Embryonic Epicardial Cells Research Articles

MiR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/β-catenin signaling.

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor β (TGF-β) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3'UTR-MUT. In epicardial cells, TGF-β-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences β-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters β-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

Full cardiac regeneration by MicroRNA and G-actin monomer binding protein

Abstract Myocardial infarction causes massive loss of cardiomyocytes, leading to high mortality rate and heart failure. Cardiac regeneration is thought to have therapeutic potential for myocardial infarction and heart failure. We recently found that Nkx2.5+ cardiac myoblasts exist in declining number in the hearts of postnatal mice. The Nkx2.5+ cardiac myoblasts reactivate after myocardial infarction. They reside mostly in the subepicardium. Using inducible Nkx2.5 enhancer-Cre/ROSA26 reporter double transgenic mice to lineage trace the fate of activated Nkx2.5+ cardiac myoblasts, we documented that the activated Nkx2.5+ cardiac myoblasts proliferate and differentiate into mature cardiomyocyte in vivo. We also documented that Nkx2.5+ cardiac myoblasts originate from the embryonic epicardial cells. Thymosin ß4 is a G-actin monomer binding protein, involved in cell proliferation, migration, and differentiation. We hypothesized that Thymosin ß4 would promote Nkx2.5+ cardiac myoblasts, an embryonic epicardial-derived cells, mobilization and cardiomyogenesis, after myocardial injury. Various microRNAs (miR) have been reported to control cardiac development and promote cardiac regeneration. We have transfected neonatal Nkx2.5 cardiac myoblasts with various microRNAs and found miR-590 markedly promotes cardiomyogenesis capacity of cardiac progenitor cells. We have proved that miR-590 plus Thymosin ß4 (intraperitoneal injection) markedly improved murine cardiac function after myocardial infarction. In the treatment group with miR-590 plus Thymosin ß4 (n=9), LVEF improved from 45% (Day 0) to 77% (Day 7), to 83% (Day 28), to 85% (Day 56) with no LV regional wall motion abnormality and minimal aneurysm formation. In comparison, the control group with miR-mimic plus PBS (n=7), LVEF was 50% (Day 0) to 51% (Day 7), to 56% (Day 28), to 51% (Day 56) with persistent hypokinesia at apex and anterior wall with impaired LV contractility and larger aneurysm was revealed (p<0.001). Heart section of the post-MI mouse receiving miR-590 plus Thymosin ß4 showed minimal fibrosis in the outer layer of myocardium. In contrast, large fibrotic area was noted in the no treatment group. The mice receiving miR-590 plus Thymosin ß4 had a 2-fold increase in survival rate than the control group. Enhanced cardiomyocytes proliferation was documented by phosphorylated histone H3 stain at treated 1-week post-MI heart section. The study applies the results of our recent studies and successfully demonstrated systemic administration of miR-590 plus Thymosin ß4 markedly enhance cardiac repair/ regeneration. We anticipate the result would promote cardiac regeneration in clinical use.

Abstract P2096: Characterization Of Unique Dystrophin Isoforms In Epicardial Cells

Duchenne muscular dystrophy (DMD) is caused by mutations that abolish dystrophin expression. Dilated cardiomyopathy, the leading cause of death in DMD patients, is characterized by the development of severe subepicardial fibrosis that precedes cardiac dysfunction. While the cellular mechanisms governing this process remain unknown, a recent study in mdx/mTR mice suggested that inflammation, in response to cardiomyocyte damage caused by dystrophin deficiency, activates the epicardium to undergo epithelial-to-mesenchymal transition into fibroblasts, thereby contributing to subepicardial fibrosis. Whether dystrophin plays an epicardial-autonomous role in this process has not been investigated. The DMD gene produces at least eight dystrophin isoforms from seven independent, tissue-specific promoters ( Fig. 1 ). We used an immortalized mouse embryonic epicardial cell line to determine which isoforms are present. We found transcripts for the full-length muscle isoform of dystrophin (Dp427m) in addition to the ubiquitous, more abundant Dp71 and Dp40 isoforms. RT-PCR and western blot analysis revealed the presence of three different Dp71 splice variants. Immunofluorescence and subcellular fractionation determined that Dp71 localizes primarily to the nucleus. Studies are ongoing to understand the role of Dp71 in epicardial cell proliferation and gene expression, as well as defining changes in dystrophin expression during the transition from the activated epicardium to myofibroblasts. Further investigation is needed to determine the role of these shorter isoforms in DMD pathology and how gene editing of full-length dystrophin may impact their function.

