. This study shown that Tβ4 significantly improved the long term neurological functional recovery at day 56 after MCAo with an optimal dose of 3.75 mg/kg. These results provide preclinical data for human clinical trials.

Topics: Acute Disease; Adenomatous Polyposis Coli; Animals; Brain; Bromodeoxyuridine; Disease Models, Animal; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Male; Myelin Basic Protein; Neuroimaging; Neurologic Examination; Rats; Stroke; Thymosin; Time Factors; Treatment Outcome; Versicans

Thymosin beta-4 (Tβ4) is known to be involved in tumorigenesis. Overexpression of this polypeptide has been observed in a wide variety of cancers, including colorectal carcinoma (CRC). Accordingly, Tβ4 has been proposed to be a novel therapeutic target for CRC, especially in its metastatic form. Although in vitro tumor-suppressive effects of Tβ4 gene silencing mediated by small hairpin RNA (shRNA) have already been demonstrated, the in vivo efficacy of such an approach has not yet been reported. Herein, we demonstrated that infection with recombinant adenovirus expressing an shRNA targeting Tβ4 markedly reduced the growth of and robustly induced apoptosis in CT-26 mouse CRC cells in culture. Additionally, tumors grown in nude mice from the CT-26 cells whose Tβ4 expression already been downregulated by virus infection were also drastically reduced. Most importantly, significant growth arrest of tumors derived from the parental CT-26 cells was observed after multiple intratumoral injections of these viruses. Together, our results show for the first time that in vivo silencing of Tβ4 expression by its shRNA generated after adenoviral infection can suppress CRC growth. These results further demonstrate the feasibility of treating CRC by a Tβ4 knockdown gene therapeutic approach.

Topics: Actins; Adenoviridae; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Models, Animal; Gene Knockout Techniques; Genetic Vectors; Humans; Mice; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thymosin; Transduction, Genetic; Tumor Burden; Xenograft Model Antitumor Assays

Pulmonary hypertension (PH) is a progressive vascular disease of pulmonary arteries that impedes ejection of blood by the right ventricle. As a result there is an increase in pulmonary vascular resistance and pulmonary arterial pressure causing right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Current therapies are limited to reverse the vascular remodeling. Investigating a key molecule is required for development of new therapeutic intervention. Thymosin beta-4 (Tβ4) is a ubiquitous G-actin sequestering protein with diverse biological function and promotes wound healing and modulates inflammatory responses. However, it remains unknown whether Tβ4 has any protective role in PH. The purpose of this study is to evaluate the whether Tβ4 can be used as a vascular-protective agent. In monocrotaline (MCT)-induced PH mouse model, we showed that mice treated with Tβ4 significantly attenuated the systolic pressure and RVH, compared to the MCT treated mice. Our data revealed for the first time that Tβ4 selectively targets Notch3-Col 3A-CTGF gene axis in preventing MCT-induced PH and RVH. Our study may provide pre-clinical evidence for Tβ4 and may consider as vasculo-protective agent for the treatment of PH induced RVH.

Topics: Animals; Cells, Cultured; Collagen Type III; Connective Tissue Growth Factor; Disease Models, Animal; Endothelial Cells; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Injections, Intraperitoneal; Lung; Male; Mice; Monocrotaline; Receptor, Notch3; Receptors, Notch; Signal Transduction; Thymosin

Previously, we found thymosin β4 (Tβ4) is upregulated in glomerulosclerosis and required for angiotensin II-induced expression of plasminogen activator inhibitor-1 (PAI-1) in glomerular endothelial cells. Tβ4 has beneficial effects in dermal and corneal wound healing and heart disease, yet its effects in kidney disease are unknown. Here we studied renal fibrosis in wild-type and PAI-1 knockout mice following unilateral ureteral obstruction to explore the impact of Tβ4 and its prolyl oligopeptidase tetrapeptide degradation product, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), in renal fibrosis. Additionally, we explored interactions of Tβ4 with PAI-1. Treatment with Ac-SDKP significantly decreased fibrosis in both wild-type and PAI-1 knockout mice, as observed by decreased collagen and fibronectin deposition, fewer myofibroblasts and macrophages, and suppressed profibrotic factors. In contrast, Tβ4 plus a prolyl oligopeptidase inhibitor significantly increased fibrosis in wild-type mice. Tβ4 alone also promoted repair and reduced late fibrosis in wild-type mice. Importantly, both profibrotic effects of Tβ4 plus the prolyl oligopeptidase inhibitor, and late reparative effects of Tβ4 alone, were absent in PAI-1 knockout mice. Thus, Tβ4 combined with prolyl oligopeptidase inhibition is consistently profibrotic, but by itself has antifibrotic effects in late-stage fibrosis, while Ac-SDKP has consistent antifibrotic effects in both early and late stages of kidney injury. These effects of Tβ4 are dependent on PAI-1.

