Epithelial-to-mesenchymal transformation alters electrical conductivity of human epicardial cells

The myocardium of the developing heart tube is covered by epicardium. These epicardial cells undergo a process of epithelial-to-mesenchymal transformation (EMT) and develop into epicardium-derived cells (EPDCs). The ingrowing EPDCs differentiate into several celltypes of which the cardiac fibroblasts form the main group. Disturbance of EMT of the epicardium leads to serious hypoplasia of the myocardium, abnormal coronary artery differentiation and Purkinje fibre paucity. Interestingly, the electrophysiological properties of epicardial cells and whether EMT influences electrical conductivity of epicardial cells is not yet known. We studied the electrophysiological aspects of epicardial cells before and after EMT in a dedicated in vitro model, using micro-electrode arrays to investigate electrical conduction across epicardial cells. Therefore, human adult epicardial cells were placed between two neonatal rat cardiomyocyte populations. Before EMT the epicardial cells have a cobblestone (epithelium-like) phenotype that was confirmed by staining for the cell-adhesion molecule β-catenin. After spontaneous EMT in vitro the EPDCs acquired a spindle-shaped morphology confirmed by vimentin staining. When comparing both types we observed that the electrical conduction is influenced by EMT, resulting in significantly reduced conductivity of spindle-shaped EPDCs, associated with a conduction block. Furthermore, the expression of both gap junction (connexins 40, Cx43 and Cx45) and ion channel proteins (SCN5a, CACNA1C and Kir2.1) was down-regulated after EMT. This study shows for the first time the conduction differences between epicardial cells before and after EMT. These differences may be of relevance for the role of EPDCs in cardiac development, and in EMT-related cardiac dysfunction.     

Epicardial epithelial-to-mesenchymal transition in injured heart

Cre-LoxP-mediated genetic lineage trace has been used to illuminate the cell fate of progenitor cells in vivo. Application of this strategy to the epicardium, a sheet of cells covering the surface of heart, revealed that it dynamically participates in both heart development and postnatal heart repair and regeneration. After myocardial infarction, epicardial cells undergo epithelial-to-mesenchymal transition (EMT) and mainly adopt myofibroblast, fibroblast and smooth muscle cell fates. Here we present the wholemount images that map epicardial EMT following myocardial infarction, taking advantage of an inducible epicardial Cre line and a double fluorescence reporter. While remote epicardium retained its epithelial cell shape, reactivated epicardium in the infarcted region showed significant EMT. This image supports active involvement of the epicardium in repair and regeneration of infarcted myocardium.     

FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation

Critical steps in coronary vascular formation include the epithelial-mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.     

心外膜的发育

心外膜是覆盖在心脏外层的间皮组织。心外膜来源于脏壁中胚层的前心外膜,后者位于心管流入极附近。心外膜的部分细胞通过上皮间充质转化进入心外膜下层,随后形成血管内皮、成纤维和平滑肌细胞,最终导致冠脉系统的形成。心外膜细胞可能形成心肌细胞,并且可能是心脏驻留干细胞的来源。因此,它在心脏修复治疗中发挥巨大作用。本文回顾了该领域的最新研究进展并且提出了目前存在的问题。     

Epicardium‐derived cells (EPDCs) in development,cardiac disease and repair of ischemia

The proepicardial-derived epicardium covers the myocardium and after a process of epithelial–mesenchymal transition (EMT) forms epicardium-derived cells (EPDCs). These cells migrate into the myocardium and show an essential role in the induction of the ventricular compact myocardium and the differentiation of the Purkinje fibres. EPDCs are furthermore the source of the interstitial fibroblast, the coronary smooth muscle cell and the adventitial fibroblast. The possible differentiation into cardiomyocytes, endothelial cells and the recently described telocyte and other cells in the cardiac stem cell niche needs further investigation. Surgically or genetically disturbed epicardial and EPDC differentiation leads to a spectrum of abnormalities varying from thin undifferentiated myocardium, which can be embryonic lethal, to a diminished coronary vascular bed with even absent main coronary arteries. The embryonic potential of EPDCs has been translated to both structural and functional congenital malformations and adult cardiac disease, like development of Ebstein’s malformation, arrhythmia and cardiomyopathies. Furthermore, the use of adult EPDCs as a stem cell source has been explored, showing in an animal model of myocardial ischemia the recapitulation of the embryonic program with improved function, angiogenesis and less adverse remodeling. Combining EPDCs and adult cardiomyocyte progenitor cells synergistically improved these results. The contribution of injected EPDCs was instructive rather than constructive. The finding of reactivation of the endogenous epicardium in ischemia with re-expression of developmental genes and renewed EMT marks the onset of a novel therapeutic focus.     