Agrin Yes-associated Protein Promotes the Proliferation of Epicardial Cells.

Embryonic epicardial cells make an important contribution to cardiac development. However, their proliferation mechanism is still unclear. Epicardial cells from E12.5 fetal hearts were used in our study. Agrin was used to treat these cells. The expression of Aurora B, Ki67, and pH3 was measured by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. The proportion of cells in G1/S/G2 phase was determined by flow cytometry. The results showed that agrin significantly increased the expression of ki67, pH3, and Aurora B in epicardial cells. Flow cytometry results showed that agrin significantly increased the proportion of epicardial cells in S phase. However, blocking yes-associated protein significantly downregulated the levels of ki67, pH3, and Aurora B and the proportion of epicardial cells in S phase. Thus, our results suggest that agrin may promote the proliferation of epicardial cells by regulating the yes-associated protein activity. This may be useful in exploring heart development mechanisms and preventing congenital heart disease.

Are microRNAs responsible for cardiac hypertrophy in fish and mammals? What we can learn in the activation process in a zebrafish ex vivo model

Recent studies have correlated dysregulated miRNA expression with diseased hearts. With the aim of developing an easily manipulated experimental model, phenylephrine (PE) was added to cultured zebrafish hearts to study the expression of miR1 and miR133a by qRT-PCR. Both miRs were downregulated, with greater downregulation leading to higher hypertrophy. The involvement of this miRs was confirmed by the in-vivo inoculation of complementary sequences (AmiR1 and AmiR133a). HSP70 (involved in transporting proteins and in anti-apoptosis processes) was increased in both treatments. Hyperplasia was observed in the epicardium based on WT1 expression (embryonic epicardial cell marker) in both the PE treatment and AmiR133a treatment. The treatment with AmiR1 showed only cardiomyocyte hypertrophy. This ex-vivo model revealed that miR1 and miR133a play a key role in activating early processes leading to myocardium hypertrophy and epicardium hyperplasia and confirmed the expected similarities with hypertrophic disease that occurs in humans.

Insulin-like growth factor 1 receptor signaling regulates embryonic epicardial cell proliferation through focal adhesion kinase pathway.

Embryonic epicardial cells (EPCs) can facilitate cardiomyocyte growth through secreting several essential growth factors, and participate in cardiac development through auto-differentiating into many cardiac cell lineages. Proper proliferation of EPCs is the precondition of these functions, so it is quite necessary to explore the mechanisms involving in EPC proliferation. In this study, we aimed to explore whether insulin-like growth factor 1 receptor (IGF1R) signaling participated in regulating the proliferation of EPCs. Our results showed that the expressions of IGF1R and its ligands IGF1 and IGF2 can be clearly spotted on the epicardium layer from E11.5d to E17.5d. Inhibition of IGF1R signaling using picropodophyllin or NVP-AEW541 significantly decreased the proliferation activity and blocked the cell cycle progression of epicardial cells in vitro. On the contrary, activating IGF1R with recombinant IGF1 and IGF2 promoted epicardial cell proliferation and cell cycle. We also found that decreased expression and phosphorylation of FAK in IGF1R inhibitor-treated cells and use of FAK inhibitor Y15 could significantly inhibit the IGFs-induced EPC proliferation. In conclusion, our results suggest that IGF1R signaling plays an important role in regulating EPC proliferation, and this effect may be mediated by FAK pathway.

Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.