Topics: Animals; Collagen; Disease Models, Animal; Fibronectins; Fibrosis; Kidney; Kidney Diseases; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Oligopeptides; Plasminogen Activator Inhibitor 1; Prolyl Oligopeptidases; Serine Endopeptidases; Serine Proteinase Inhibitors; Thymosin; Time Factors; Ureteral Obstruction; Urological Agents

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin β4 (Tβ4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tβ4-induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tβ4-stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tβ4 promoted proliferation, migration and trans-differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell-based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm-setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields.

Topics: Animals; Biomarkers; Carotid Artery Injuries; Cell Differentiation; Cell Lineage; Cell Movement; Cell Proliferation; Cell- and Tissue-Based Therapy; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Mice; Mice, Inbred ICR; Myocytes, Smooth Muscle; Omentum; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Stem Cells; Thymosin; Wound Healing

Thymosin β4 (Tβ4) was recently found at high concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with lung involvement. It has been hypothesized that Tβ4 may exert a cyto-protective effect during lung injury because lower Tβ4 levels were associated with interstitial lung disease progression. Moreover, Tβ4 treatment prevented profibrotic gene expression in cardiac cells in vitro and in vivo.. In this study, we explored a putative Tβ4 protective role in lung damage by utilizing a well-known in vivo model of lung fibrosis. C57BL/6 mice were treated with bleomycin (BLEO, 1 mg/kg) in the absence or presence of Tβ4 (6 mg/kg delivered intraperitoneally on the day of BLEO treatment and for two additional doses). After sacrifice 1 week later, measurement of fluid and collagen content in the lung, BALF analysis, myeloperoxidase (MPO) activity assay, lung histology and IHC were performed.. Compared with BLEO-treated mice, BLEO-treated mice who received Tβ4 did not lose as much weight and had a higher survival rate. Moreover, BLEO-induced inflammation and lung damage were substantially reduced by Tβ4 treatment, as demonstrated by the significant reduction in oedema, total collagen content, lung infiltration by leucocytes, MPO activity in lung homogenates, and histological evidence of the ongoing lung fibrosis. Results of IHC show a strong reactivity for Tβ4 in the lung tissue of Tβ4-treated mice.. This is the first report that shows a Tβ4 protective role in lung toxicity associated with BLEO in a mouse model. Future studies are needed to assess its putative antifibrotic properties.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Disease Models, Animal; Lung Injury; Male; Mice; Mice, Inbred C57BL; Peroxidase; Protective Agents; Pulmonary Edema; Pulmonary Fibrosis; Random Allocation; Thymosin; Weight Loss

Peripheral neuropathy is one of the most common complications of diabetes mellitus. Using a mouse model of diabetic peripheral neuropathy, we tested the hypothesis that thymosin β4 (Tβ4) ameliorates diabetes-induced neurovascular dysfunction in the sciatic nerve and promotes recovery of neurological function from diabetic peripheral neuropathy. Tβ4 treatment of diabetic mice increased functional vascular density and regional blood flow in the sciatic nerve, and improved nerve function. Tβ4 upregulated angiopoietin-1 (Ang1) expression, but suppressed Ang2 expression in endothelial and Schwann cells in the diabetic sciatic nerve. In vitro, incubation of Human Umbilical Vein Endothelial Cells (HUVECs) with Tβ4 under high glucose condition completely abolished high glucose-downregulated Ang1 expression and high glucose-reduced capillary-like tube formation. Moreover, incubation of HUVECs under high glucose with conditioned medium collected from Human Schwann Cells (HSCs) treated with Tβ4 significantly reversed high glucose-decreased capillary-like tube formation. PI3K/Akt signaling pathway is involved in Tβ4-regulated Ang1 expression on endothelial and Schwann cells. These data indicate that Tβ4 likely acts on endothelial cells and Schwann cells to preserve and/or restore vascular function in the sciatic nerve which facilitates improvement of peripheral nerve function under diabetic neuropathy. Thus, Tβ4 has potential for the treatment of diabetic peripheral neuropathy.

Topics: Animals; Blotting, Western; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Neuropathies; Disease Models, Animal; Electrophysiology; Endothelial Cells; Humans; Immunohistochemistry; Laser-Doppler Flowmetry; Mice; Neuroprotective Agents; Real-Time Polymerase Chain Reaction; Regional Blood Flow; Reverse Transcriptase Polymerase Chain Reaction; Schwann Cells; Sciatic Nerve; Signal Transduction; Thymosin

Thymosin β4 (Tβ4) is a peptide known for its abilities to protect and facilitate regeneration in a number of tissues following injury. Its cardioprotective effects have been evaluated in different animal models and, currently, a clinical trial is being planned in patients suffering from acute myocardial infarction. This paper focuses on the effects of Tβ4 on cardiac function in animal studies utilizing different imaging modalities for outcome measurements.

Topics: Animals; Disease Models, Animal; Heart Failure; Humans; Myocardial Infarction; Thymosin

Clinical administration of bone marrow-derived stem cells in the setting of acute myocardial infarction (AMI) leads to improved left ventricular ejection fraction. Thymosin beta-4 (TB4) and vascular endothelial growth factor (VEGF) are linked to adult epicardial progenitor cell mobilization and neovascularization and is cardioprotective after myocardial ischemia. This study investigated the time course of TB4 and VEGF during AMI, cardiac arrest, and resuscitation. Fifteen anesthetized and instrumented domestic swine underwent balloon occlusion of the proximal left anterior descending coronary artery. During occlusion, venous blood samples were collected from the right atrium at 5-min intervals until 15 min after the onset of cardiopulmonary resuscitation (CPR). Plasma levels of TB4, VEGF, and matrix metalloproteinase-9 (MMP-9, selected as a marker for remodeling and repair) were measured by ELISA. Generalized linear mixed models were employed to model the time-dependent change in plasma concentration. All variables were natural log transformed, except TB4 values, to normalize distributions. Fifteen animals successfully underwent balloon occlusion of the left anterior descending coronary artery and samples were collected from these subjects. The average onset of spontaneous ventricular fibrillation was 28 min. TB4, VEGF, and MMP-9 demonstrated a statistically significant, time-dependent increase in concentration during ischemia. Following arrest and throughout the first 15 min of resuscitation, MMP-9 had an unchanged rate of rise when compared with the prearrest, ischemic period, with VEGF showing a deceleration in its time-dependent concentration trajectory and TB4 demonstrating an acceleration. Endogenous TB4 and VEGF increase shortly after the onset of AMI and increase through cardiac arrest and resuscitation in parallel to remodeling proteases. These markers continue to rise during successful resuscitation and may represent an endogenous mechanism to recruit undifferentiated stem cells to areas of myocardial injury.

Topics: Animals; Balloon Occlusion; Biomarkers; Cell Differentiation; Cell Movement; Disease Models, Animal; Heart Arrest; Humans; Matrix Metalloproteinase 9; Myocardial Ischemia; Regenerative Medicine; Resuscitation; Stem Cell Transplantation; Swine; Thymosin; Up-Regulation; Vascular Endothelial Growth Factor A; Ventricular Remodeling

In recent years, various types of stem cells have been characterized and their potential for cardiac regeneration has been investigated. We have previously described the isolation of broadly multipotent cells from amniotic fluid, defined as amniotic fluid stem (AFS) cells. The aim of this study was to investigate the therapeutic potential of human AFS cells (hAFS) in a model of acute myocardial infarction. Wistar rats underwent 30 min of ischemia by ligation of the left anterior descending coronary artery, followed by administration of hAFS cells and 2 h of reperfusion. Infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining and planimetry. hAFS cells were also analyzed by enzyme-linked immunosorbent assay to detect secretion of putative paracrine factors, such as the actin monomer-binding protein thymosin β4 (Tβ4). The systemic injection of hAFS cells and their conditioned medium (hAFS-CM) was cardioprotective, improving myocardial cell survival and decreasing the infarct size from 53.9%±2.3% (control animals receiving phosphate-buffered saline injection) to 40.0%±3.0% (hAFS cells) and 39.7%±2.5% (hAFS-CM, P<0.01). In addition, hAFS cells were demonstrated to secrete Tβ4, previously shown to be both cardioprotective and proangiogenic. Our results suggest that AFS cells have therapeutic potential in the setting of acute myocardial infarction, which may be mediated through paracrine effectors such as Tβ4. Therefore, AFS cells might represent a novel source for cell therapy and cell transplantation strategies in repair following ischemic heart disease, with a possible paracrine mechanism of action and a potential molecular candidate for acute cardioprotection.

