Epicardial Cell Differentiation Research Articles

Transcription factor 21 rs12190287 polymorphism is related to stable angina and ST elevation myocardial infarction in a Chinese Population.

Background: Transcription factor 21 (TCF21, epicardin, capsuling, pod-1) is expressed in the epicardium and is involved in the regulation of cell fate and differentiation via epithelial-mesenchymal transformation during development of the heart. In addition, TCF21 can suppress the differentiation of epicardial cells into vascular smooth muscle cells and promote cardiac fibroblast development. This study aimed to explore whether TCF21 gene (12190287G/C) variants affect coronary artery disease risk. Methods: We enrolled 381 patients who had stable angina, 138 with ST elevation myocardial infarction (STEMI), and 276 healthy subjects. Genotyping of rs12190287 of the TCF21 gene was performed. Results: Higher frequencies of the CC genotype were found in the patients with stable angina/STEMI than in the healthy controls. After adjusting for diabetes mellitus, hypertension, age, sex, smoking, body mass index and hyperlipidemia, the patients with the CC genotype of the TCF21 gene were associated with 2.49- and 9.19-fold increased risks of stable angina and STEMI, respectively, compared to the patients with the GG genotype. Furthermore, TCF21 CC genotypes showed positive correlations with both stable angina and STEMI, whereas TCF21 GG genotypes exhibited a negative correlation with STEMI. Moreover, the stable angina and STEMI patients with the CC genotype had significantly elevated high-sensitivity C-reactive protein levels than those with the GG genotype. In addition, significant associations were found between type 2 diabetes mellitus, hypertension, and hyperlipidemia with TCF21 gene polymorphisms (p for trend < 0.05). Conclusion: TCF21 gene polymorphisms may increase susceptibility to stable angina and STEMI.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their invivo counterparts, which enabled optimization of invitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. Invitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

The Role of Transcription Factor 21 in Epicardial Cell Differentiation and the Development of Coronary Heart Disease.

Transcription factor 21 (TCF21) is specific for mesoderm and is expressed in the embryos’ mesenchymal derived tissues, such as the epicardium. It plays a vital role in regulating cell differentiation and cell fate specificity through epithelial-mesenchymal transformation during cardiac development. For instance, TCF21 could promote cardiac fibroblast development and inhibit vascular smooth muscle cells (VSMCs) differentiation of epicardial cells. Recent large-scale genome-wide association studies have identified a mass of loci associated with coronary heart disease (CHD). There is mounting evidence that TCF21 polymorphism might confer genetic susceptibility to CHD. However, the molecular mechanisms of TCF21 in heart development and CHD remain fundamentally problematic. In this review, we are committed to providing a detailed introduction of the biological roles of TCF21 in epicardial fate determination and the development of CHD.

Hypoxia Supports Epicardial Cell Differentiation in Vascular Smooth Muscle Cells through the Activation of the TGFβ Pathway.

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). Results: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. Conclusion: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals.

Lack of Genetic Interaction between Tbx18 and Tbx2/Tbx20 in Mouse Epicardial Development.

The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.

A glimpse of Cre-mediated controversies in epicardial signalling.

Among the many important aspects of cardiac development, formation of the epicardium and the underlining signalling pathways that direct the mobilization and differentiation of epicardial cells have been one of the highly pursued research subjects within the past decade. These epicardium-derived cells (EPDCs) are reported to contribute to several critical cell types which include cardiac myofibroblasts and coronary smooth muscle contributing to the formation of the coronary vasculature network within the developing embryonic heart.1 Recent data suggesting that epicardial progenitor cells (EPCs) can contribute to newly formed cardiomyocytes in injured hearts2,3 has drawn further attention into the mechanisms of epicardiogenesis, as the successful use of epicardial cells in cell replacement therapies will undoubtedly have a high clinical impact on treating heart disease. To achieve this goal, a better understanding of the molecular mechanism that drive epicardiogenesis will be required. As the outermost cell layer of the vertebrate heart, the epicardium is derived from the proepicardial organ (PE), which originates from splanchnic mesenchyme within the septum transversum that is juxtaposed near the venous pole of the embryonic day 9.5 (E9.5) mouse heart (E22 for human). EPCs detach from the PE, migrate into pericardial cavity, and adhere to the myocardial surface where they rapidly spread to form a mesothelial monolayer covering the atria, atrioventricular canal, and ventricles, and in doing so, establish a primary epicardium by E10.5 in the mouse. Two days later, at E12.5, some of the primary epicardial cells undergo an epithelial to mesenchymal transition to convert into highly mobile and developmentally plastic EPDCs ( Figure 1 ). These EPDCs proceed to invade the subepicardial matrix and subsequently migrate into the myocardium, where they differentiate into interstitial …

