Research Items (59)
- Jan 2019
Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic switch which promotes atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 may additionally function to maintain healthy vasculature and protect against disease throughout postnatal life. This was supported by identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Tβ4-null mice display aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. We confirmed predisposition to disease in models of atherosclerosis and AAA. Diseased vessels and plaques were characterised by accelerated contractile-synthetic VSMC switching and augmented PDGFRβ signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with dysregulated endocytosis, leading to increased recycling of LRP1-PDGFRβ and reduced lysosomal targeting. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight which may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression.
- Oct 2018
Identification of cellular surface markers that distinguish tumorous from normal vasculature is important for the development of tumor vessel-targeted therapy. Here, we show that Apj, a G protein-coupled receptor, is highly enriched in tumor endothelial cells but absent from most endothelial cells of adult tissues in homeostasis. By genetic targeting using Apj-CreER and Apj-DTRGFP-Luciferase, we demonstrated that hypoxia-VEGF signaling drives expansion of Apj⁺ tumor vessels and that targeting of these vessels, genetically and pharmacologically, remarkably inhibits tumor angiogenesis and restricts tumor growth. These in vivo findings implicate Apj⁺ vessels as a key driver of pathological angiogenesis and identify Apj⁺ endothelial cells as an important therapeutic target for the anti-angiogenic treatment of tumors. Angiogenesis underpins tumor growth and metastasis, but current targets are suboptimal. Zhao et al. report that Apj is a cell surface marker of pathological angiogenesis and a tractable therapeutic target for tumors.
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.
Rationale: Organs of the body require vascular networks to supply oxygen and nutrients and maintain physiological function. The blood vessels of different organs are structurally and functionally heterogeneous in nature. To more precisely dissect their distinct in vivo function in individual organs, without potential interference from off-site targets, it is necessary to genetically target them in an organ-specific manner. Objective: To generate a genetic system that targets vascular endothelial cells in an organ- or tissue- specific manner and to exemplify the potential application of intersectional genetics for precise, target-specific gene manipulation in vivo. Methods and Results: We took advantage of two orthogonal recombination systems, Dre-rox and Cre-loxP, to create a genetic targeting system based on intersectional genetics. Using this approach, Cre activity was only detectable in cells that had expressed both Dre and Cre. Applying this new system, we generated a coronary endothelial cell specific Cre (CoEC-Cre) and a brain endothelial cell specific Cre (BEC-Cre). Through lineage tracing, gene knockout and over-expression experiments, we demonstrated that CoEC-Cre and BEC-Cre efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of VEGFR2 using BEC-Cre, we showed that VEGF signaling regulates angiogenesis in the central nervous system and also controls the integrity of the blood-brain barrier. Conclusions: We provide two examples to illustrate the use of intersectional genetics for more precise gene targeting in vivo, namely manipulation of genes in blood vessels of the heart and brain. More broadly, this system provides a valuable strategy for tissue-specific gene manipulation that can be widely applied to other fields of biomedical research.
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.
Survival rates following myocardial infarction have increased in recent years but current treatments for post-infarction recovery are inadequate and cannot induce regeneration of damaged hearts. Regenerative medicine could provide disease-reversing treatments by harnessing modern concepts in cell and developmental biology. A recently-established paradigm in regenerative medicine is that regeneration of a tissue can be achieved by reactivation of the coordinated developmental processes that originally formed the tissue. In the heart, the epicardium has emerged as an important regulator of cardiac development and reactivation of epicardial developmental processes may provide a means to enable cardiac regeneration. Indeed, in adult mouse hearts, treatment with thymosin β4 and other drug-like molecules reactivates the epicardium and improves outcomes after myocardial infarction by inducing regenerative paracrine signalling, neovascularisation and de novo cardiomyocyte production. However, there are considerable limitations to current methods of epicardial reactivation that prevent direct translation into clinical practice. Here, we describe the rationale for targeting the epicardium and the successes and limitations of this approach. We consider how several recent advances in epicardial biology could be used to overcome these limitations. These advances include insight into epicardial signalling and heterogeneity, epicardial modulation of inflammation and epicardial remodelling of extracellular matrix.
High temporal resolution cine-MRI to assess global functional and morphological parameters in +/Y (WT) and −/Y (KO) mice. Significantly reduced left ventricular end-diastolic volumes in −/Y mice are apparent, shown here in mid-ventricular short axis (KO1, middle panel, compared with WT, left panel). Distinct regional variability in % fractional wall thickening was apparent in −/Y hearts, consistent with the patchy distribution of cardiomyocytes with shorter sarcomeres. Variability in wall motion contributed to a more pronounced twisting motion in −/Y hearts upon contracion (particularly evident in KO2, right panel).