Topics: Amniotic Fluid; Animals; Antigens, Differentiation; Apoptosis; Cells, Cultured; Disease Models, Animal; Female; Humans; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Pregnancy; Rats; Rats, Wistar; Stem Cell Transplantation; Stem Cells; Thymosin

Thymosin beta4 (Tbeta4) is a polypeptide involved in cellular proliferation, differentiation, and migration, over-expressed in several tumor entities. We evaluated its expression and function in 298 newly diagnosed multiple myeloma patients and the murine 5TMM model. Mean Tbeta4 expression was significantly lower in myeloma cells compared to normal plasma cells (P<0.001). The same observation can be made in the 5TMM-mouse model by qRT-PCR and ELISA. Here, Tbeta4 overexpression by lentiviral transduction of 5T33MMvt-cells led to significantly decreased proliferative and migratory capacities and increased sensitivity to apoptosis-induction. Mice injected with Tbeta4 over-expressing myeloma cells showed a longer survival compared to mice injected with controls (88,9 vs. 65,9 days, P<0.05). In 209 MM patients treated with high-dose therapy and autologous stem cell transplantation, expression of Tbeta4 below the median was associated with a significantly shorter event free survival (37.6 vs. 26.2 months, P<0.05). In conclusion, our results indicate a possible tumor suppressive function of Tbeta4.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mice, Inbred C57BL; Multiple Myeloma; Prognosis; Survival Rate; Thymosin; Tumor Suppressor Proteins

Thymosin beta4 (Tbeta4) is an actin-binding peptide whose expression in developing brain correlates with migration and neurite extension of neurons. Here, we studied the effects of the downregulation of Tbeta4 expression on growth and differentiation of murine neural progenitor cells (NPCs), using an antisense lentiviral vector. In differentiation-promoting medium, we found twice the number of neurons derived from the Tbeta4-antisense-transduced NPCs, which showed enhanced neurite outgrowth accompanied by increased expression of the adhesion complex N-cadherin-beta-catenin and increased ERK activation. Importantly, when the Tbeta4-antisense-transduced NPCs were transplanted in vivo into a mouse model of spinal cord injury, they promoted a significantly greater functional recovery. Locomotory recovery correlated with increased expression of the regeneration-promoting cell adhesion molecule L1 by the grafted Tbeta4-antisense-transduced NPCs. This resulted in an increased number of regenerating axons and in sprouting of serotonergic fibers surrounding and contacting the Tbeta4-antisense-transduced NPCs grafted into the lesion site. In conclusion, our data identify a new role for Tbeta4 in neuronal differentiation of NPCs by regulating fate determination and process outgrowth. Moreover, NPCs with reduced Tbeta4 levels generate an L1-enriched environment in the lesioned spinal cord that favors growth and sprouting of spared host axons and enhances the endogenous tissue-repair processes.

Topics: Animals; Axons; beta Catenin; Cadherins; Cell Communication; Disease Models, Animal; DNA, Antisense; Down-Regulation; Mice; Nerve Regeneration; Neural Cell Adhesion Molecule L1; Neurogenesis; Neurons; Spinal Cord Injuries; Stem Cell Transplantation; Stem Cells; Telencephalon; Thymosin

