thymosin-beta(4) and Myocardial-Infarction

Our dream of defeating the processes of organ damage and aging remains a challenge scientists pursued for hundreds of years. Although the goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. Since initial approaches utilizing only progenitor cells appear limited, we propose interconnecting our collective knowledge regarding aging and embryonic development may lead to the discovery of molecules which provide alternatives to effectively reverse cellular damage. In this review, we introduce and summarize our results regarding Thymosin beta-4 (TB4) to support our hypothesis using the heart as model system. Accordingly, we investigated the developmental expression of TB4 in mouse embryos and determined the impact of the molecule in adult animals by systemically injecting the peptide following acute cardiac infarction or with no injury. Our results proved, TB4 is expressed in the developing heart and promotes cardiac cell migration and survival. In adults, the peptide enhances myocyte survival and improves cardiac function after coronary artery ligation. Moreover, intravenous injections of TB4 alter the morphology of the adult epicardium, and the changes resemble the characteristics of the embryo. Reactivation of the embryonic program became equally reflected by the increased number of cardiac vessels and by the alteration of the gene expression profile typical of the embryonic state. Moreover, we discovered TB4 is capable of epicardial progenitor activation, and revealed the effect is independent of hypoxic injury. By observing the above results, we believe, further discoveries and consequential postnatal administration of developmentally relevant candidate molecules such as TB4 may likely result in reversing aging processes and accelerate organ regeneration in the human body.

Topics: Aging; Animals; Humans; Mice; Myocardial Infarction; Peptides; Pericardium; 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

The burden of cardiovascular disease is a growing worldwide issue that demands attention. While many clinical trials are ongoing to test therapies for treating the heart after myocardial infarction (MI) and heart failure, there are few options doctors able to currently give patients to repair the heart. This eventually leads to decreased ventricular contractility and increased systemic disease, including vascular disorders that could result in stroke. Small peptides such as thymosin β4 (Tβ4) are upregulated in the cardiovascular niche during fetal development and after injuries such as MI, providing increased neovasculogenesis and paracrine signals for endogenous stem cell recruitment to aid in wound repair. New research is looking into the effects of in vivo administration of Tβ4 through injections and coatings on implants, as well as its effect on cell differentiation. Results so far demonstrate Tβ4 administration leads to robust increases in angiogenesis and wound healing in the heart after MI and the brain after stroke, and can differentiate adult stem cells toward the cardiac lineage for implantation to the heart to increase contractility and survival. Future work, some of which is currently in clinical trials, will demonstrate the in vivo effect of these therapies on human patients, with the goal of helping the millions of people worldwide affected by cardiovascular disease.

Topics: Cardiovascular Diseases; Cardiovascular System; Cell Differentiation; Humans; Myocardial Infarction; Stroke; Thymosin; Tissue Scaffolds

Despite recent improvements in interventional medicine, cardiovascular disease still represents the major cause of morbidity worldwide, with myocardial infarction being the most common cardiac injury. This has sustained the development of several regenerative strategies based on the use of stem cells and tissue engineering approaches in order to achieve cardiac repair and regeneration by enhancing coronary neovascularization, modulating acute inflammation and supporting myocardial regeneration to provide new functional muscle.. The actin monomer binding peptide, Thymosin β4 (Tβ4), has recently been described as a powerful regenerative agent with angiogenic, anti-inflammatory and cardioprotective effects on the heart and which specifically acts on its resident cardiac progenitor cells. In this review we will discuss the state of the art regarding the many roles of Tβ4 in preserving and regenerating the mammalian heart, with specific attention to its ability to activate the quiescent adult epicardium and specific subsets of epicardial progenitor cells for repair.. The therapeutic potential of Tβ4 for the treatment of cardiac failure is herein evaluated alongside existing, emerging and prospective novel treatments.