A glimpse of Cre-mediated controversies in epicardial signalling

Among the many important aspects of cardiac development, formation of the epicardium and the underlining signalling pathways that direct the mobilization and differentiation of epicardial cells have been one of the highly pursued research subjects within the past decade. These epicardium-derived cells (EPDCs) are reported to contribute to several critical cell types which include cardiac myofibroblasts and coronary smooth muscle contributing to the formation of the coronary vasculature network within the developing embryonic heart.1 Recent data suggesting that epicardial progenitor cells (EPCs) can contribute to newly formed cardiomyocytes in injured hearts2,3 has drawn further attention into the mechanisms of epicardiogenesis, as the successful use of epicardial cells in cell replacement therapies will undoubtedly have a high clinical impact on treating heart disease. To achieve this goal, a better understanding of the molecular mechanism that drive epicardiogenesis will be required. As the outermost cell layer of the vertebrate heart, the epicardium is derived from the proepicardial organ (PE), which originates from splanchnic mesenchyme within the septum transversum that is juxtaposed near the venous pole of the embryonic day 9.5 (E9.5) mouse heart (E22 for human). EPCs detach from the PE, migrate into pericardial cavity, and adhere to the myocardial surface where they rapidly spread to form a mesothelial monolayer covering the atria, atrioventricular canal, and ventricles, and in doing so, establish a primary epicardium by E10.5 in the mouse. Two days later, at E12.5, some of the primary epicardial cells undergo an epithelial to mesenchymal transition to convert into highly mobile and developmentally plastic EPDCs ( Figure 1 ). These EPDCs proceed to invade the subepicardial matrix and subsequently migrate into the myocardium, where they differentiate into interstitial …

Epicardial function of canonical Wnt-, Hedgehog-, Fgfr1/2-, and Pdgfra-signalling

The embryonic epicardium is a source of smooth muscle cells and fibroblasts of the coronary vasculature and of the myocardium, but the signalling pathways that control mobilization and differentiation of epicardial cells are only partly known. We aimed to (re-)evaluate the relevance of canonical Wnt-, Hedgehog (Hh)-, Fibroblast growth factor receptor (Fgfr)1/2-, and platelet-derived growth factor receptor alpha (Pdgfra)-signalling in murine epicardial development. We used a T-box 18 (Tbx18)(cre)-mediated conditional approach to delete and to stabilize, respectively, the downstream mediator of canonical Wnt-signalling, beta-catenin (Ctnnb1), to delete and activate the mediator of Hh-signalling, smoothened (Smo), and to delete Fgfr1/Fgfr2 and Pdgfra in murine epicardial development. We show that epicardial loss of Ctnnb1, Smo, or Fgfr1/Fgfr2 does not affect cardiac development, whereas the loss of Pdgfra prevents the differentiation of epicardium-derived cells into mature fibroblasts. Epicardial expression of a stabilized version of Ctnnb1 results in the formation of hyperproliferative epicardial cell clusters; epicardial expression of a constitutively active version of Smo leads to epicardial thickening and loss of epicardial mobilization. Canonical Wnt-, Hh-, and Fgfr1/Fgfr2-signalling are dispensable for epicardial development, but Pdgfra-signalling is crucial for the differentiation of cardiac fibroblasts from epicardium-derived cells.

Tbx18 function in epicardial development

The embryonic epicardium is a source of smooth muscle cells and fibroblasts of the coronary vasculature and of the myocardium, but the molecular circuits that direct the temporal and spatial generation of these cell types from epicardium-derived cells are only partly known. We aimed to elucidate the functional significance of the conserved epicardial expression of the T-box transcription factor gene Tbx18 using transgenic technology in the mouse. We show by cellular and molecular analyses that in Tbx18-deficient mice the epicardium is formed normally and that epicardial cells undergo an epithelial-mesenchymal transition, differentiate into smooth muscle cells and fibroblasts, and form a normal coronary vasculature and fibrous skeleton. Prolonged expression of Tbx18 in epicardium-derived cells by a transgenic approach in vivo does not affect the differentiation and migratory behaviour of these cells. In contrast, epicardial misexpression of a transcriptional activator version of Tbx18, Tbx18VP16, results in premature smooth muscle differentiation of epicardial cells. Inhibition of Notch and transforming growth factor beta receptor signalling in Tbx18VP16 expressing epicardial cells in explant cultures reverts this phenotype. Tbx18 is dispensable for epicardial development, yet a repressive T-box function may be required to prevent premature smooth muscle cell differentiation by repressing transforming growth factor beta receptor and Notch signalling in the embryonic epicardium.

Application of Small Organic Molecules Reveals Cooperative TGFβ and BMP Regulation of Mesothelial Cell Behaviors

Epicardial development is a process during which epithelial sheet movement, single cell migration, and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement, and single cell migration by Transforming Growth Factor β (TGFβ). With the DM analogue DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.

Notch Signaling Regulates Smooth Muscle Differentiation of Epicardium-Derived Cells

The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. To define the role of Notch signaling in murine epicardial development. Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor β signaling in SM differentiation of EPDCs. Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.

Bves and NDRG4 modulate epicardial cell differentiation

Bves and NDRG4 modulate epicardial cell differentiation

TGFβ2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.