To explore the mechanism of recombinant thymosin beta4 (Tbeta4) accelerating skin wound healing in rats by regulating laminin 5 expression.. Two full thickness 8 mm punch wounds were made at the costovertebral angle on dorsal surface of each adult male rats weighing 200-250 g. Sixty rats were randomized into the control group (n = 15) and the experimental group (n = 45), which was subdivided into low, medium and high dose groups (n = 15). Tbeta4 was applied topically at 2, 6, 18 microg in 50 microL PBS for every 12 hours after model making in the experimental group. The identical amounts of phosphate buffered saline was applied in the control group. Wound healing was observed after model making and immunohistochemical observation was conducted 2, 4 and 7 days after operation.. Seven days after operation, wound contracted obviously and most of the wounds connected well with the margin. In the control group, low dose group, medium dose group and high dose group, the wound healing rate were 7.67% +/- 5.46%, 29.01% +/- 7.43%, 26.54% +/- 11.49% and 10.39% +/- 3.96% respectively 2 days after operation; 28.16% +/- 13.76%, 37.99% +/- 13.05%, 42.00% +/- 9.56% and 39.58% +/- 12.74% respectively 4 days after operation; 59.08% +/- 19.02%, 64.15% +/- 17.92%, 77.39% +/- 8.45% and 69.78% +/- 8.45% respectively 7 days after operation. At 2 days after operation, significant differences were notified in healing rats between 3 sub-experimental groups and the control group (P < 0.05). Immunohistochemistry staining showed that there was a little more positive expression of laminin 5 2 days after operation that beneficial to promote the proliferation and differentiation of cell in every group, including positive cells and ECM. But in medium group there was fewer expression, only at the borderline and bottom of the wound, while the expression significantly increased 4 days after operation (P < 0.05) and there was a relative high expression 7 days after operation (P < 0.01).. Tbeta4 can inhibit the expression of laminin 5 early, and then up-regulate laminin 5 expression to moderate the reformation of ECM, promote the migration of epidemic cell and accelerate skin wound healing.

Topics: Animals; Cell Adhesion Molecules; Disease Models, Animal; Kalinin; Male; Rats; Rats, Sprague-Dawley; Skin; Soft Tissue Injuries; Thymosin; Wound Healing

Human thymosin beta 4 (TB4) is a small acidic peptide involved in angiogenesis, wound healing, cancer metastasis and cardiac repair. Currently human TB4 is synthesized chemically for research and this is costly. In order to obtain sufficient biologically active human TB4 economically, we cloned and overexpressed this protein in an Escherichia coli system. We also developed a one-step affinity purification method to purify this fusion protein. After the fusion tag was removed from the fusion protein through autohydrolysis by dithiothreitol (DTT), the biological activity and function of this recombinant human TB4 was evaluated by cell proliferation assay using prepared spleen cells and wound assay using a mouse model, respectively. Our data demonstrated that human recombinant TB4 can promote lymphocyte proliferation and differentiation. Further, it can also promote full-thickness cutaneous wound healing in BALB/c mice. To our knowledge, this is the first report of recombinant human TB4 with the ability to promote wound healing.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cells, Cultured; Disease Models, Animal; Escherichia coli; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Recombinant Proteins; Skin; Skin Physiological Phenomena; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymosin; Wound Healing

Corneal alkali injury is highly caustic, and present clinical therapies are limited. The purpose of this study was to investigate the ability of thymosin-beta4 (Taubeta4) to promote healing in an alkali injury model and the mechanisms involved in that process.. Corneas of BALB/c mice were injured with NaOH, irrigated copiously with PBS, and treated topically with either Tbeta4 or PBS twice daily. At various time points after injury (PI), corneas from the Tbeta4- versus the PBS-treated group were examined for polymorphonuclear leukocyte (PMN) infiltration, chemokine, and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) expression.. Tbeta4-treated corneas demonstrated improved corneal clarity at day 7 PI. Whereas Tbeta4 decreased corneal MMP-2 and -9 and MT6-MMP levels after alkali injury, no change in TIMP-1 and -2 expression was detected. Tbeta4 treatment also decreased corneal KC (CXCL1) and macrophage inflammatory protein (MIP)-2 chemokine expression and PMN infiltration. Immunohistochemistry studies demonstrated MMP-9 expression at the leading edge of the epithelial wound, in the the limbus (containing stem cells), and in stromal PMNs.. Tbeta4 treatment decreases corneal inflammation and modulates the MMP/TIMP balance and thereby promotes corneal wound repair and clarity after alkali injury. These results suggest that Tbeta4 may be useful clinically to treat severe inflammation-mediated corneal injuries.

Topics: Animals; Burns, Chemical; Chemokines; Corneal Diseases; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Burns; Matrix Metalloproteinases; Mice; Mice, Inbred BALB C; Neutrophil Infiltration; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; Sodium Hydroxide; Thymosin; Tissue Inhibitor of Metalloproteinases; Wound Healing