All- trans-retinoic acid (RA), a vitamin A metabolite, is an important signaling molecule required for the proper development of the heart. The epicardium is the main source of RA in the embryonic heart, yet the cardiogenic functions of epicardial-produced RA are not fully understood. Here, we investigated the roles of RA signaling in the embryonic epicardium using in vivo and in vitro models of excess or deficiency of RA. Our results suggested that RA signaling facilitates the cytoskeletal rearrangement required for the epicardial-to-mesenchymal transition of epicardial cells. In vivo treatment with an inhibitor of RA synthesis delayed the migration of epicardial-derived precursor cells (EPDCs) into the myocardium; the opposite was seen in the case of dehydrogenase/reductase superfamily (DHRS)3-deficient embryos, a mouse model of RA excess. Analysis of the behavior of epicardial cells exposed to RA receptor agonists or inhibitors of RA synthesis in vitro revealed that appropriate levels of RA are important in orchestrating the platelet-derived growth factor-induced loss of epithelial character, cytoskeletal remodeling, and migration, necessary for the infiltration of the myocardium by EPDCs. To understand the molecular mechanisms by which RA regulates epicardial cytoskeletal rearrangement, we used a whole transcriptome profiling approach, which in combination with pull-down and inhibition assays, demonstrated that the Ras homolog gene family, member A (RhoA) pathway is required for the morphologic changes induced by RA in epicardial cells. Collectively, these data demonstrate that RA regulates the cytoskeletal rearrangement of epicardial cells via a signaling cascade that involves the RhoA pathway.-Wang, S., Yu, J., Jones, J. W., Pierzchalski, K., Kane, M. A., Trainor, P. A., Xavier-Neto, J., Moise, A. R. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.

Biological characteristics of embryonic epicardial cells in vitro correlate with embryonic day.

The epicardial cell (EpiC) culture system plays an important role in investigating the specific mechanisms and signaling molecules that are involved in the development of EpiCs. From this early formation until adulthood, EpiCs undergo dynamic changes in the expression of embryonic genes that correlate with changes in the embryonic EpiC properties. The differences of embryonic EpiC properties may affect the related results of experiments in which EpiC culture system is used; however, these differences have not been explored. Therefore, in this study we examined the differences in the biological characteristics of EpiCs on different embryonic days in vitro EpiCs were isolated from embryonic ventricle explants on embryonic day (E) 11.5, E13.5, and E15.5. The differences in the migration, proliferation and differentiation were studied in EpiCs of different embryonic day by scratch assay, cell cycle analysis and platelet derived growth factor-bb (PDGF-BB) treatment. The results showed that EpiCs were successfully cultured from E11.5, E13.5, and E15.5 embryonic ventricle explants. The time windows of E11.5, E13.5, and E15.5 EpiC isolation out of the explants were different. The migration abilities of E11.5, E13.5, and E15.5 EpiCs decreased during embryonic development. Smooth muscle cell differentiation potential of early stage EpiCs was better than that of the later stage EpiCs. Although the proliferation ability of E11.5 EpiCs was significantly weaker than those of E13.5 and E15.5 EpiCs, the proliferation abilities of E13.5 and E15.5 EpiCs did not differ. These results suggest that the biological characteristics of EpiCs correlate with the timing of embryonic development, and different embryonic stage of ventricle should be properly chosen for culturing EpiCs depending on the purposes of the specific experiments.

Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFβR3 In Vitro.

The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFβ1 (515), TGFβ2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFβ1-TGFβ2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFβ and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFβ2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFβ2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFβR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.

<em>In Vitro</em> Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-β1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-β1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

Developmental origin of postnatal cardiomyogenic progenitor cells.

Aim:To trace the cell origin of the cells involved in postnatal cardiomyogenesis.Materials & methods:Nkx2.5 enhancer-eGFP (Nkx2.5 enh-eGFP) mice were used to test the cardiomyogenic potential of Nkx2.5 enhancer-expressing cells. By analyzing Cre excision of activated Nkx2.5-eGFP+ cells from different lineage-Cre/Nkx2.5 enh-eGFP/ROSA26 reporter mice, we traced the developmental origin of Nkx2.5 enhancer-expressing cells.Results:Nkx2.5 enhancer-expressing cells could differentiate into striated cardiomyocytes both in vitro and in vivo. Nkx2.5-eGFP+ cells increased remarkably after experimental myocardial infarction (MI). The post-MI Nkx2.5-eGFP+ cells originated from the embryonic epicardial cells, not from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells or perinatal/postnatal epicardial cells.Conclusion:Postnatal Nkx2.5 enhancer-expressing cells are cardiomyogenic progenitor cells and originate from embryonic epicardium-derived cells.

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.

