Thymosin β4 and the vasculature: multiple roles in development, repair and protection against disease

Introduction: Formation of the vasculature is a complex process, defects in which can lead to embryonic lethality or disease in later life. Understanding mechanisms of vasculogenesis may facilitate the treatment of developmental defects and may be extrapolated to promote wound healing and tissue repair. Thymosin β4 (Tβ4) is an actin monomer binding protein with recognized roles in vascular development, neovascularization and protection against disease. Areas covered: Vascular network assembly is complex, regulated by multiple signals and cell types; Tβ4 functions in many of the underlying processes, including vasculogenesis, angiogenesis, arteriogenesis, endothelial-mesenchymal transition and extracellular matrix remodeling. Loss of Tβ4 perturbs vessel growth and stability, whereas exogenous application enhances capillary formation and pericyte recruitment, during development and in injury models. Expert opinion: Although vascular functions for Tβ4 have been well documented, the underlying molecular mechanisms remain obscure. While Tβ4-induced cytoskeletal remodeling likely mediates the directional migration of endothelial cells, paracrine roles have also been implicated in migration and differentiation of smooth muscle cells. Moreover, nuclear functions of Tβ4 have been described but remain to be explored in the vasculature. Delineati+ng the molecular pathways impacted by Tβ4 to promote vascular growth and remodeling may reveal novel targets for prevention and treatment of vascular disease

Epicardial progenitor cells in cardiac regeneration and neovascularisation.

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

Thymosin β4 protein therapy for cardiac repair.

Clinical interventions leading to improved survival in patients with acute myocardial infarction have, paradoxically, increased the need for cardiac regenerative strategies as more people are living with heart failure. Over the last 10-15 years there have been significant advances in our understanding of cell-based therapy for cardiac repair. Evidence that paracrine stimulation largely underlies the functional benefits in cell transplantation has led to a paradigm shift in regenerative medicine: from cell therapy to factor/protein-based therapy. Although, future regenerative approaches may likely involve a synergistic protein cocktail, this review will focus on the role of a promising candidate, thymosin beta 4 (Tβ4) in cardioprotection, neovascularization, tissue regeneration and inflammation - all essential components in cardiac repair

Essential role for thymosin β4 in regulating vascular smooth muscle cell development and vessel wall stability.

RATIONALE: Compromised development of blood vessel walls leads to vascular instability that may predispose to aneurysm with risk of rupture and lethal hemorrhage. There is currently a lack of insight into developmental insults that may define the molecular and cellular characteristics of initiating and perpetrating factors in adult aneurismal disease. OBJECTIVE: To investigate a role for the actin-binding protein thymosin β4 (Tβ4), previously shown to be proangiogenic, in mural cell development and vascular wall stability. METHODS AND RESULTS: Phenotypic analyses of both global and endothelial-specific loss-of-function Tβ4 mouse models revealed a proportion of Tβ4-null embryos with vascular hemorrhage coincident with a reduction in smooth muscle cell coverage of their developing vessels. Mechanistic studies revealed that extracellular Tβ4 can stimulate differentiation of mesodermal progenitor cells to a mature mural cell phenotype through activation of the transforming growth factor-beta (TGFβ) pathway and that reduced TGFβ signaling correlates with the severity of hemorrhagic phenotype in Tβ4-null vasculature. CONCLUSIONS: Tβ4 is a novel endothelial secreted trophic factor that functions synergistically with TGFβ to regulate mural cell development and vascular wall stability. These findings have important implications for understanding congenital anomalies that may be causative for adult-onset vascular instability

Dynamic haematopoietic cell contribution to the developing and adult epicardium

The epicardium is a cellular source with the potential to reconstitute lost cardiovascular tissue following myocardial infarction. Here we show that the adult epicardium contains a population of CD45+ haematopoietic cells (HCs), which are located proximal to coronary vessels and encased by extracellular matrix (ECM). This complex tertiary structure is established during the regenerative window between post-natal days 1 and 7. We show that these HCs proliferate within the first 24 h and are released between days 2 and 7 after myocardial infarction. The ECM subsequently reforms to encapsulate HCs after 21 days. Vav1-tdTomato labelling reveals an integral contribution of CD45+ HCs to the developing epicardium, which is not derived from the proepicardial organ. Transplantation experiments with either whole bone marrow or a Vav1+ subpopulation of cells confirm a contribution of HCs to the intact adult epicardium, which is elevated during the first 24 weeks of adult life but depleted in aged mice

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerisation in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescencebased sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction

Amniotic fluid stem cells are cardioprotective following acute myocardial infarction.

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

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin β4 knockout mice

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerisation in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescencebased sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction