Nicola Smart

Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization.

Cardiac failure has a principal underlying aetiology of ischaemic damage arising from vascular insufficiency. Molecules that regulate collateral growth in the ischaemic heart also regulate coronary vasculature formation during embryogenesis. Here we identify thymosin β4 (Tβ4) as essential for all aspects of coronary vessel development in mice, and demonstrate that Tβ4 stimulates significant outgrowth from quiescent adult epicardial explants, restoring pluripotency and triggering differentiation of fibroblasts, smooth muscle cells and endothelial cells. Tβ4 knockdown in the heart is accompanied by significant reduction in the pro-angiogenic cleavage product N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). Although injection of AcSDKP was unable to rescue Tβ4 mutant hearts, it significantly enhanced endothelial cell differentiation from adult epicardially derived precursor cells. This study identifies Tβ4 and AcSDKP as potent stimulators of coronary vasculogenesis and angiogenesis, and reveals Tβ4-induced adult epicardial cells as a viable source of vascular progenitors for continued renewal of regressed vessels at low basal level or sustained neovascularization following cardiac injury.

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 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.

The stem cell movement

Stem or progenitor cell–based strategies to combat ischemic heart disease and myocardial infarction, whether autologous transplantation or stimulation of resident populations, not only require detailed insight into transdifferentiation potential and functional coupling, but the efficacy of this approach is underpinned by the need to induce appropriate migration and homing to the site of injury. This review focuses on existing insights into the trafficking of stem cells in the context of cardiac regenerative therapy, with particular focus on the wide variety of potential sources of cells, critical factors that may regulate their migration, and how extrapolating from embryonic stem/progenitor cell behavior during cardiogenesis may reveal pathways implicit in the adult heart postinjury.

Thymosin beta4 and angiogenesis: modes of action and therapeutic potential.

Here we review the mechanisms by which Thymosin β4 (Tβ4) regulates angiogenesis, its role in processes, such as wound healing and tumour progression and we discuss in more detail the role of Tβ4 in the cardiovascular system and significant recent findings implicating Tβ4 as a potential therapeutic agent for ischaemic heart disease.

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.

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

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 β4 (Tβ4) 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 Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.

Thymosins in Health and Disease: 2nd International Symposium

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 β4 (Tβ4) 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 Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.

Nucleolar release of Hand1 acts as a molecular switch to determine cell fate

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. Here we implicate Hand1 as a molecular switch that determines whether a trophoblast stem cell continues to proliferate or commits to differentiation. We identify a novel interaction of Hand1 with a protein that contains an I-mfa (inhibitor of myogenic factor) domain that anchors Hand1 in the nucleolus where it negatively regulates Hand1 activity. In the trophoblast stem-cell line Rcho-1, nucleolar sequestration of Hand1 accompanies sustained cell proliferation and renewal, whereas release of Hand1 into the nucleus leads to its activation, thus committing cells to a differentiated giant-cell fate. Site-specific phosphorylation is required for nucleolar release of Hand1, for its dimerization and biological function, and this is mediated by the non-canonical polo-like kinase Plk4 (Sak). Sak is co-expressed in Rcho-1 cells, localizes to the nucleolus during G2 and phosphorylates Hand1 as a requirement for trophoblast stem-cell commitment to a giant-cell fate. This study defines a novel cellular mechanism for regulating Hand1 that is a crucial step in the stem-cell differentiation pathway.

Hand1 regulates cardiomyocyte proliferation versus differentiation in the developing heart.

The precise origins of myocardial progenitors and their subsequent contribution to the developing heart has been an area of considerable activity within the field of cardiovascular biology. How these progenitors are regulated and what signals are responsible for their development are, however, much less well understood. Clearly, not only is there a need to identify factors that regulate the transition from proliferation of cardioblasts to differentiation of cardiac muscle, but it is also necessary to identify factors that maintain an adequate pool of undifferentiated myocyte precursors as a prerequisite to preventing organ hypoplasia and congenital heart disease. Here, we report how upregulation of the basic helix-loop-helix (bHLH) transcription factor Hand1, restricted exclusively to Hand1-expressing cells, brings about a significant extension of the heart tube and extraneous looping caused by the elevated proliferation of cardioblasts in the distal outflow tract. This activity is independent of the further recruitment of extracardiac cells from the secondary heart field and permissive for the continued differentiation of adjacent myocardium. Culture studies using embryonic stem (ES) cell-derived cardiomyocytes revealed that, in a Hand1-null background, there is significantly elevated cardiomyocyte differentiation, with an apparent default mesoderm pathway to a cardiomyocyte fate. However, Hand1 gain of function maintains proliferating precursors resulting in delayed and significantly reduced cardiomyocyte differentiation that is mediated by the prevention of cell-cycle exit, by G1 progression and by increased cell division. Thus, this work identifies Hand1 as a crucial cardiac regulatory protein that controls the balance between proliferation and differentiation in the developing heart, and fills a significant gap in our understanding of how the myocardium of the embryonic heart is established.

Thymosin beta-4 is essential for coronary vessel development and promotes neovascularization via adult epicardium.

Abstract:  Ischemic heart disease leading to myocardial infarction causes irreversible cell loss and scarring and is a major cause of morbidity and mortality in humans. Significant effort in the field of cardiovascular medicine has been invested in the search for adult cardiac progenitor cells that may replace damaged muscle cells and/or contribute to new vessel formation (neovascularization) and in the identification of key factors, which may induce such progenitor cells to contribute to myocardial repair and collateral vessel growth. We recently demonstrated that the actin monomer‐binding protein, thymosin β‐4 (Tβ‐4), when secreted from the myocardium provides a paracrine stimulus to the cells of the epicardium‐derived cells (EPDCs) to promote their inward migration and differentiation into endothelial and smooth muscle cells to form the coronary vasculature. Translating this essential role for Tβ‐4 in coronary vessel development to the adult, we found that treatment of cultured adult explants with Tβ‐4 stimulated extensive outgrowth of epicardin‐positive epicardial cells, which, as they migrated away from the explant, differentiated into procollagen type I, SMαA, and Flk1‐positive cells indicative of fibroblasts, smooth muscle, and endothelial cells; thus releasing the adult epicardium from a quiescent state and restoring pluripotency. The ability of Tβ‐4 to promote coronary vessel development and potentially induce new vasculature in the adult is essential for cardiomyocyte survival and could contribute significantly toward the reported Tβ4‐induced cardioprotection and repair in the adult heart. Tβ‐4 is currently subject to multicenter phase 1 clinical trials for treatment of cardiovascular disease (http://www.regenerx.com), therefore, insight into the repair mechanism(s) induced by Tβ‐4 is an essential step toward harnessing therapeutic survival, migration, and repair properties of the peptide in the context of acute myocardial damage.