Abstract 16189: Lineage Tracing of Postnatal Cardiomyogenic Progenitor Cells

Recent studies report that postnatal mammalian hearts undergo cardiomyocyte refreshment. While the exact origin of the cells involved in postnatal cardiomyogenesis remains unclear. Here, we identified a pool of Nkx2.5 enhancer expressing cells in the postnatal mouse heart with cardiomyogenic differentiation potential in vitro. We tracked the expression of a cardiac-specific enhancer of Nkx2.5 using inducible Nkx2.5 enhancer-Cre mice from embryonic development to adulthood and post-myocardial infarction (MI) and documented the Nkx2.5 enhancer expressing cells directly contribute to postnatal cardiomyogenesis in vivo. Upon genetic ablation of these activated progenitors after myocardial injury, the cardiac function deteriorated. Transcriptomic analysis of Nkx2.5 enhancer expressing cells showed high expression of heart development genes. To trace the developmental origin of the activated Nkx2.5 cardiomyogenic progenitor cells, we created different lineage-Cre/Nkx2.5 enh-eGFP/ROSA26 reporter triple transgenic mice. Post-MI Nkx2.5 cardiomyogenic progenitor cells originated from the embryonic epicardial cells, not from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, or perinatal/postnatal epicardial cells. Together, this study confirmed that cardiac lineage-specific progenitor cells, which originate from embryonic epicardium-derived cells, contribute to postnatal mammalian cardiomyogenesis.

WTIP interacts with ASXL2 and blocks ASXL2-mediated activation of retinoic acid signaling

The Asx-like (ASXL) family proteins are chromatin factors that play dual roles in transcriptional activation and repression. ASXL2 is highly expressed in the heart and is required for proper heart development and function. Here, we identify a novel ASXL2-binding partner, the LIM domain-containing protein WTIP. Genetic and biochemical assays show a direct interaction between ASXL2 and WTIP. In HeLa cells, ASXL2 enhances retinoic acid-dependent luciferase activity, while WTIP represses it. Furthermore, WTIP blocks ASXL2’s stimulatory effect on transcription. In addition, we found that ASXL2 and WTIP are expressed in mouse embryonic epicardial cells, a tissue that is regulated by retinoic acid signaling. Together, these results implicate ASXL2 and WTIP in regulation of retinoic acid signaling during heart development.

Re-Activated Adult Epicardial Progenitor Cells Are a Heterogeneous Population Molecularly Distinct from Their Embryonic Counterparts

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 which becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate adult epicardium-derived cells (EPDCs) after myocardial infarction (MI), to proliferate, and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterization 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. Wt1(GFPCre/+) mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1(+) GFP(+) EPDCs were fluorescence-activated cell sorted (FACS) at 2, 4, and 7 days after MI. Embryonic WT1(+) GFP(+) EPDCs were isolated from embryonic hearts (E12.5) by FACS, and sorted cells were characterized by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunostaining. Adult WT1(+) GFP(+) EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of stem cell antigen-1 (Sca-1), CD44, and CD90, we identified different subpopulations of EPDCs of varying cardiovascular potential, according to marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, adult WT1(+) GFP(+) cells are a heterogeneous population that 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 that discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.

Abstract 19099: Epicarial Progenitors Contribute to Neonatal Mammalian Heart Regeneration

Heart failure is a costly and deadly disease affecting over 5 million Americans. At the core of the pathophysiology of heart failure is the inability of the adult mammalian heart to regenerate following injury. In sharp contrast to the adult mammalian heart, we recently showed that the neonatal mammalian heart is capable of complete regeneration, including re-vascularization of the myocardium, following resection of the ventricular apex or myocardial infarction (MI). However, this remarkable regenerative capacity is lost by 7 days post natally. The regenerative response of the neonatal mouse heart is mediated mostly by proliferation of pre-existing cardiomyocytes, which is in line with the mechanism of zebrafish heart regeneration. We also have shown that neonatal heart injury is associated with epicardial activation, which is also a hallmark of zebrafish heart regeneration. However, the lineage contribution of epicardial cells during neonatal mouse heart regeneration remains to be determined. We performed genetic lineage tracing data using Capsulin (Cap)MerCreMer knock-in mouse crossed with Rosa26/tdTomato reporter mouse, in which epicardial cells are specifically and irreversibly labeled by tamoxifen pulse at postnatal day 0, revealed the contribution of neonatal epicardium to multiple lineages of the heart, including the majority of interstitial fibroblasts, vascular endothelial and smooth muscle cells both during normal post natal growth and after injury.. Considering that embryonic epicardial cells contribute to fibroblasts, vascular endothelial/smooth muscle cells and in contrast adult epicardium only contribute to interstitial fibroblasts and very rarely to vascular smooth muscle cells after the injury, it appears that lineage contribution of epicardial cells is highly correlated with the capacity for neovascularization following cardiac injury. These findings uncover a novel and important role of Capsulin positive epicardium derived cells in vasculogenesis during neonatal heart development and regeneration, and opens the door for future studies to better understand the dynamics of Capsulin expression and function in the post-natal mammalian heart.