Epithelial‐to‐mesenchymal transition in epicardium is independent of snail1

The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial‐to‐mesenchymal transition (EMT) to generate mesenchymal epicardial‐derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1‐ and Tbx18‐Cre drivers. Unexpectedly, epicardial‐specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT. genesis 51:32–40, 2013. © 2012 Wiley Periodicals, Inc.     

Nf1 limits epicardial derivative expansion by regulating epithelial to mesenchymal transition and proliferation

The epicardium is the primary source of coronary vascular smooth muscle cells (cVSMCs) and fibroblasts that reside in the compact myocardium. To form these epicardial-derived cells (EPDCs), the epicardium undergoes the process of epithelial to mesenchymal transition (EMT). Although several signaling pathways have been identified that disrupt EMT, no pathway has been reported that restricts this developmental process. Here, we identify neurofibromin 1 (Nf1) as a key mediator of epicardial EMT. To determine the function of Nf1 during epicardial EMT and the formation of epicardial derivatives, cardiac fibroblasts and cVSMCs, we generated mice with a tissue-specific deletion of Nf1 in the epicardium. We found that mutant epicardial cells transitioned more readily to mesenchymal cells in vitro and in vivo. The mesothelial epicardium lost epithelial gene expression and became more invasive. Using lineage tracing of EPDCs, we found that the process of EMT occurred earlier in Nf1 mutant hearts, with an increase in epicardial cells entering the compact myocardium. Moreover, loss of Nf1 caused increased EPDC proliferation and resulted in more cardiac fibroblasts and cVSMCs. Finally, we were able to partially reverse the excessive EMT caused by loss of Nf1 by disrupting Pdgfrα expression in the epicardium. Conversely, Nf1 activation was able to inhibit PDGF-induced epicardial EMT. Our results demonstrate a regulatory role for Nf1 during epicardial EMT and provide insights into the susceptibility of patients with disrupted NF1 signaling to cardiovascular disease.     

Inhibition of alpha4-integrin stimulates epicardial-mesenchymal transformation and alters migration and cell fate of epicardially derived mesenchyme

Epithelial-mesenchymal transformation of the embryonic epicardium produces the subepicardial mesenchyme that is essential for normal coronary vascular development. Gene targeting experiments in mice have demonstrated an essential role for alpha4-integrin in normal epicardial development, but the precise cellular consequences of alpha4-integrin loss remain uncertain. To better understand the function of alpha4-integrin in epicardial development, we constructed a replication-incompetent adenovirus (AdlacZalpha4AS) that expresses antisense chicken alpha4-integrin as the 3' untranslated region of a lacZ reporter gene. This construct effectively labeled cells while greatly reducing levels of alpha4-integrin mRNA and protein. In quail chick chimeras, transplanted epicardial cells infected with AdlacZalpha4AS adhered to the heart and were incorporated into the epicardium, but 4 days after grafting, were largely absent from the epicardial epithelium, recapitulating the defect in alpha4-null mice. This did not result from epicardial cell apoptosis or anomalous migration of epicardial cells to extracardiac sites. Rather, AdlacZalpha4AS-infected epicardial cells were particularly invasive, being three to four times more likely to migrate to the interstitium of the myocardium than AdlacZ-infected epicardial cells. Accelerated epicardial-mesenchymal transformation and migration of alpha4-negative epicardium was observed in an organ culture system that does not require prior culture of epicardial cells. Remarkably, AdlacZalpha4AS infection also prevented targeting of epicardially derived mesenchyme to the media of developing coronary vasculature in the myocardial interstitium. This study provides evidence that epicardial alpha4-integrin normally restrains epicardial-mesenchymal transformation, invasion, and migration and is essential for correct targeting of epicardially derived mesenchyme to the developing coronary vasculature.     