Topics: Animals; Cardiotonic Agents; Heart Failure; Humans; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Pericardium; Regeneration; Thymosin; Tissue Engineering

Epicardial resident stem cells are known to differentiate into cardiomyocytes during cardiac development, amongst other cell types. Whether epicardium-derived progenitor cells (EPDCs) retain this plasticity in the adult heart has been the topic of heated scientific debate. Priming with thymosin beta 4, a peptide which has been suggested to be critical for cardiac development and to have cardio-protective properties, was recently shown to induce differentiation of EPDCs into cardiomyocytes in a small animal model of myocardial infarction. This finding is in stark contrast to another recent study in which thymosin beta 4 treatment following myocardial infarction did not induce cardiomyocyte differentiation of EPDCs. While EPDCs seem to exhibit overall cardio-protective effects on the heart following myocardial infarction, they have not been shown to differentiate into cardiomyocytes in a clinically relevant setting. It will be important to understand why the ability of one therapeutic agent to induce cardiomyocyte differentiation of EPDCs seemingly depends on a single variable, i.e. the time of administration. Furthermore, in light of a recent report, it appears that thymosin beta 4 may be dispensable for cardiac development.

Topics: Cell Differentiation; Heart; Humans; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Regeneration; Stem Cell Transplantation; Stem Cells; Thymosin

The mammalian heart loses its regenerative capacity during early postnatal stages; consequently, individuals surviving myocardial infarction are at risk of heart failure due to excessive fibrosis and maladaptive remodeling. There is an urgent need, therefore, to develop novel therapies for myocardial and coronary vascular regeneration. The epicardium-derived cells present a tractable resident progenitor source with the potential to stimulate neovasculogenesis and contribute de novo cardiomyocytes. The ability to revive ordinarily dormant epicardium-derived cells lies in the identification of key stimulatory factors, such as Tβ4, and elucidation of the molecular cues used in the embryo to orchestrate cardiovascular development. myocardial infarction injury signaling reactivates the adult epicardium; understanding the timing and magnitude of these signals will enlighten strategies for myocardial repair.

Topics: Coronary Vessels; Extracellular Matrix; Humans; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Pericardium; Regenerative Medicine; Signal Transduction; Stem Cells; Thymosin; Vascular Endothelial Growth Factor A

Thymosin β4 (Tβ4) is a peptide with multiple biological functions. Here, we focus on the role of Tβ4 in vascularization, and review our studies of the controlled delivery of Tβ4 through its incorporation in biomaterials. Tβ4 promotes vascularization through VEGF induction and AcSDKP-induced migration and differentiation of endothelial cells. We developed a collagen-chitosan hydrogel for the controlled release of Tβ4 over 28 days. In vitro, the Tβ4-encapsulated hydrogel increased migration of endothelial cells and tube formation from epicardial explants that were cultivated on top of the hydrogel, compared to Tβ4-free hydrogel and soluble Tβ4 in the culture medium. In vivo, subcutaneously injected Tβ4-containing collagen-chitosan hydrogel in rats led to enhanced vascularization compared to Tβ4-free hydrogel and collagen hydrogel with Tβ4. Furthermore, the injection of the Tβ4-encapsulated hydrogel in the infarct region improved angiogenesis, reduced tissue loss, and retained left ventricular wall thickness after myocardial infarction in rats.

Topics: Animals; Biocompatible Materials; Hydrogel, Polyethylene Glycol Dimethacrylate; Myocardial Infarction; Neovascularization, Physiologic; Rats; Regenerative Medicine; Thymosin; Tissue Engineering

Thymosin beta4 - an endogenously occurring 43 amino acid peptide - has recently been shown to possess cardioprotective properties in the setting of acute myocardial infarction. This review focuses on the reported mechanisms of action through which Thymosin beta4 might accomplish this effect and the clinical prospects for its use as a therapeutic agent.