Tβ4 -/Y mice depend upon increasing heart rate and ejection fraction to maintain an effectively normal cardiac output. Bolus infusion of the β1-selective antagonist esmolol slowed heart rate and increased LV volumes in both +/Y (WT) and −/Y (KO) hearts. Whereas esmolol treatment markedly reduced SV in WT mice, KO mice were unaffected; cardiac output dropped significantly in KO mice.
Tβ4 knockout mice subjected to dobutamine stress testing, with high temporal cine MR imaging. The inotropic effects of dobutamine increase cardiac output via elevated heart rate and enhanced myocardial contractility. +/Y control mice (WT) decreased ESV with dobutamine treatment, as evidence of a normal contractile reserve. In contrast, −/Y (KO) mice already display small ESV at baseline, and this is only modestly reduced by dobutamine treatment. This test reveals that Tβ4 −/Y mice possess a limited contractile reserve.
- Nov 2016
Neovascularisation of the ischaemic myocardium post-infarction is necessary to restore blood flow to vulnerable cardiomyocytes and will be indispensable for prospective regenerative strategies, to perfuse newly formed myocardium. Therapeutic attempts to enhance new vessel formation have, to date, yielded modest clinical benefits and innovative approaches are now needed. Intrinsic mechanisms are initiated by the heart in an attempt to rebuild injured vessels but these are poorly understood. Insight into the underlying mechanisms may reveal targets for therapeutically augmenting this low-level neovascular response. Starting from a limited number of descriptive studies, this review summarises what is known of coronary neovascularisation and explores putative mechanisms and cellular sources which may endogenously contribute, or that may be pharmacologically triggered, to support vasculo- or angiogenesis. As injury responses in the adult frequently recapitulate embryological processes, a particular focus is placed on the developmental mechanisms of coronary vessel formation. An understanding of the cellular sources and the regulatory pathways used by the embryo may reveal novel targets for reactivating coronary vessel and myocardial regeneration. This article is protected by copyright. All rights reserved.
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 polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 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 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.
- Jun 2015
Introduction: 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. Areas covered: 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. Expert opinion: The therapeutic potential of Tβ4 for the treatment of cardiac failure is herein evaluated alongside existing, emerging and prospective novel treatments.
The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo we have generated a novel transgenic reporter mouse expressing a Green Fluorescent Protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115+ monocytes of adult blood, spleen and bone marrow and we took advantage of this to directly compare the trafficking of bone marrow derived CD68-GFP monocytes to that of CX3CR1GFP monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1GFP monocytes, which down regulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high level GFP expression for 72 hours following differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse-line represents a powerful resource for analysing monocyte mobilisation and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation.
- Aug 2014
You are probably thinking ‘if this series is so straightforward, why does it need an article to guide me through it?’ and you would be right, it does not. The series is self-explanatory and will be useful to basic scientists and clinicians because the articles are written by international experts who have been selected for their ability to communicate clearly and enjoyably. However, there is a hidden purpose …
Cardiovascular disease remains the major cause of mortality and cardiac cell therapy has recently emerged as a paradigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during development contributes to different cardiovascular lineages, including cardiomyocytes, but becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate the adult epicardium-derived cells (EPDCs) following myocardial infarction (MI) to proliferate and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterisation of the adult EPDCs relative to the embryonic epicardial lineage and to determine prospective cell fate biases within the activated adult population during cardiovascular repair. Wt1GFPCre/+ mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1+ GFP+ EPDCs were FACS-sorted 2, 4 and 7 days after MI. Embryonic WT1+ GFP+ EPDCs were isolated from embryonic hearts (E12.5) by FACS and sorted cells were characterised by real time qRT-PCR and immunostaining. Adult WT1+ GFP+ EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of Sca-1, CD44 and CD90 we identified different subpopulations of EPDCs of varying cardiovascular potential, based on marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, the activated adult WT1+ GFP+ cells are a heterogeneous population which when activated can restore an embryonic gene programme but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.
In recent years cardiac regenerative medicine has emerged as a fast-developing exploratory field with tremendous potential to treat end-stage heart disease. Different approaches have been investigated for the repair of cardiovascular ischemic injuries such as myocardial infarction, in order to improve heart performance in the long term. In this scenario, stem cell-based medicine has received a lot of attention, and several stem sources have been evaluated to identify the most suitable therapeutic approach. Cardiac regeneration has become a multidisciplinary research area based primarily on different stem cell- and tissue engineering-based strategies, with the ultimate goal of preventing or reversing heart failure. Amniotic fluid stem (AFS) cells are broadly multipotent and clonogenic cells which have emerged as a potent therapeutic agent in regenerative medicine and which can be easily obtained throughout pregnancy from surplus samples taken for prenatal diagnostic procedures. In this chapter we will discuss the most significant findings in the field of stem cell therapy for cardiac regeneration, focusing on the recent results using AFS cells.