bves: A novel gene expressed during coronary blood vessel development

We have used a subtractive method to clone novel messages enriched in the heart. Here we show that one such message, bves (blood vessel/epicardial substance) is a novel protein that is highly conserved between chicken and mouse. The bves message is detected at high levels in early chick hearts. Using anti-Bves antibodies, we show expression in cells of the proepicardial organ, migrating epicardium, epicardial-derived mesenchyme, and smooth muscle of the developing intracardiac arterial system, including the coronary arteries. Our data suggest that Bves is an early marker of developing vascular smooth muscle cells. In addition, the expression pattern of Bves protein reveals the patterning of intracardiac vascular smooth muscle and possible insights into the cellular regulation of smooth muscle differentiation during vasculogenesis.     

基因谱系示踪技术揭示小鼠Tbx18+心脏祖细胞向心系细胞分化的潜能

心脏祖细胞(cardiac progenitor cells,CPCs)的研究对阐明先天性心脏病的机制及治疗心血管疾病具有重要意义.哺乳动物的心脏组织由多种不同CPCs分化形成.转录因子Tbx18在发育中的心外膜中表达,对心脏的发育形成起重要的调节作用.为了在组织及活体细胞水平检测和阐明Tbx18+CPC的分化潜能,应用Cre-LoxP系统建立Tbx18+CPCs基因命运谱系示踪模型:Tbx18-Cre/Rosa26R-EYFP和Tbx18-Cre/Rosa26R-LacZ双杂合基因敲入小鼠.该双杂合基因敲入小鼠通过Cre的表达能有效地示踪Tbx18+细胞在胚胎和成年小鼠中的分化命运.Tbx18-Cre/Rosa26R-EYFP双杂合小鼠心脏能非常容易地利用流式细胞分选系统(FACS)分离出YFP+细胞,也可在倒置共聚焦显微镜下观察.应用X-gal染色分析其表达模式,揭示Tbx18命运谱系参与心房肌、室间隔、心室肌、冠状动脉、瓣膜等的形成.应用免疫荧光技术初步揭示Tbx18+CPCs向心脏肌钙蛋白T(cTNT)阳性心肌细胞和平滑肌肌球蛋白重链11(MYH11)阳性血管平滑肌细胞分化的潜能.心脏是一个由多种肌肉和非肌肉组织细胞构成的复杂器官.推测Tbx18可能在心脏祖细胞向肌源性细胞分化的信号通路中起重要调节作用.在上述研究中应用基因谱系示踪技术,验证Tbx18可作为一类CPCs的标志,为更深入揭示心脏祖细胞向心系细胞的分化潜能打下基础.     

BMP and FGF regulate the differentiation of multipotential pericardial mesoderm into the myocardial or epicardial lineage

Proepicardial cells give rise to epicardium, coronary vasculature and cardiac fibroblasts. The proepicardium is derived from the mesodermal lining of the prospective pericardial cavity that simultaneously contributes myocardium to the venous pole of the elongating primitive heart tube. Using proepicardial explant cultures, we show that proepicardial cells have the potential to differentiate into cardiac muscle cells, reflecting the multipotency of this pericardial mesoderm. The differentiation into the myocardial or epicardial lineage is mediated by the cooperative action of BMP and FGF signaling. BMP2 is expressed in the distal IFT myocardium and stimulates cardiomyocyte formation. FGF2 is expressed in the proepicardium and stimulates differentiation into the epicardial lineage. In the base of the proepicardium, coexpression of BMP2 and FGF2 inhibits both myocardial and epicardial differentiation. We conclude that the epicardial/myocardial lineage decisions are mediated by an extrinsic, inductive mechanism, which is determined by the position of the cells in the pericardial mesoderm.