Topics: Animals; Cardiotonic Agents; Humans; Myocardial Infarction; Oligopeptides; Thymosin

Despite the numerous advances made in the prevention and treatment of cardiovascular diseases, there is a need for new strategies to repair and/or regenerate the myocardium after ischemia and infarction in order to prevent maladaptive remodeling and cardiac dysfunction. This article compiles and analyzes the available experimental data regarding the potential therapeutic effects of thymosin-beta4 and its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) in cardiac healing after myocardial infarction (MI) as well as discussing the possible mechanisms involved. The healing properties of thymosin-beta4 have been described in different types of tissues, such as the skin and cornea, and more recently it has been shown that thymosin-beta4 facilitates cardiac repair after infarction by promoting cell migration and myocyte survival. Additionally, the tetrapeptide Ac-SDKP was reported to reduce left ventricular fibrosis in hypertensive rats, reverse fibrosis and inflammation in rats with MI, and stimulate both in vitro and in vivo angiogenesis. Ac-SDKP also reduced cardiac rupture rate in mice post-MI. Some of the effects of Ac-SDKP, such as the enhancement of angiogenesis and the decrease in inflammation and collagenase activity, are similar to those described for thymosin-beta4. Thus, it is possible that Ac-SDKP could be mediating some of the beneficial effects of its precursor. Although the experimental evidence is very promising, there are no data available from a clinical trial supporting the use of thymosin-beta(4) or Ac-SDKP as means of healing the myocardium after MI in patients.

Topics: Animals; Heart; Humans; Models, Biological; Myocardial Infarction; Myocardium; Oligopeptides; Thymosin; Ventricular Remodeling

Inflammation is a critical factor in the development and progression of myocardial infarction and cardiac fibrosis. Thymosin. Real-time quantitative reverse-transcription PCR (qRT-PCR), immunohistochemistry (IHC), and Western blot were used to analyze T. T. AAV-T

Topics: Animals; Fibrosis; Hydrogen Peroxide; Inflammation; Mice; Myocardial Infarction; Myocytes, Cardiac; Thymosin

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.

Topics: Animals; Cell Movement; Cell Proliferation; Dependovirus; Gene Expression Regulation; Humans; Ischemia; Mice, Knockout; Models, Biological; Myocardial Infarction; Myocytes, Cardiac; Neovascularization, Physiologic; Protein Precursors; RNA, Messenger; Thymosin; Zinc Finger E-box Binding Homeobox 2

The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin β4 (Tβ4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium.

Topics: Animals; Cell Differentiation; Cell Movement; Cell Proliferation; Endothelial Cells; Lymphangiogenesis; Mice; Mice, Transgenic; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Peptides; Pericardium; Thymosin

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

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

The survival of transplanted stem cells in ischemic tissue is poor. In the present study, the effects of thymosin β4 (Tβ4) on the survival and angiogenesis of endothelial progenitor cells (EPCs) and improvement in cardiac functions after transplantation of Tβ4-treated EPCs in the infarcted myocardium were investigated. EPCs were isolated from bone marrow of adult male rats and incubated in Endothelial Basal Medium-2. Then the cells were treated with Tβ4 at different concentrations (0.05, 0.1 and 0.2 µM), and cells incubated with DMEM were set as controls. MTT assay, Transwell assay and tube formation in Matrigel were used to detect cell viability, migration and angiogenesis, respectively. For examining the protective effect of Tβ4 on EPCs, the cells were also incubated in the condition of hypoxia and serum deprivation. p-Akt expression was also examined using western blot analysis. Rat models of myocardial infarction (MI) were established by ligation of the anterior descending branch of the left coronary artery. At four weeks after intramyocardial injection of Tβ4-treated EPCs, the changes in cardiac functions, size of the scar tissue and density of microvessels were examined by echocardiography, Masson's trichrome staining, immunohistochemistry and fluorescence in situ hybridization (FISH) for the Y-chromosome. Tβ4 enhanced EPC viability, protected the cells from apoptosis in hypoxia and serum deprivation, and promoted the proliferation and migration of the cells and formation of capillary-like structures in the cells. Moreover, Tβ4 increased p-Akt expression in the cells. The cytoprotective and proangiogenic effects of Tβ4 were in a dose-dependent manner. Tβ4-treated EPCs improved cardiac function, enhanced the repair of the infarcted myocardium, and promoted angiogenesis after transplantation in the infarcted myocardium. In conclusion, pretreatment of EPCs with Tβ4 is a novel strategy for the repair of ischemic tissue after transplantation in MI.

Topics: Angiogenesis Inducing Agents; Animals; Cell Movement; Cell Survival; Cytoprotection; Endothelial Progenitor Cells; Male; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats, Sprague-Dawley; Thymosin

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin β4 (Tβ4) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin-remodelling complex, is required for expression of Wilms' tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with Tβ4 and is recruited by CCAAT/enhancer-binding protein β (C/EBPβ) to discrete regulatory elements in the Wt1 locus. BRG1-Tβ4 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin-remodelling in the activation of EPDCs during cardiovascular development and repair.

Topics: Animals; Base Sequence; CCAAT-Enhancer-Binding Protein-beta; Chromatin Assembly and Disassembly; Conserved Sequence; DNA Helicases; Epigenesis, Genetic; Gene Expression Regulation; Genes, Wilms Tumor; Heart; HEK293 Cells; Humans; Mice; Mice, Transgenic; Myocardial Infarction; Nuclear Proteins; Pericardium; Regulatory Elements, Transcriptional; Thymosin; Transcription Factors

Restoring blood flow after myocardial infarction (MI) is essential for survival of existing and newly regenerated tissue. Endogenous vascular repair processes are deployed following injury but are poorly understood. We sought to determine whether developmental mechanisms of coronary vessel formation are intrinsically reactivated in the adult mouse after MI. Using pulse-chase genetic lineage tracing, we establish that de novo vessel formation constitutes a substantial component of the neovascular response, with apparent cellular contributions from the endocardium and coronary sinus. The adult heart reverts to its former hypertrabeculated state and repeats the process of compaction, which may facilitate endocardium-derived neovascularization. The capacity for angiogenic sprouting of the coronary sinus vein, the adult derivative of the sinus venosus, may also reflect its embryonic origin. The quiescent epicardium is reactivated and, while direct cellular contribution to new vessels is minimal, it supports the directional expansion of the neovessel network toward the infarcted myocardium. Thymosin β4, a peptide with roles in vascular development, was required for endocardial compaction, epicardial vessel expansion, and smooth muscle cell recruitment. Insight into pathways that regulate endogenous vascular repair, drawing on comparisons with development, may reveal novel targets for therapeutically enhancing neovascularization.

Topics: Adult Stem Cells; Animals; Coronary Sinus; Coronary Vessels; Endothelial Cells; Heart Failure; Male; Mice; Myocardial Infarction; Myocytes, Smooth Muscle; Neovascularization, Pathologic; Pericardium; Regeneration; Thymosin

Cardiac patch is considered a promising strategy for enhancing stem cell therapy of myocardial infarction (MI). However, the underlying mechanisms for cardiac patch repairing infarcted myocardium remain unclear. In this study, we investigated the mechanisms of PCL/gelatin patch loaded with MSCs on activating endogenous cardiac repair. PCL/gelatin patch was fabricated by electrospun. The patch enhanced the survival of the seeded MSCs and their HIF-1α, Tβ4, VEGF and SDF-1 expression and decreased CXCL14 expression in hypoxic and serum-deprived conditions. In murine MI models, the survival and distribution of the engrafted MSCs and the activation of the epicardium were examined, respectively. At 4 weeks after transplantation of the cell patch, the cardiac functions were significantly improved. The engrafted MSCs migrated across the epicardium and into the myocardium. Tendency of HIF-1α, Tβ4, VEGF, SDF-1 and CXCL14 expression in the infarcted myocardium was similar with expression in vitro. The epicardium was activated and epicardial-derived cells (EPDCs) migrated into deep tissue. The EPDCs differentiated into endothelial cells and smooth muscle cells, and some of EPDCs showed to have differentiated into cardiomyocytes. Density of blood and lymphatic capillaries increased significantly. More c-kit

Topics: Animals; Biocompatible Materials; Capillaries; Cell Differentiation; Cell Survival; Chemokines; Cytoprotection; Gelatin; Gene Expression Regulation; Heart Function Tests; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphangiogenesis; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Polyesters; Proto-Oncogene Proteins c-kit; Rats, Sprague-Dawley; Regeneration; Thymosin; Vascular Endothelial Growth Factor A