Migration and differentiation of epicardial cells stimulated by TGFβ1, TGFβ2, or BMP‐2 do not require the Type III Transforming Growth Factor β receptor

Transforming Growth Factor β (TGFβ) Receptor III (TGFβR3) binds all three TGFβ ligands, inhibin, and Bone Morphogenic Protein‐2 (BMP‐2). Since TGFβ causes epicardial cell differentiation into smooth muscle in vitro we asked if Tgfbr3 null epicardial cells differentiate in response to TGFβ or BMP‐2. Wiltype and Tgfbr3−/− immortalized mouse epicardial cells (ED 11.5) were cultured in the presence or absence of ligands for 72 hours and scored by immunohistochemistry, QT‐PCR, or a reportor assay for Smooth Muscle 22 alpha (SM22α). Wildtype and Tgfbr3−/− cells lose epithelial character and undergo smooth muscle differentiation in response to 250 pM TGFβ1 or TGFβ2. Induction of SM22α in wildtype cells occurred with an EC50 of 12.5 pM. BMP‐2 (5 nM) induced the loss of Zonula Occludens‐1 (ZO‐1) but failed to induce the smooth muscle markers SM22α, smooth muscle α‐actin (SMαA) or calponin. Inhibition of activin receptor‐like kinase (ALK) 5 with 2.5 μM SB431542 blocked the effects of TGFβ1 and TGFβ2, but not BMP‐2. Pre‐ and co‐incubation of cells with 5 nM BMP‐2 did not block the ability of 250 pM TGFβ1 or TGFβ2 to induce smooth muscle markers. These data demonstrate that BMP2 induces epicardial cell motility without smooth muscle differentiation and that TGFβR3 is not required for epicardial cell responsiveness to TGFβ or BMP‐2.Funding: HL085708, NIGMS 5K12GM068543Grant Funding Source: NIH HL

Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFβ

Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor beta (TGFbeta) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGFbeta1 or TGFbeta2 lose epithelial character and undergo smooth muscle differentiation. To further study TGFbeta Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor 1 (WT1). TGFbeta induced the loss of zonula occludens-1 (ZO-1) and the appearance of SM22alpha and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGFbeta while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGFbeta induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation.

TGFβ Induces Loss of Epithelial Character and Smooth Muscle Cell Differentiation in Epicardial Cells

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature including smooth muscle cells. We tested the hypothesis that transforming growth factor β (TGFβ) stimulates EMT and smooth muscle differentiation of epicardial cells. To test this hypothesis, we cultured explanted epicardium from 4 days in ovo chick hearts on three dimensional collagen gels or collagen coated chamber slides. In epicardial explants, TGFβ1 and TGFβ2 induce loss of epithelial morphology, cytokeratin and membrane-associated Zonula Occludens-1 (ZO-1), and increase the smooth muscle markers, calponin and caldesmon. TGFβ activates a family of membrane bound serine-threonine kinases, including activin receptor-like kinase (ALK) 5, resulting in the stimulation of a number of downstream effectors. Inhibition of ALK5 kinase activity blocks these effects while overexpression of constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFβ while only inhibition of p160 rho kinase blocked TGFβ–stimulated caldesmon expression. These data demonstrate that TGFβ stimulates loss of epithelial character and smooth muscle differentiation in epicaridal cells via a mechanism that requires ALK5 and p160 rho kinase. NIH HL67105; Vanderbilt MSTP 5 T32 GM07347.

Transforming growth factor‐β induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature, including smooth muscle cells. We tested the hypothesis that transforming growth factor-beta (TGFbeta) stimulates EMT and smooth muscle differentiation of epicardial cells. In epicardial explants, TGFbeta1 and TGFbeta2 induce loss of epithelial morphology, cytokeratin, and membrane-associated Zonula Occludens-1 and increase the smooth muscle markers calponin and caldesmon. Inhibition of activin receptor-like kinase (ALK) 5 blocks these effects, whereas constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFbeta, whereas only inhibition of p160 rho kinase blocked TGFbeta-stimulated caldesmon expression. These data demonstrate that TGFbeta stimulates loss of epithelial character and smooth muscle differentiation in epicardial cells by means of a mechanism that requires ALK5 and p160 rho kinase.

Epicardial/Mesothelial cell line retains vasculogenic potential of embryonic epicardium.

Recent work has demonstrated the importance of the epicardium in the development of the heart. During embryogenesis, these epithelial cells provide the progenitors for the epicardium, coronary smooth muscle, endothelium, and cardiac fibroblasts. The epicardium sends important signals to the developing myocardium. Still, analysis of these epithelial cells has lagged behind that of other cardiac cell types largely because of the lack of a defined experimental cell system in which epicardial cell differentiation can be studied. The present report examines the developmental potential of a cell line derived from rat epicardial mesothelial cells. These analyses demonstrate that the cell line retains many characteristics of the intact epithelium, including the ability to form a polarized epithelium and express many epicardial genes. Our data show for the first time that these cells retain the ability to produce mesenchyme in response to specific growth factors and, importantly, to generate smooth muscle cells. Thus, this study provides evidence that these cells can serve as an important model system for the analysis of the cellular and molecular mechanisms that govern epicardial development and function.