Cardiac lymphatics are heterogeneous in origin and respond to injury.

The lymphatic vasculature is a blind-ended network crucial for tissue-fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. By contrast, here we show that cardiac lymphatic vessels in mice have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple Cre­lox-based lineage tracing revealed a potential contribution from the putative haemogenic endothelium during development, and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C, resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

Aberrant developmental titin splicing and dysregulated sarcomere length in Thymosin ß4 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 ß4 (Tß4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tß4 null mice, immunofluorescence-based 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 Tß4, whereby normal sarcomere length was restored. Our data suggest that Tß4 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 Tß4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction.

De novo cardiomyocytes from within the activated adult heart after injury.

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 ß4, 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.

Essential role for thymosin ß4 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 ß4 (Tß4), 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 Tß4 mouse models revealed a proportion of Tß4-null embryos with vascular hemorrhage coincident with a reduction in smooth muscle cell coverage of their developing vessels. Mechanistic studies revealed that extracellular Tß4 can stimulate differentiation of mesodermal progenitor cells to a mature mural cell phenotype through activation of the transforming growth factor-beta (TGFß) pathway and that reduced TGFß signaling correlates with the severity of hemorrhagic phenotype in Tß4-null vasculature. CONCLUSIONS: Tß4 is a novel endothelial secreted trophic factor that functions synergistically with TGFß 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.

Thymosin ß4 protects against aortic aneurysm via endocytic regulation of growth factor signaling.

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic modulation which increases susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesized that Thymosin ß4 (Tß4) may function to maintain healthy vasculature throughout postnatal life. This was supported by the identification of an interaction with low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of platelet-derived growth factor BB (PDGF-BB) signaling and VSMC proliferation. LRP1 variants have been implicated by genome-wide association studies with risk of AAA and other arterial diseases. Tß4-null mice displayed aortic VSMC and elastin defects that phenocopy those of LRP1 mutants, and their compromised vascular integrity predisposed them to Angiotensin II-induced aneurysm formation. Aneurysmal vessels were characterized by enhanced VSMC phenotypic modulation and augmented PDGFR-ß signaling. In vitro, enhanced sensitivity to PDGF-BB upon loss of Tß4 was associated with dysregulated endocytosis, with increased recycling and reduced lysosomal targeting of LRP1-PDGFR-ß. Accordingly, the exacerbated aneurysmal phenotype in Tß4-null mice was rescued upon treatment with the PDGFR-ß antagonist Imatinib. Our study identifies Tß4 as a key regulator of LRP1 for maintaining vascular health, and provides insights into the mechanisms of growth factor-controlled VSMC phenotypic modulation underlying aortic disease progression.

Coronary vessel development and insight towards neovascular therapy.

Formation of the coronary arteries consists of a precisely orchestrated series of morphogenetic and molecular events which can be divided into three distinct processes: vasculogenesis, angiogenesis and arteriogenesis (Risau 1997; Carmeliet 2000). Even subtle perturbations in this process may lead to congenital coronary artery anomalies, as occur in 0.2-1.2% of the general population (von Kodolitsch et al. 2004). Contrary to the previously held dogma, the process of vasculogenesis is not limited to prenatal development. Both vasculogenesis and angiogenesis are now known to actively occur within the adult heart. When the need for regeneration arises, for example in the setting of coronary artery disease, a reactivation of embryonic processes ensues, redeploying many of the same molecular regulators. Thus, an understanding of the mechanisms of embryonic coronary vasculogenesis and angiogenesis may prove invaluable in developing novel strategies for cardiovascular regeneration and therapeutic coronary angiogenesis.

Thymosin ß4 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 ß4 (Tß4) 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; Tß4 functions in many of the underlying processes, including vasculogenesis, angiogenesis, arteriogenesis, endothelial-mesenchymal transition and extracellular matrix remodeling. Loss of Tß4 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 Tß4 have been well documented, the underlying molecular mechanisms remain obscure. While Tß4-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 Tß4 have been described but remain to be explored in the vasculature. Delineati+ng the molecular pathways impacted by Tß4 to promote vascular growth and remodeling may reveal novel targets for prevention and treatment of vascular disease.

Recapitulation of developmental mechanisms to revascularize the ischemic heart.

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 ß4, 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.

BRG1-SWI/SNF-dependent regulation of the Wt1 transcriptional landscape mediates epicardial activity during heart development and disease.

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin ß4 (Tß4) 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 Tß4 and is recruited by CCAAT/enhancer-binding protein ß (C/EBPß) to discrete regulatory elements in the Wt1 locus. BRG1-Tß4 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.

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.

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 ß4 (Tß4), restores the quiescent adult epicardium to its pluripotent embryonic state. Tß4 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, Tß4 facilitates an epicardial contribution of mature de novo cardiomyocytes, structurally and functionally coupled with resident myocardium, which may contribute towards the functional improvement of Tß4-treated hearts post-MI.

Thymosin ß4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing.

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 ß4-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 ß4-sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, thymosin ß4-sulfoxide is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

Thymosin ß4 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 (Tß4) in cardioprotection, neovascularization, tissue regeneration and inflammation - all essential components in cardiac repair.

Myocardial regeneration: expanding the repertoire of thymosin ß4 in the ischemic heart.

Efficient cardiac regeneration postinfarction (MI) requires the replacement of lost cardiomyocytes, formation of new coronary vessels and appropriate modulation of the inflammatory response. However, insight into how to stimulate repair of the human heart is currently limited. Using the embryonic paradigm of regeneration, we demonstrated that the actin-binding peptide thymosin ß4 (Tß4), required for epicardium-derived coronary vasculogenesis, can recapitulate its embryonic role and activate quiescent adult epicardial cells (EPDCs). Once stimulated, EPDCs facilitate neovascularization of the ischemic adult heart and, moreover, contribute bona fide cardiomyocytes. EPDC-derived cardiomyocytes structurally and functionally integrate with resident muscle to regenerate functional myocardium, limiting pathological remodeling, and effecting an improvement in cardiac function. Alongside pro-survival and anti-inflammatory properties, these regenerative roles, via EPDCs, markedly expand the range of therapeutic benefits of Tß4 to sustain and repair the myocardium after ischemic damage.

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 ß4 (Tß4). 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 Tß4, 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 Tß4. 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.

Identification of Thymosin ß4 as an effector of Hand1-mediated vascular development.

The bHLH transcription factor Hand1 (Heart and neural crest-derived transcript-1) has a fundamental role in cardiovascular development; however, the molecular mechanisms have not been elucidated. In this paper we identify Thymosin ß4 (Tß4/Tmsb4x), which encodes an actin monomer-binding protein implicated in cell migration and angiogenesis, as a direct target of Hand1. We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tß4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tß4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors. Hand1-mediated activation of Tß4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1-null embryos. Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.