Repairing defective cardiac cells is important towards improving heart function. Due to the frequency and severity of ischemic heart disease, management of patients featuring this type of cardiac failure receives significant interest. Previously we discovered that Thymosin β4 (TB4), a 43 amino-acid secreted actin sequestering peptide, is beneficial for myocardial cell survival and coronary re-growth after infarction in adult mammals. Considering the regenerative potential of full-length TB4 in the heart, and that minimal structural variations alter TB4's influence on actin assembly and cell movement, we investigated how various TB4 domains affect cardiac cell behavior and post-ischemic mammalian heart function. We synthesized 17 domain combinations of full-length TB4 and analyzed their impact on embryonic cardiac cells in vitro, and after cardiac infarction in vivo. We discovered the domains of TB4 affect cardiac cell behavior distinctly. We revealed TB4 specific C-terminal tetrapeptide, AGES, increases embryonic cardiac cell migration and myocyte beating in culture, and improves adult mammalian heart function following ischemia. Investigating the molecular background and mechanism we discovered systemic injection of AGES enhances early myocyte survival by activating Akt-mediated signaling mechanisms, increases coronary vessel growth and inhibits inflammation in mice and pigs. Biodistribution analyses revealed cardiomyocytes uptake AGES efficiently in vitro and in vivo projecting a potential independent clinical utilization for the tetrapeptide. Our comprehensive domain investigations also suggest, preservation and/or restoration of cardiomyocyte communication is a target of TB4 and AGES, and critical to improve post-ischemic heart function in pigs. In summary, we identified the C-terminal four amino-acid variable end of TB4 as the essential and responsible domain for the molecule's full benefits in the hypoxic heart. Additionally, we introduced AGES as a novel, systemically applicable drug candidate to aid cardiac infarction in adult mammals.

Topics: Amino Acid Motifs; Animals; Cell Proliferation; Cell Survival; Coronary Vessels; Gene Expression Regulation, Developmental; Humans; Mice; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Peptides; Swine; Thymosin

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that thymosin β4-sulfoxide lies downstream of hydrogen peroxide in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T-cell/CD14+ monocyte co-cultures, thymosin β4-sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, thymosin β4-sulfoxide is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

Topics: Amino Acid Sequence; Animals; Cell Adhesion; Cell Death; Cell Movement; Humans; Hydrogen Peroxide; Inflammation; Leukocytes; Macrophages; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Monocytes; Myocardial Infarction; Myocardium; Reactive Oxygen Species; Thymosin; Wound Healing; Zebrafish

Thymosin β4 (Tβ4) has been shown to enhance the survival of cultured cardiomyocytes. Here, we investigated whether the cytoprotective effects of Tβ4 can increase the effectiveness of transplanted swine mesenchymal stem cells (sMSCs) for cardiac repair in a rat model of myocardial infarction (MI).. Under hypoxic conditions, cellular damage (lactate dehydrogenase leakage), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelingc cells), and caspase-8 activity were significantly lower, whereas B-cell lymphoma-extra large protein expression was significantly higher, in sMSCs cultured with Tβ4 (1 μg/mL) than in sMSCs cultured without Tβ4, and Tβ4 also increased sMSC proliferation. For in vivo experiments, animals were treated with basal medium (MI: n=6), a fibrin patch (Patch: n=6), a patch containing sMSCs (sMSC: n=9), or a patch containing sMSCs and Tβ4 (sMSC/Tβ4: n=11); Tβ4 was encapsulated in gelatin microspheres to extend Tβ4 delivery. Four weeks after treatment, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly better (P<0.05) in sMSC/Tβ4 animals (left-ventricular ejection fraction=51.7 ± 1.1%; fractional shortening=26.7 ± 0.7%) than in animals from MI (39 ± 3%; 19.5 ± 1.7%) and Patch (43 ± 1.4%; 21.6 ± 0.9%) groups. Histological assessment of infarct wall thickness was significantly higher (P<0.05) in sMSC/Tβ4 animals (50%, [45%, 80%]) than in animals from MI (25%, [20%, 25%]) group. Measurements in sMSC (left-ventricular ejection fraction=45 ± 2.6%; fractional shortening=22.9 ± 1.6%; TH = 43% [25%, 45%]), Patch, and MI animals were similar. Tβ4 administration also significantly increased vascular growth, the retention/survival of the transplanted sMSCs, and the recruitment of endogenous c-Kit(+) progenitor cells to the infarcted region.. Extended-release Tβ4 administration improves the retention, survival, and regenerative potency of transplanted sMSCs after myocardial injury.

Topics: Animals; Cell Proliferation; Cells, Cultured; Female; Mesenchymal Stem Cell Transplantation; Microfilament Proteins; Myocardial Infarction; Random Allocation; Rats; Rats, Nude; Swine; Thymosin; Up-Regulation

Thymosin β4 (Tβ4), a small G-actin sequestering peptide, mediates cell proliferation, migration, and angiogenesis. Whether embryonic stem (ES) cells, overexpressing Tβ4, readily differentiate into cardiac myocytes in vitro and in vivo and enhance cardioprotection following transplantation post myocardial infarction (MI) remains unknown. Accordingly, we established stable mouse ES cell lines, RFP-ESCs and Tβ4-ESCs, expressing RFP and an RFP-Tβ4 fusion protein, respectively. In vitro, the number of spontaneously beating embryoid bodies (EBs) was significantly increased in Tβ4-ESCs at day 9, 12 and 15, compared with RFP-ESCs. Enhanced expression of cardiac transcriptional factors GATA-4, Mef2c and Txb6 in Tβ4-EBs, as confirmed with real time-PCR analysis, was accompanied by the increased number of EB areas stained positive for sarcomeric α-actin in Tβ4-EBs, compared with the RFP control, suggesting a significant increase in functional cardiac myocytes. Furthermore, we transplanted Tβ4-ESCs into the infarcted mouse heart and performed morphological and functional analysis 2 weeks after MI. There was a significant increase in newly formed cardiac myocytes associated with the Notch pathway, a decrease in apoptotic nuclei mediated by an increase in Akt and a decrease in levels of PTEN. Cardiac fibrosis was significantly reduced, and left ventricular function was significantly augmented in the Tβ4-ESC transplanted group, compared with controls. It is concluded that genetically modified Tβ4-ESCs, potentiates their ability to turn into cardiac myocytes in vitro as well as in vivo. Moreover, we also demonstrate that there was a significant decrease in both cardiac apoptosis and fibrosis, thus improving cardiac function in the infarcted heart.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line; Embryonic Stem Cells; Fibrosis; Heart; Mice; Mice, Inbred C57BL; Muscle Development; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Notch; Signal Transduction; Stem Cell Transplantation; Thymosin; Transcription Factors; Ventricular Function, Left; Ventricular Remodeling

While cardiovascular diseases remain the major worldwide cause of mortality and morbidity, there is an urgent need to tackle the clinical and economic burden of heart failure. Since the mammalian heart is unable to adequately regenerate beyond early postnatal stages, individuals surviving acute myocardial infarction are at risk of heart failure. Understanding the embryonic mechanisms of vasculogenesis and cardiogenesis, as well as the mechanisms retained for regeneration in species such as the zebrafish, will inform on strategies for human myocardial repair. Due to their fundamental role in heart development, epicardium-derived cells (EPDCs) have emerged as a population with potential to restore myocardium and coronary vasculature. The ability to revive ordinarily dormant EPDCs lies in the identification of key molecular cues used in the embryo to orchestrate cardiovascular development. One such stimulatory factor, Thymosin β4 (Tβ4), restores the quiescent adult epicardium to its pluripotent embryonic state. Tβ4 treatment of infarcted hearts induces dramatic EPDC proliferation and formation of a network of perfused, functional vessels to enhance blood flow to the ischaemic myocardium. Moreover, Tβ4 facilitates an epicardial contribution of mature de novo cardiomyocytes, structurally and functionally coupled with resident myocardium, which may contribute towards the functional improvement of Tβ4-treated hearts post-MI.

