Original article
Thymosin β4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo

https://doi.org/10.1016/j.yjmcc.2009.01.017Get rights and content

Abstract

Hypoxic heart disease is a predominant cause of disability and death worldwide. Since adult mammalian hearts are incapable of regeneration after hypoxia, attempts to modify this deficiency are critical. As demonstrated in zebrafish, recall of the embryonic developmental program may be the key to success. Because thymosin β4 (TB4) is beneficial for myocardial cell survival and essential for coronary development in embryos, we hypothesized that it reactivates the embryonic developmental program and initiates epicardial progenitor mobilization in adult mammals. We found that TB4 stimulates capillary-like tube formation of adult coronary endothelial cells and increases embryonic endothelial cell migration and proliferation in vitro. The increase of blood vessel/epicardial substance (Bves) expressing cells accompanied by elevated VEGF, Flk-1, TGF-β, Fgfr-2, Fgfr-4, Fgf-17 and β-Catenin expression and increase of Tbx-18 and Wt-1 positive myocardial progenitors suggested organ-wide recall of the embryonic program in the adult epicardium. TB4 also positively regulated the expression and phosphorylation of myristoylated alanine-rich C-kinase substrate (Marcks), a direct substrate and indicator of protein kinase C (PKC) activity in vitro and in vivo. PKC inhibition significantly reduced TB4 initiated epicardial thickening, capillary growth and the number of myocardial progenitors. Our results demonstrate that TB4 is the first known molecule capable of organ-wide activation of the embryonic coronary developmental program in the adult mammalian heart after systemic administration and that PKC plays a significant role in the process.

Introduction

Defects in the coronary vascular system have significant impact on heart function and disease. Ischemic myocardial infarctions cause irreversible cell loss and scarring and are major source of morbidity and mortality in humans. A proper angiogenic response after infarction is critical for healing and repair.

A variety of stimuli can initiate the formation of new blood vessels in the heart, presumably through common downstream signaling cascades that trigger quiescent endothelial or other progenitor cells to form nascent tubular structures [1]. Although many of the cellular and molecular mechanisms of embryonic coronary development are well investigated, the molecular basis of angiogenesis in the embryo seems to differ from the pathological vessel regeneration in adults [2]. Blood vessels in the embryo form primarily through vasculogenesis, a differentiation of precursor cells (angioblasts) to endothelial cells that assemble into a vascular network. The critical cellular events include the formation of the primordium, proepicardial organ and epicardium, generation of subepicardial space and mesenchymal cells and development and remodeling of the vascular plexus [3], [4]. New vessels in adults arise mainly through angiogenic sprouting, although vasculogenesis may also occur [2]. Many studies have attempted to reveal the molecular and cellular mechanisms that support cardiac regeneration in adult hearts and to identify progenitor cells capable of cardiac repair [5], [6], [7], [8], [9], [10], [11]. In zebrafish, epicardial cells invade the myocardium and create a vascular network likely to encourage cardiac regeneration in adults [12]. Thus, the injured adult zebrafish heart can recall signaling pathways essential during embryonic coronary development, and the ability to mobilize epicardial cells may be the primary reason they effectively regenerate myocardium. Since adult mammalian hearts typically show insufficient neovascularization after myocardial infarction, experimental attempts to modify this deficiency by directly utilizing epicardial cells or their progenitors could prove favorable for cardiac regeneration.

We previously demonstrated that thymosin β4 (TB4), a 43-amino-acid G-actin-sequestering peptide is expressed in the embryonic heart, stimulates cardiomyocyte migration in vitro and increases cardiac function while promoting the survival of cardiomyocytes in adult mice in vivo[13]. More recently, analysis of heart-specific TB4 knockdown mouse embryos revealed a critical role for the peptide in epicardial development and coronary artery formation [14]. However, the effects of TB4 on the adult epicardium and coronary growth in vivo have not been discussed.

Here we show that TB4 initiates capillary-like tube formation of adult coronary endothelial cells, induces endothelial cell migration and proliferation in embryonic cardiac explants in vitro and supports revascularization in vivo. Importantly, it induces an organ-wide epicardial thickening and progenitor cell activation in adults similar to the changes in developing embryos and in regenerating adult zebrafish, while initiating the expression of numerous pro-angiogenic developmental genes. TB4 initiated protein kinase C (PKC) activity revealed to be essential for the epicardial activation.

Thus, TB4 supports cardiac regeneration not only by inhibiting myocardial cell death after infarction, but also through induction of vessel growth, myocardial progenitor mobilization and by reactivating the embryonic developmental program in adult epicardium in vivo.

Section snippets

Materials

TB4 was synthesized by the Department of Biochemistry and by the Protein Chemistry Technology Center at UT Southwestern Medical Center (Dallas, TX). Human coronary endothelial cells (HCEC) were purchased from PromoCell GmbH (Heidelberg, Germany). Rat tail collagen was purchased from Roche Diagnostics Corporation (Indianapolis, IN). Tissue culture reagents were purchased from Gibco/Invitrogen (Carlsbad, CA) and Promo Cell GmbH (Heidelberg, Germany). Primary antibodies for Western blot and

TB4 induces embryonic endothelial cell migration and proliferation and initiates capillary structure formation of adult coronary endothelial cells in vitro

We tested the effects of TB4 on cardiac endothelial cell migration by embryonic heart explant assays on rat tail collagen [18]. Since TB4 is expressed in endothelial and myocardial cells and is absent in cardiac mesenchyme (Supplemental Fig. 1 and [13]) we used TB4 primary antibody to identify endothelial cells. Exogenously administered TB4 significantly increased embryonic endothelial cell migration (Figs. 1a, e) by facilitating the number of round actively moving cells in vitro (Fig. 1c

Discussion

This study shows that TB4 increases or activates proteins and epicardial progenitors important for myocardial regeneration or vascular growth. TB4 initiates organ-wide activation of the adult epicardium reminiscent of embryonic coronary development by re-stimulation of signaling pathways essential during embryogenesis. Our results reveal a novel role for PKC in this process. Additionally, we present some evidence that p-Marcks positive epicardial cells may serve as endothelial and smooth muscle

Acknowledgments

Authors wish to thank H. Ball and LJ. Burdine for providing TB4 peptide, HR. Garner for financial support to perform microarrays, A. Mobely for FACS separation of progenitor cells and C. Nolen for animal work. We are grateful to JE. Marquette for helping with visual graphics and manuscript review.

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