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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.
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 . 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 . 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, the proepicardial organ and the epicardium, generation of the 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 . 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 . 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 . More recently, analysis of heart-specific TB4 knockdown mouse embryos revealed a critical role for the peptide in epicardial development and coronary artery formation . 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.
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 immunohistochemistry were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Cell Signaling Technology Inc. (Danvers, MA) and Abnova Corporation (Taipei, Taiwan). TB4 primary antibody was a gift of H. Yin at UT Southwestern Medical Center (Dallas, TX) .
Myocardial infarctions were produced in C57BL/6J male mice at 16 weeks of age (25–30 g) by ligation of the left anterior descending (LAD) coronary artery as described [13, 16]. All animal protocols were reviewed and approved by the University of Texas Southwestern Medical School Institutional Animal Care Advisory Committee and were in compliance with the rules governing animal use as published by the NIH. Mice were sedated in an isoflurane chamber (5%) for 60 seconds until self-designed coaxial mask could be safely applied. The mask supplied continuous isoflurane (2.0%) and oxygen (98.0%) under positive pressure from a Harvard small animal respirator throughout the procedures. Immediately after ligation, half of the mice were injected intraperitoneally with 150μg of TB4 in 300 μl PBS and half with 300 μl PBS, as described . The effective dose of synthesized TB4 was determined by in vitro cell migration assays (Supplemental Figure 2 k) and in vivo biodistribution of TB4 [13, 17]. For in vivo inhibition of PKC activity mice were treated systemically with 10μg of Bisindolylmaleinimide-I hydrochloride in 100 μl PBS (Chalbiochem, San Diego, CA) and with 150μg of TB4 in 300 μl PBS or 300 μl PBS in two boluses after myocardial infarction. We administered buprenorphine (0.05 mg/kg) for post-operative pain control. Hearts were removed 1, 3, or 14 days after ligation and processed for further investigations. Exposure time for immunohistological analysis was 3 sec.
RNA from the core and remote areas of four TB4 treated, and four PBS treated hearts were isolated 24 h after treatment using Trizol (Invitrogen, Carlsbad, CA) reagent following the manufacturer's protocol. The quality and quantity of RNA were determined and processed with Mouse Affymetrix Genome 430 2.0 arrays at the Microarray Core Facility at UT Southwestern Medical Center. Four independent experimental and four control arrays were analyzed with GeneChip operating software (GCOS, Affymetrix), GeneSpring 7.0 (Silicon Genetics), significance analysis of microarrays (SAM, Stanford University, Palo Alto, California, USA), and Spotfire Decision Site 8.2 (Spotfire). Computational hierarchical cluster analysis was performed with Spotfire and CLUSFAVOR 6.0 (Baylor College of Medicine, Houston, Texas, USA). Analysis of variance was performed with Spotfire. The data were normalized by mean values (for Spotfire pair-wise comparisons and SAM two-class comparisons) or percentile values (for GeneSpring analyses). Gene expression changes were considered significant if the p value was less than 0.05, the fold change at least 1.5, and gene expression was altered in all replicate comparisons. Genes expressed at different levels in untreated controls were excluded from analysis, as they most likely represent experimental variation between samples.
Adult epicardial cells were isolated (Supplemental Methods) and directly cultured on Matrigel (BD Biosciences, San Jose, CA) or immunostained and separated by FACS sorting (Supplemental Methods). Sorted cells were cultured on gelatin coated culture dishes for 0, 7 and 14 days in endothelial cell growth medium MW (PromoCell, Germany). Cultured cells were analyzed by immunocytochemistry.
Statistical calculations were performed using a standard t-test of variables with 95% confidence intervals.
We tested the effects of TB4 on cardiac endothelial cell migration by embryonic heart explant assays on rat tail collagen . Since TB4 is expressed in endothelial and myocardial cells and is absent in cardiac mesenchyme (Supplemental Figure 1 and ) we used TB4 primary antibody to identify endothelial cells. Exogenously administered TB4 significantly increased embryonic endothelial cell migration (Figure 1 a, e) by facilitating the number of round actively moving cells in vitro (Figure 1 c arrowheads) (Supplemental Figure 1B). We did not detect increase in cellular death (data not shown). Phospho-histone H3 staining and immunocytochemistry after BrdU administration indicated that TB4 significantly affects endothelial cell proliferation. (Figure 1 f-k). An explanation for increased proliferation could be the activation or up-regulation of β-Catenin expression [19, 20] by TB4 in vitro (Supplemental Figure 1C).
Human umbilical vein endothelial cells (HUVEC) form capillary structures when plated on matrigel [21, 22]. To analyze the effect of TB4 on cardiac vessel formation we checked whether adult human coronary endothelial cells (HCEC) behave similarly to HUVECs and if TB4 may alter the process in vitro. Our results indicated that HCECs are capable of capillary structure formation when cultured on matrigel and that TB4 expedites this process when compared to PBS treated controls (Figure 1 l-t).
Because of our in vitro observations on cardiac and vessel endothelial cells, we assessed the effect of TB4 on coronary vascular growth in adult mice after hypoxia. We created cardiac infarctions by ligating the LAD coronary artery in adult mice followed by immediate systemic TB4 or PBS administration. Simultaneous staining with platelet endothelial cell adhesion molecule-1 (Pecam-1) and smooth muscle α-actin (sm α-actin) specific primary antibodies revealed significant increase in capillary density three days after TB4 injection at the infarction border zone and non infarcted remote areas of the hearts (Figure 2).
Coronary vessels are believed to originate from the epicardium during development. Recent work from Lepilina et al.  demonstrated that an elaborate sequence of organ-wide and local responses by epicardial cells increases cardiac regeneration and revascularization in adult zebrafish. The changes of the epicardium in adult fish are similar to the alterations in developing embryos. Mammalian hearts however are incapable of such neovascularization or general regeneration after cardiac injury. Thus, experimental attempts to activate the embryonic coronary developmental program in mammals could enhance cardiac regeneration. To determine if TB4 may stimulate an epicardial response, we analyzed the changes in blood vessel/epicardial substance (Bves) expression 24 and 72 hours after systemic TB4 administration. Bves is widely expressed in the developing coronary vascular system [23, 24] and is also used as one of the markers of epicardial cells or cells of epicardial origin in adult and embryonic tissues. In our experiments, we observed elevated Bves expression 24 hours after TB4 treatment, and an increase in Bves positive cells with general organ-wide thickening of the adult epicardium 72 hours after peptide administration in the non-infarcted remote regions of the hearts (Figure 3). We found that most of the adult epicardial cells also express sm α-actin (Figure 2 c, d) (Supplemental Figure 2 a-h) and that the number of sm α-actin positive cells significantly increases proximal to the thickened epicardium after TB4 treatment (Supplemental Figure 2 i, j), indicating direct connection between the thickened epicardium and new capillary outgrowths.
Our in vivo results suggested that TB4 might activate the adult epicardium and initiate vessel growth. To support this hypothesis we investigated the expression of proteins essential during coronary development in embryos by Western blot and by immunohistochemistry 24 and 72 hours after systemic TB4 injection (Figure 4, Supplemental Figure 3). Our data indicate that TB4 affects developmental gene expression as early as 24 hours after systemic injection (Figure 4 A) while alterations in epicardial morphology were first observed three days after the initial peptide treatment (Figure 4 B). We detected significant increase in VEGF, VEGF receptor-2 (Flk-1) and TGF-β expressions and moderate elevation in FGF-17, FGF receptor-2 (FGFR-2) and FGFR-4 levels by Western blot after 24 hours of treatment. Immunohistochemistry after 3 days of TB4 injection indicated that these changes are primarily manifested in the thickened epicardium (Figure 4B). The alterations in gene expression were consistent with the findings in regenerating adult zebrafish hearts . Since FGF and WNT signaling pathways can function jointly to sustain mesenchymal growth or to coordinate epithelial morphogenesis during development [25, 26], and epicardium-derived progenitor cells also require β-Catenin for coronary artery formation , we asked whether TB4 may also alter β-Catenin expression in vivo. Our findings revealed an increase in β-Catenin expression in the thickened epicardium and in developing vasculature of the adult mouse hearts, which may indicate a role for β-Catenin and a regulatory convergence for FGF and WNT signaling in the epicardial initiation process.
In our previous work, we showed that TB4 significantly reduces scar volume by inhibiting myocardial cell death 24 hours after infarction . Because of recent studies suggesting that the epicardium serves as a source of cardiomyocytes in the embryo [9, 10] we asked whether TB4 could also initiate long term post ischemic muscle regeneration by myocardial progenitor activation in adult mouse hearts. We found that TB4 raises the expression of Tbx-18 and Wt-1 in the non infarcted remote areas after 24 hours (Figure 4 A) and significantly increases the number of Tbx-18 and Wt-1 positive cells after three days (Figure 4 B), (Supplemental Figure 3 B). Tbx-18 positive cells were distributed equally in the epicardium and myocardium, while Wt-1 positive cells were primarily located in the subepicardial space (Figure 4 B white arrowheads), suggesting that Wt-1 and Tbx-18 may mark different progenitor populations in the activated epicardium. Finally, the analysis of additional known regenerative proteins revealed that TB4 significantly increases Jun N-terminal kinase (JNK) expression while p38 expression and p38 and JNK activation were significantly reduced. We detected minor alterations in extracellular signal regulated kinase1/2 (Erk1/2) activation, inducible isoform of nitric oxide synthase (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) levels in the non infarcted cardiac tissue in the first 24 hours of treatment (Supplemental Figure 3 A). These observations strongly suggest an early molecular support for new vessel formation and myocardial regeneration by initiation of the embryonic epicardial developmental program and by activation of myocardial progenitors in adult mouse hearts after TB4 injection in vivo.
