Proepicardial Cells Research Articles

Transcription factor heterogeneity in proepicardial and epicardium-derived cell populations during heart development

Transcription factor heterogeneity in proepicardial and epicardium-derived cell populations during heart development

A migratory role for EphrinB ligands in avian epicardial mesothelial cells

Little is known about the molecules that mediate the attachment of proepicardial cells to the heart. Ephrins are cell surface ligands for Eph tyrosine kinase receptors, molecules known to play a role in cell adhesion and migration. Here, we detected EphrinB ligands in proepicardial and epicardial mesothelial cells (EMCs) using reverse transcriptase-polymerase chain reaction, immunoblotting, immunolocalization, and EphB-Fc binding. Aggregated EphB-Fc fragments clustered ephrinB1 ligands on living EMCs indicating that they are cell surface expressed. In vitro assays demonstrated that ephrinB ligands participate in EMC migration but not cell adhesion. Localization studies in hearts at Hamburger and Hamilton stage 30 and older revealed that ephrinB1 is expressed in the epicardium and subepicardial mesenchyme of the atrioventricular sulcus. EMCs treated with platelet-derived growth factor-BB expressed smooth muscle markers but not ephrinB1. Our study supports an early role for ephrinB ligands for migration of epicardial cells and a later role in perivascular fibroblasts of coronary vessels in the atrioventricular sulcus.

The Role of Serum Response Factor in Early Coronary Vasculogenesis

Defects in vascular development are a major cause of fetal demise and congenital cardiovascular disease. Knowledge is scarce concerning both embryonic vascular cell progenitors and the underlying regulatory mechanisms that determine their commitment to various vessel cell lineages. During early cardiovascular development, the proepicardium (PE) is the major source of vascular progenitors. The PE is a transient embryonic structure found at stages 16 and 17 in the chick, at 9.5 days postcoitum (dpc) in the mouse, and at approximately 5 weeks in the human that consists of mesothelial and mesenchymal cells overlying the septum transversum (ST). Cells within the PE undergo a complex process of development and differentiation that involves at least two directed waves of migration and epithelial-to-mesenchymal transitions (EMT). The PE cells make contact with the inferior surface of the developing myocardium, migrate over the surface of the heart forming the epicardium, and penetrate into the myocardium and give rise to subepicardial mesenchymal cells (SEMCs). The SEMCs subsequently migrate throughout the developing heart giving rise to the entire coronary vasculature consisting of fibroblasts as well as endothelial and smooth muscle cells [32]. Serum response factor (SRF) is essential for mesoderm formation during embryonic development [2]. A necessary role for SRF in the development of vascular smooth muscle from yet to be characterized PE-derived coronary vascular precursor cells has been demonstrated in avian explant models [15]. The role of SRF in vivo during some of the earliest steps in mammalian PE-mediated vascular development, however, is less well studied, and our recent observations suggest that it likely is more complex. This brief review aims to discuss the role of SRF during early coronary vasculogenesis as it relates to development of the PE.

Effects of endothelial growth media on proepicardial cell gene expression and morphogenesis in 3D collagen matrices

Proepicardial cells (PE) contribute to embryonic coronary vessel and epicardial development. Cells from the PE region can differentiate into coronary vascular smooth muscle cells and fibroblasts in vitro, but the endothelial specification capability of these cells is controversial. We sought to examine the effects of endothelial cell growth media on gene expression and the morphogenic properties of proepicardial cells in three-dimensional (3D) matrices. A primary culture of avian PE cells was subjected to molecular characterization with selected endothelial specific markers. Morphogenic properties of PE cells were assessed by in vitro assays of coronary vasculogenesis and invasion, which utilized highly defined, serum free, three-dimensional matrix conditions. PE cells maintained mixed cell population properties in the culture based on morphogenic features, immunohistochemistry, and the gene expression data. When suspended in a 3D vasculogenesis in vitro assay, PE cells formed intracellular vacuoles and assembled into multicellular tubes. Further, ultrastructural analysis revealed the presence of pinocytic vacuoles, intercellular junctions, and endothelial specific Weibel Palade bodies. In the invasion assay, PE cells spontaneously invaded control matrices. This invasion was markedly enhanced by lysophosphatidic acid (94±9.6 vs. 285.6±54.9, p<0.05) and was completely blocked with synthetic broad-spectrum metalloproteinase inhibitor GM6001. Isolated PE cells grown in endothelial cell media represent mixed-cell population, characterized by both smooth muscle and endothelial gene expression. When placed in 3D in vitro assays, PE cells manifest morphogenic properties, including multicellular tube assembly and invasion.