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.
- Feb 2013
We thank you for the opportunity to respond to the commentary from Banerjee et al, regarding our publication “Essential Role for Thymosin β4 in Regulating Vascular Smooth Muscle Cell Development and Vessel Wall Stability.” See Research Commentary, p e25 The discrepancies between our findings and those of Banerjee et al were outlined in our original article1 and plausible explanations provided, which were acceptable to the reviewers of our article. We reiterate below our rationale and data, which support a role for thymosin β4 (Tβ4) in mural cell development (in addition to those presented in our original publication). We did not, as stated in the Banerjee commentary, report impaired mural cell migration.
The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimeriasation or LIFRβ/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20 min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr(701) and STAT3 Tyr(705) were enhanced by the p38-MAPK inhibitor SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box1 and Box2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independently of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.
- Oct 2012
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 β4 (Tβ4), 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 Tβ4 to sustain and repair the myocardium after ischemic damage.
- Aug 2012
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.
- Jun 2012
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. 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. 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. 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.
Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5. To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease. A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction. Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.
- Feb 2012
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.
- Jan 2012
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.
- Jun 2011
As the developing heart grows and the chamber walls thicken, passive diffusion of oxygen and nutrients is replaced by a vascular plexus which remodels and expands to form a mature coronary vascular system. The coronary arteries and veins ensure the continued development of the heart and facilitate cardiac output with progression towards birth. Many aspects of coronary vessel development are surprisingly not well understood and recently there has been much debate surrounding both the developmental origin and tissue contribution of cardiovascular cells alongside the specific signals that determine their fate and function. What is clear is that an understanding of the cellular and molecular cues to vascularize the heart of the embryo has significant implications for adult heart disease and regeneration, as we move towards targeted cell-based therapies for neovascularization and coronary bypass engraftment. This review will focus on the proposed cellular origins for the coronary endothelium with due consideration to the pro-epicardial organ/epicardium, sinus venosus and endocardium as potential sources, and we will explore the outstanding questions and technical limitations with respect to accurate labelling and lineage tracing of the developing coronaries. We will briefly document canonical vascular signalling that induces vessels in the heart alongside a focus on the potential for developmental reprogramming and putative mechanisms underpinning venous vs. arterial cell fate. Finally, we will extrapolate directly from development to address adult maintenance of the coronaries, vascular homeostasis and remodelling in response to pathology, aligned with the potential for revascularizing the injured adult heart.
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.
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.
- Feb 2011
Stem cell therapy has recently emerged as an innovative strategy over conventional cardiovascular treatments to restore cardiac function in patients affected by ischemic heart disease. Various stem cell populations have been tested and their potential for cardiac repair has been analyzed. Embryonic stem cells retain the greatest differentiation potential, but concerns persist with regard to their immunogenic and teratogenic effects. Although adult somatic stem cells are not tumourigenic and easier to use in an autologous setting, they exist in small numbers and possess reduced differentiation potential. Traditionally the heart was considered to be a post-mitotic organ; however, this dogma has recently been challenged with the identification of a reservoir of resident stem cells, defined as cardiac progenitor cells (CPCs). These endogenous progenitors may represent the best candidates for cardiovascular cell therapy, as they are tissue-specific, often pre-committed to a cardiac fate, and display a greater propensity to differentiate towards cardiovascular lineages. This review will focus on current research into the biology of CPCs and their regenerative potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
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 β4 (Tβ4/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 Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 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 Tβ4 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.
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.
The inability of the human heart to effectively repair itself after acute ischaemic injury has driven the search for efficacious means of promoting cardiac regenerative growth. Central to this has been the emergence of cell-based strategies to stimulate and augment both myocardial regeneration and neovascularization. Autologous cell transplantation of a variety of adult progenitor cells has been taken forward in clinical trials and, in parallel, investigators have begun to focus on the activation of resident cardiac cell populations as a means to stimulate endogenous repair. The latter approach depends on characterizing native progenitors with self-renewal, clonality, multipotency and arguably an analogous embryological counterpart. Recently, we have focused on adult EPDCs (epicardium-derived progenitor cells), which, when induced by the actin monomer-binding protein Tbeta4 (thymosin beta4), are able to revert to their embryonic phenotype and give rise to endothelial cells and vascular smooth muscle cells ex vivo. Studies are ongoing to determine whether activated adult EPDCs can contribute to bona fide neovascularization in the injured adult mammalian heart proper, as a therapeutic means to support surviving cardiac muscle cells and sustain regenerating myocardium.