Topics: Adult; Animals; Cell Proliferation; Heart Failure; Humans; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Pericardium; Regeneration; Stem Cells; Thymosin

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality world-wide. Whether endogenous repair and regenerative ability could be augmented by drug administration is an important issue for generation of novel therapeutic approach. Recently it was reported that in mice pretreated with thymosin beta 4 (TB4) and subsequently subjected to experimental MI, a subset of epicardial cells differentiated into cardiomyocytes. In clinical settings, epicardial priming with TB4 prior to MI is impractical. Here we tested if TB4 treatment after MI could reprogram epicardium into cardiomyocytes and augment the epicardium's injury response. Using epicardium genetic lineage trace line Wt1(CreERT2/+) and double reporter line Rosa26(mTmG/+), we found post-MI TB4 treatment significantly increased the thickness of epicardium and coronary capillary density. However, epicardium-derived cells did not adopt cardiomyocyte fate, nor did they migrate into myocardium to become coronary endothelial cells. Our result thus indicates that TB4 treatment after MI does not alter epicardial cell fate to include the cardiomyocyte lineage, providing both cautions and insights for the full exploration of the potential benefits of TB4 in the clinical settings. This article is part of a Special Issue entitled 'Possible Editorial'.

Topics: Animals; Cell Differentiation; Mice; Myocardial Infarction; Myocytes, Cardiac; Pericardium; Thymosin

Topics: Animals; Myocardial Infarction; Myocytes, Cardiac; Pericardium; Thymosin

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

Topics: Animals; Biomarkers; Cell Lineage; Cell Transdifferentiation; Cellular Reprogramming; Cicatrix; Female; Fibroblasts; GATA4 Transcription Factor; Gene Expression Regulation; Genetic Vectors; Heart; Male; MEF2 Transcription Factors; Mice; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Myogenic Regulatory Factors; Regenerative Medicine; T-Box Domain Proteins; Thymosin

Acute myocardial infarction (MI) leads to fibrosis and severe left ventricular wall thinning. Enhancing vascularization within the infarct reduces cell death and maintains a thick left ventricular wall, which is essential for proper cardiac function. Here, we evaluated the controlled delivery of thymosin β4 (Tβ4), which supports cardiomyocyte survival by inducing vascularization and upregulating Akt activity, in the treatment of MI.. We injected collagen-chitosan hydrogel with controlled release of Tβ4 into the infarct after performing left anterior descending artery ligation in rats.. Tβ4-encapsulated hydrogel (thymosin) significantly reduced tissue loss post-MI (13 ± 4%), compared with 58 ± 3% and 30 ± 8% tissue loss for no treatment (MI only) and Tβ4-free hydrogel (control). Significantly more Factor VIII-positive blood vessels with diameter >50 µm were in the thymosin group compared with both MI only and control (p < 0.0001), showing Tβ4-induced vascularization. Wall thickness was positively correlated with the mature blood vessel density (r = 0.9319; p < 0.0001).. Controlled release of Tβ4 within the infarct enhances angiogenesis and presence of cardiomyocytes that are necessary for cardiac repair.

Topics: Animals; Blood Vessels; Chitosan; Collagen; Delayed-Action Preparations; Factor VIII; Hydrogels; Injections; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Physiologic; Rats; Rats, Inbred Lew; Staining and Labeling; 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

Heart disease is a leading cause of death in newborns and in adults. We previously reported that the G-actin-sequestering peptide thymosin β4 promotes myocardial survival in hypoxia and promotes neoangiogenesis, resulting in cardiac repair after injury. More recently, we showed that reprogramming of cardiac fibroblasts to cardiomyocyte-like cells in vivo after coronary artery ligation using three cardiac transcription factors (Gata4/Mef2c/Tbx5) offers an alternative approach to regenerate heart muscle. We have combined the delivery of thymosin β4 and the cardiac reprogramming factors to further enhance the degree of cardiac repair and improvement in cardiac function after myocardial infarction. These findings suggest that thymosin β4 and cardiac reprogramming technology may synergistically limit damage to the heart and promote cardiac regeneration through the stimulation of endogenous cells within the heart.