Pro-epicardial and inflow myocardial cells share common lineage in avian

Pro-epicardial and inflow myocardial cells share common lineage in avian

Development of the Cardiac Conduction System

The conduction system of the heart initiates and coordinates the electric signal that causes the rhythmic and synchronized contractions of the atria and ventricles. In higher vertebrates, this system comprises the sinuatrial (SAN) and atrioventricular nodes (AVN) and the “wiring” of the ventricles. The latter comprises the atrioventricular bundle (AVB), the left and right bundle branches (BBs), and the peripheral ventricular conduction system (PVCS). Failure in the function of the nodes or bundles leads to arrhythmias, requiring electronic pacemaker implantation. On the other side, ectopic pacemaker or conductive activity may cause arrhythmias requiring intervention. Recent progress in molecular-genetic and developmental biology has lead to novel insights into the processes underlying the formation of the conduction system. These insights provide a framework that helps to understand pathologies of the conduction system. In this review, we focus on the cellular origin of the conduction system components, the molecular-genetic mechanisms that control their differentiation, and their impact on arrhythmias. The distinct components of the cardiac conduction system of the heart are essentially myocardial (Figure 1).1–4 The hypothesis that the conduction system may have a neural crest origin has not been supported by evidence. Retroviral labeled single cardiac cells of the early embryonic chicken heart (HH15-17, ±30 somites) were found to give rise to multicellular clones containing both working myocardium and myocardial cells of the conduction system.5,6 Moreover, in both chicken and mouse, labeled neural crest or proepicardial cells were never traced in the conduction system components, even though their descendants were found in close association with these components.6 Figure 1. Schematic overview of heart development in higher vertebrates. The early heart tube has a primitive phenotype (light purple). Chamber myocardium (gray) expands from the outer curvatures of the primary heart tube, whereas nonchamber myocardium (purple) of the inflow tract …

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.

Coronary endothelial proliferation and morphogenesis are regulated by a VEGF‐mediated pathway

Though development of the coronary vasculature is a critical event during embryogenesis, the molecular mechanisms that regulate its formation are not well characterized. Two unique approaches were used to investigate interactions between cardiac myocytes and proepicardial (PE) cells, which are the coronary anlagen. One of these experimental approaches used a 3-D collagen scaffold system on which specific cell-cell and cell-matrix interactions were studied. The other approach used a whole heart culture system that allowed for the analysis of epicardial to mesenchymal transformation (EMT). The VEGF signaling system has been implicated previously as an important regulator of coronary development. Our results demonstrated that a specific isoform of VEGF-A, VEGF(164), increased PE-derived endothelial cell proliferation and also increased EMT. However, VEGF-stimulated endothelial cells did not robustly coalesce into endothelial tubes as they did when cocultured with cardiac myocytes. Interestingly, blocking VEGF signaling via flk-1 inhibition reduced endothelial tube formation despite the presence of cardiac myocytes. These results indicate that VEGF signaling is complex during coronary development and that combinatorial signaling by other VEGF-A isoforms or other flk-1-binding VEGFs are likely to regulate endothelial tube formation.

Development of the proepicardium in Xenopus laevis

The proepicardium (PE) is an embryonic progenitor cell population, which provides the epicardium, the majority of the cardiac interstitium, the coronary vasculature and possibly some cardiomyocytes. Recent studies have documented (1) the presence of bilaterally paired PE anlagen in several vertebrates, and (2) species-specific differences in the fate of the left and right PE anlagen. Here, we document PE development in Xenopus laevis (stages 37-46). The PE appears at stage 41 in the form of a cone-shaped accumulation of mesothelial cells covering the pericardial surface of the right horn of the sinus venosus. No such structure appears on the left sinus horn. At the end of stage 41, the tip of the PE establishes a firm contact with the developing ventricle. A secondary tissue bridge is established facilitating the transfer of PE cells to the heart. During stages 41-46, this tissue bridge is visible in vivo through the transparent body wall. Corresponding to the morphological data, the PE marker gene Tbx18 is expressed only on the right sinus horn suggesting a right-sided origin of the PE. Left-right lineage tracing has confirmed this idea. These results show that Xenopus PE development proceeds in a bilaterally asymmetric pattern as previously observed in chicks. We speculate that asymmetric PE development is controlled by signals from left-right signaling pathways and that the PE is an indicator for right-sidedness in Xenopus embryos. Xenopus might be a good model to uncover the role of left-right signaling pathways in the control of asymmetric PE development.

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton. Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns. We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff–Parkinson White syndrome with persistent AV myocardial connections.