The developmental origin of postnatal cardiac regeneration

Recent studies have shown that postnatal mammalian hearts undergo cardiomyocyte refreshment. However, evidence is lacking for the cell origin of these postnatal cardiomyogenesis. We first confirmed that myocardial injury triggered the expression of embryonic cardiogenesis genes at the external part of the heart. To determine whether cardiac lineage-specific progenitor cells exist in the postnatal mammalian heart and have cardiomyogenic potential, we created coronary artery ligation on the Nkx2.5 enhancer-eGFP transgenic mice. The number of Nkx2.5 enhancer-eGFP cells was increasing after myocardial injury and peaked at 2 weeks post injury. The activated eGFP+ cells expressed cardiac precursor markers. The eGFP signaling was not expressed in mature cardiomyocytes, hematopoietic cells or fibroblasts. These activated Nkx2.5 enhancer-eGFP cells would differentiate into striated cardiomyocytes in vitro. We further confirmed that cardiac progenitor cells contributed directly to postnatal cardiomyogenesis by lineage tracing using inducible Cre system. To trace the developmental origin of the activated Nkx2.5 enhancer-eGFP cells, we created different lineage-Cre/Nkx2.5 enhancer-eGFP/ ROSA26 reporter triple transgenic mice. The post-injury Nkx2.5 enhancer-eGFP+ cells originated from the embryonic epicardial cells, but did not originate from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, and perinatal/ postnatal epicardial cells. Taken together, our data confirmed that cardiac-lineage progenitor cells, which originated from embryonic epicardium-derived cells, contributed to postnatal mammamian cardiomyogenesis. The discovery of cardiomyogenic cell population in the postnatal heart enables the future cell therapy for cardiac regeneration.

2 Characterising the progenitor cell potential of adult mesothelial cells

Organs situated within coelomic cavities, that is, heart, lungs and intestinal organs, are surrounded by mesothelial cells throughout life. We and others have shown previously that mesothelial cells contribute to...

The regulation of lymphangiogenic signaling in the epicardium

We have demonstrated that the cardiac lymphatics are derived in part from mesothelial cells in the embryonic epicardium, and that their development is impacted by VEGFR-3 inhibition. In this study, we explored the mechanisms that may affect lymphatic differentiation in pluripotent epicardial cells. Adult and embryonic epicardial cells were treated with the lymphangiogenic growth factor VEGF-C, resulting in a dramatic increase in nuclear expression for Prox-1, a master lymphatic regulatory gene, within 5 min of VEGF-C administration. The nuclear accumulation of Prox-1 was not observed after FGF or VEGF-A treatment. The ERK inhibitor UO126 induced a decrease in Prox-1 expression in nuclei in both untreated and VEGF-C treated cells, suggesting that activated ERK may regulate Prox-1 nuclear accumulation. Inhibiting ERK function in the epicardium also inhibited cell migration and differentiation. Our studies suggest that the lymphangiogenic growth factor VEGF-C binds to VEGFR-3 and acts via ERK to stimulate nuclear accumulation of Prox-1, in adult and embryonic epicardial cells. Preliminary findings suggest that tissue oxygen levels, mediated by the hypoxia inducible factor HIF-1, may also regulate lymphangiogenic signaling in the epicardium. Source of Research Support: R01 HL091171(ARRA), ES013507, AHA 0715153B. Grant Funding Source: NIH