To further identify molecules that respond to TB4 and are expressed in the adult epicardium, we analyzed adult mouse hearts with Mouse Genome 430 2.0 Affymetrix cDNA microarrays 24 h after cardiac infarction (Supplemental Figure 4). While focusing on genes significant in angiogenesis, myristoylated alanine-rich C-kinase substrate (Marcks) was up-regulated 2.8-fold after TB4 administration (Figure 5 a). These results were confirmed by real-time RT-PCR (Figure 5 i). Marcks is a prominent intracellular substrate for PKC, a regulator of angiogenesis , and is distributed in numerous cell types, including vascular endothelial cells . It mediates PKC signaling through its phosphorylation , resulting in a release of Marcks from the cell membrane to the cytosol. These responses are commonly used to indicate PKC activity in vitro .
To determine if TB4 affects PKC activation in cell culture, we investigated Marcks phosphorylation and localization in adult HCECs by Western blot and immunocytochemistry after TB4 treatment. Our results indicate that external administration of TB4 increases Marcks expression, phosphorylation and translocation from the cell membrane to the cytosol suggesting that TB4 modulates PKC activity (Figure 5 b, c, h). PKC lies on the signal transduction pathways by which VEGF augments development and angiogenesis during initial and later stages of vessel development . Since VEGF expression was significantly increased after TB4 treatment (Figure 4) and PKC is known to activate Akt by Ser473 phosphorylation [1, 32], we speculate that VEGF mediated PKC activation represents an ILK-independent means of Akt activation by TB4 in cardiac cells.
To confirm our in vitro results, we examined the effect of TB4 on Marcks expression and phosphorylation after cardiac ligation in adult mice. As shown by Western blot (Figure 5 j) and immunohistochemistry, the expression (Figure 5 d, e, j) and phosphorylation (Figure 5 f, g, j) of Marcks increased, especially in the thickened epicardium, indicating a role for PKC in epicardial activation and coronary re-growth in adults after TB4 treatment in vivo. Additionally, analysis of Bves, TB4, VEGF and p-Marcks expression showed an increase and co-localization of Bves, TB4 and VEGF in the upper layer of the thickened epicardium (Supplemental Figure 5 A a, e, white arrowheads) after TB4 treatment, while p-Marcks-positive cells were mainly found in the subepicardium (Supplemental Figure 5 A c, g, white arrowheads). This is consistent with an indirect activation of PKC by TB4, possibly through increasing VEGF expression in the primary epicardial cells.
Recent results suggest that the epicardium is highly heterogenous and serves as a source of various progenitors in the embryonic heart [9, 10]. Consistent with these observations, immunohistochemistry on adult hearts showed several p-Marcks positive cells lacking VEGF while expressing Bves in wild type or in TB4 activated adult epicardium (Supplemental Figure 5 A g, l white arrowheads, c, j). Because of these findings, we asked whether p-Marcks positive cells might represent a distinct epicardial cell population of the adult heart. First, we cultured non-separated or FACS sorted p-Marcks positive and p-Marcks negative adult mouse epicardial cells (Supplemental Figure 5 C) on matrigel or gelatin coated dishes. Our data revealed that purified p-Marcks positive epicardial cells are small, have narrow cytoplasm and express both smooth muscle and/or endothelial markers in cell culture (Figure 6 a-c), while p-Marcks negative epicardial cells differentiate into large sm α-actin positive and Cytokeratin negative lineages (Supplemental Figure 5 C b, c). Consistent with our in vitro results, co-immunostaining with p-Marcks, sm α-actin, Cytokeratin and Pecam-1 antibodies indicated an overlap of smooth muscle and early and late endothelial markers in the newly developing capillaries (Figure 6 d-i white arrowheads) and in the endothelial and smooth muscle layers of the mature vessel wall (Figure 6 j-l). Similar to p-Marcks and VEGF expression, the adult epicardium contained a mixed population of p-Marcks, sm α-actin and Cytokeratin positive cells but did not stain for the late endothelial marker Pecam-1 (Figure 6 h, i). Also, external administration of TB4 to non-separated adult epicardial cultures resulted in a large number of p-Marcks and Cytokeratin specific endothelial colonies when compared to PBS treated controls (Supplemental Figure 5 B white arrowheads). Thus, our in vitro and in vivo results suggest that p-Marcks positive epicardial cells may be a source for smooth muscle and endothelial cells of the newly developing vessels in the injured adult heart.
To define whether PKC activity may be regulatory for TB4 induced epicardial progenitor activation, we tested the effect of Bisindolylmaleimide-I (PKC inhibitor) on HCECs and adult epicardial cells in vitro, and injected 10μg of PKC inhibitor intraperitoneally with or without TB4 into infarcted adult mice. Animals were harvested and hearts were processed for further analysis three days after injection. Our in vitro experiments indicated that PKC inhibition alters HCEC capillary structure formation (Figure 7 A a-d; Supplemental Figure 6 k) and inhibits epicardial cell differentiation (Figure 7 A i-l) on matrigel. The newly formed structures contained irregular sm α-actin positive cells and showed disordered morphology (Figure 7 A e-h). In live animals immunohistochemistry with Bves (Supplemental Figure 6 a-d), VEGF (Supplemental Figure 6 e-h) or p-Marcks (Figure 7 e-h) specific primary antibodies showed reduction of epicardial thickening. We also observed a significant decrease in capillaries and sm α-actin positive cells at the non infarcted remote areas (Figure 7 B a-d, i, j) or borders of the infarction (Supplemental Figure 6 i, j) after Bisindolylmaleimide-I injection. This suggests reduction of coronary outgrowths from the epicardium in the TB4+PKC inhibitor treated infarcted hearts. In addition, Bisindolylmaleimide-I significantly suppressed the number of TB4 activated Tbx-18 and Wt-1 positive progenitor cells in the non infarcted remote areas of the hearts (Figure 7 k, l). Our results suggest that TB4 mediated direct or indirect PKC activation is essential for epicardial cell transformation and migration in the adult mouse heart in vivo (Supplemental Figure 6 l, m).
This study shows that TB4 increases or activates proteins and epicardial progenitors important for myocardial regeneration or vascular growth and initiates organ-wide activation of the adult epicardium reminiscent of embryonic coronary development by re-stimulation of signaling pathways essential during embryogenesis. It reveals a novel role for PKC in this process. Additionally, it presents some evidence that p-Marcks positive epicardial cells may serve as endothelial and smooth muscle progenitors for future capillaries in the adult mammalian heart.
Despite the high specificity of the signaling systems triggering angiogenic responses in epicardial endothelial cells, there are no specific signaling pathways or transcription factors known that regulate the entire process . Given the roles of Pecam-1, VEGF, MAP kinases, ILK, Akt, FGFs, NOSes, β-Catenin, and PKC in vessel formation, our findings suggest that TB4 may initiate broad angiogenic events that promote cardiac regeneration after cardiac injury in adults. This is consistent with the recent observations that TB4 regulates epicardial development in embryonic mice .
Our findings also suggest that TB4 augments cardiac regeneration and increases cardiac function in the adult hypoxic heart through at least two steps. First, it inhibits myocardial cell death 24 h after cardiac ligation as previously described . Second, TB4 can initiate signaling pathways responsible for late-phase or chronic regeneration, such as vascular re-growth or progenitor cell activation. This process may be initiated by organ-wide epicardial thickening and activation of endothelial-mesenchymal transformation of adult epicardial cells, is most likely regulated by PKC, and is first visible 3 days after TB4 administration. Since TB4 is well conserved during evolution, it might be one of the factors responsible for epicardial activation during cardiac regeneration in zebrafish .
Coronary vessel growth is independent of cells outside the heart once the epicardium is formed . Thus the potential for TB4 to activate dormant cardiac stem cells that exist in the adult mammalian heart is critical for cardiac regeneration. Furthermore, since TB4 also inhibits inflammation [11, 34, 35], this property may be also supportive during cardiac regeneration in adults.
The concept of developing novel therapies that recapitulate embryonic events to achieve coupled myocardial and vascular regeneration is expanding . Recall of the embryonic developmental program in the adult heart may be the key to success. Our findings reveal the cellular and molecular changes initiated by TB4 during adult coronary remodeling in vivo. TB4 is the first molecule capable of initiating the embryonic coronary developmental program in adult mammalian hearts by systemic administration while protecting the myocardium after infarction. Since the discovery of innovative ways to enhance cardiac regeneration will be important for future therapies, the continued investigation of molecular signals initiated by TB4 could be critical.
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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