- Jul 2009
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.
- Mar 2009
The epicardium has, like the other cell lineages of the terminally differentiated adult heart, long been regarded as quiescent, incapable of migration or differentiation. In contrast, the embryonic epicardium possesses an innate ability to proliferate, migrate, and differentiate into a number of mature cardiovascular cell types, including vascular smooth muscle cells, fibroblasts, cardiomyocytes, and, arguably, some endothelial cells. In recapitulating its essential developmental role, we recognized the ability of the actin-binding peptide thymosin beta4 (Tbeta4) to induce epicardium-derived progenitor cell (EPDC) migration from adult heart and noted the derivation of cell types originating from embryonic epicardium. This protocol provides a means of enabling adult EPDC outgrowth and culture. We establish a model system in which to study the ability of factors to influence the migration of vascular precursors and their differentiation and to move towards screening of small molecules ex vivo prior to clinical trials of therapeutic cardiac repair.
- Jun 2008
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.
- Feb 2008
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.
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.
- Oct 2007
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.
Here we review the mechanisms by which Thymosin beta4 (Tbeta4) regulates angiogenesis, its role in processes, such as wound healing and tumour progression and we discuss in more detail the role of Tbeta4 in the cardiovascular system and significant recent findings implicating Tbeta4 as a potential therapeutic agent for ischaemic heart disease.
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 beta4 (Tbeta4) as essential for all aspects of coronary vessel development in mice, and demonstrate that Tbeta4 stimulates significant outgrowth from quiescent adult epicardial explants, restoring pluripotency and triggering differentiation of fibroblasts, smooth muscle cells and endothelial cells. Tbeta4 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 Tbeta4 mutant hearts, it significantly enhanced endothelial cell differentiation from adult epicardially derived precursor cells. This study identifies Tbeta4 and AcSDKP as potent stimulators of coronary vasculogenesis and angiogenesis, and reveals Tbeta4-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.
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.
- Feb 2006
Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is a prognostic marker associated with left ventricular contractile dysfunction and heart failure. On the other hand, IL-6 activates signalling pathways which mediate delayed ischemic preconditioning. We have therefore studied the cellular mechanisms of IL-6-induced cardioprotection. Inducible nitric oxide synthase (iNOS) expression, cardiomyocyte calcium handling, mitochondrial energetics, and the activation of protective signalling pathways in response to IL-6 were studied in a model of simulated ischemia/reperfusion (sI/R) in isolated neonatal rat ventricular cardiomyocytes. Reperfusion after sI/R induced a rise in cytosolic [Ca2+], a loss of cell morphology and integrity, and a transient increase in mitochondrial potential (Deltapsi m), followed by mitochondrial swelling and collapse of Deltapsi m. Pre-treatment of cardiomyocytes with 10 ng/ml IL-6 for 6 h, 24 h prior to sI/R prevented the secondary rise in cytosolic [Ca2+] and induced expression of iNOS and NO-dependent protection against sI/R injury. The protection against sI/R was concomitant with a NO-dependent reduction in the amplitude of cytosolic Ca2+ transients. IL-6 induced an increase in inner mitochondrial membrane polarisation and increased mitochondrial Ca2+ loading (rhod-2 fluorescence) at baseline, but prevented the reperfusion-induced changes in mitochondrial function. IL-6 pre-treatment also resulted in activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and both iNOS induction and IL-6-dependent protection were blocked by the PI 3-kinase inhibitor wortmannin. IL-6 induces a PI 3-kinase and NO-dependent protection of cardiomyocytes, which is associated with alterations in mitochondrial Ca2+ handling, inhibition of reperfusion-induced mitochondrial depolarisation, swelling and loss of structural integrity, and suppression of cytosolic Ca2+ transients.
RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin beta4 (Tbeta4); determined their effectiveness at silencing Tbeta4 and have both excluded off-target silencing of the Tbeta4 homologue Thymosin beta10 (Tbeta10) and demonstrated partial knockdown of Tbeta10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.
Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects. Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse, which has been associated with mutations in at least five loci including bl. As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.
- Mar 2003
The bHLH transcription factor, Hand1 has been implicated in cardiac looping in the mouse, however its function in the developing heart remains unknown. To investigate the mechanism(s) through which Hand1 might function, we screened for potential downstream target genes using representational difference analysis. Thymosin beta4 was found to be down-regulated whereas cystatin C and alphaCA were up-regulated in Hand1-null embryoid bodies. Whole-mount in situ hybridisation on wild type embryos (E8.0-E10.5) and Hand1 homozygous-mutant embryos (E8.0) confirmed co-expression of the putative targets with Hand1 in the heart and their aberrant expression in a Hand1-null background.