Topics: Animals; Heart; Humans; Mice; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Regeneration; Thymosin; Wound Healing

We present use of a synthetic, injectable matrix metalloproteinase (MMP)-responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin β4 (Tβ4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + ∼7%, P < 0.001; 6 wk ejection faction, + ∼12%, P < 0.001). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue.

Topics: Animals; Biocompatible Materials; Cell Survival; Embryonic Stem Cells; Humans; Hydrogels; Materials Testing; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Rats; Regeneration; Thymosin; Tissue Engineering; Transplants

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

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression--the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cardiomyopathy, Dilated; DNA-Binding Proteins; Humans; LIM Domain Proteins; Membrane Proteins; Mice; Microfilament Proteins; Myocardial Infarction; NF-kappa B; Protein Serine-Threonine Kinases; Thymosin

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Topics: Adult Stem Cells; Animals; Cell Differentiation; Cellular Reprogramming; Gene Expression Regulation; Heart Injuries; Mice; Myocardial Infarction; Myocytes, Cardiac; Thymosin; WT1 Proteins

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans (http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html). After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin beta4 (Tbeta4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC-derived endothelial-restricted neovascularization constitutes suboptimal "endogenous repair," following injury, which is significantly augmented by Tbeta4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tbeta4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tbeta4, have the capacity to sustain the myocardium after ischemic damage.

Topics: Adult; Humans; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Neovascularization, Pathologic; Regeneration; Thymosin; Wound Healing

Topics: Animals; Cell Movement; Clinical Trials as Topic; Corneal Injuries; Eye Injuries; Humans; Myocardial Infarction; Neoplasms; Neovascularization, Physiologic; Thymalfasin; Thymosin; Wound Healing

Heart disease is a leading cause of death in newborns and in adults. Efforts to promote cardiac repair by introduction or recruitment of exogenous stem cells hold promise but typically involve isolation and introduction of autologous or donor progenitor cells. We have found that the G-actin-sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture was also enhanced by thymosin beta4. We found that thymosin beta4 formed a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt/PKB, which was necessary for thymosin beta4's effects on cardiomyocytes. After coronary artery ligation in mice, thymosin beta4 treatment resulted in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival, and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte and endothelial migration, survival, and repair and may be a novel therapeutic target in the setting of acute myocardial damage.

Topics: Cardiotonic Agents; Cell Movement; Cell Survival; Enzyme Activation; Humans; Myocardial Infarction; Proto-Oncogene Proteins c-akt; Thymosin

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (Akt-MSCs) inhibits ventricular remodeling and restores cardiac function measured 2 wk after myocardial infarction. Here, we report that the functional improvement occurs in < 72 h. This early remarkable effect cannot be readily attributed to myocardial regeneration from the donor cells. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be important mechanisms of tissue repair and functional improvement after injection of the Akt-MSCs. Indeed, in the current study we demonstrate that conditioned medium from hypoxic Akt-MSCs markedly inhibits hypoxia-induced apoptosis and triggers vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When injected into infarcted hearts, the Akt-MSC conditioned medium significantly limits infarct size and improves ventricular function relative to controls. Support to the paracrine hypothesis is provided by data showing that several genes, coding for factors (VEGF, FGF-2, HGF, IGF-I, and TB4) that are potential mediators of the effects exerted by the Akt-MSC conditioned medium, are significantly up-regulated in the Akt-MSCs, particularly in response to hypoxia. Taken together, our data support Akt-MSC-mediated paracrine mechanisms of myocardial protection and functional improvement.

Topics: Animals; Cytoprotection; Female; Fibroblast Growth Factors; Gene Expression Regulation; Hepatocyte Growth Factor; Insulin-Like Growth Factor I; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Paracrine Communication; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Thymosin; Up-Regulation; Vascular Endothelial Growth Factor A