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart

The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

A myocardial lineage derives from Tbx18 epicardial cells

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

The influence of proepicardial cells on the osteogenic potential of marrow stromal cells in a three-dimensional tubular scaffold

It is well established that the process of neovascularization or neoangiogenesis is coupled to the development and maturation of bone. Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) comprise a heterogeneous population of cells that can be differentiated in vitro into both mesenchymal and non-mesenchymal cell lineages. When both rat BMSCs and quail proepicardia (PEs) were seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned collagen type I strands and co-cultured in osteogenic media, the maturation and co-differentiation into osteoblastic and vascular cell lineages were observed. In addition, these cells produced abundant mineralized extracellular matrix materials and vessel-like structures. BMSCs were seeded at a density of 2 × 10 6 cells/15 mm tube and cultured in basal media for 3 days. Subsequently, on day 3, PEs were seeded onto the same tubes and the co-culture was continued for another 3, 6 or 9 days either in basal or in osteogenic media. Differentiated cells were subjected to immunohistochemical, cytochemical and biochemical analyses. Phenotypic induction was analyzed at mRNA level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Immunolocalization of key osteogenic and vasculogenic lineage specific markers were examined using confocal scanning laser microscopy. In osteogenic tube cultures, both early and late osteogenic markers were observed and were reminiscent of in vivo expression pattern. Alkaline phosphatase activity and calcium content significantly increased over the observed period of time in osteogenic medium. Abundant interlacing fascicles of QCPN, QH1, isolectin and α-smooth muscle actin (α-SMA) positive cells were observed in these tube cultures. These cells formed extensive arborizations of nascent capillary-like structures and were seen amidst the developing osteoblasts in osteogenic cultures. The 3-D culture system not only generated de novo vessel-like structures but also augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, this novel co-culture system provides a useful in vitro model to investigate the functional role and effects of neovascularization in the proliferation, differentiation and maturation of BMSC derived osteoblasts.

Embryonic Epicardial Cell Lineages: Making and Unmaking a Heart

The interaction of heart muscle precursors with other non‐muscular endodermal, ectodermal and mesodermal cell populations (i.e. the pharyngeal endoderm, the neural crest ectomesenchyme, the endocardium and the epicardium, respectively) is critical to the proper morphogenesis of the heart, including the complete differentiation of myocardial progenitors into a variety of muscle types that are neither equal in origin nor equivalent in their biomechanical properties. Moreover, normal cardiac development also depends on the restriction of myocardial domains after the inhibition of the muscular potentials of the pericardiac mesoderm. Proepicardial cells, which constitute a part of the pericardiac mesoderm located at the heart inflow, and their epicardial derivatives are most relevant to heart development and viability since they give rise to both coronary blood vessels and cardiac interstitial cells, but also modulate chamber myocardial proliferation. Most interestingly, proepicardial cells can differentiate into myocardium in vitro but not under normal in vivo conditions. It is the main aim of this presentation to provide information on the transcriptional and signaling networks involved in the definition of epicardial developmental potential and fate and to discuss on their embryological significance.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus

The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

The epicardium has potential in cardiac healing and regeneration in mammals

The proepicardium is a small transient structure that develops at the base of the heart, adjacent to the septum transversum. It is made up of pluripotent cells that migrate to the developing heart to give rise to fibroblasts, vascular support cells, cardiomyocytes, and the epicardium. It is unclear if and when proepicardial cells lose their pluripotential.After an ischemic event, such as a heart attack, the heart compensates for cardiomyocyte loss with fibrosis to maintain the integrity of the cardiac wall, preventing rupture. The result is a stiffer and less effective pump.These experiments show the ability of embryonic epicardial cells to contribute to myocardial healing In Vitro after transmural laceration. Following injury, the whole embryonic heart is explanted and cultured. Further studies in adult mice show that epicardial cells are present at the site of injury in a surgical model of cardiac damage. Further studies will show the fate of cells arising from the epicardium that contribute to the healing response following cardiac injury.These preliminary results suggest that epicardial cells are able to migrate and differentiate in response to injury. The epicardium may be a source of pluripotent cells that, with the right encouragement, may have the ability to contribute new vasculature as well as new myocytes after an ischemic event.

Embryonic myocyte‐derived factors regulate early coronary vessel development

Many heart problems are associated with defects and disease of the coronary vessels; however coronary vessel development is not well understood. Coronary vessels originate from epicardial cells which come from the proepicardium (PE). At E9.0 in mice, PE cells migrate to and spread over the heart to form the epicardium. A subset of epicardial cells undergoes an epithelial to mesenchymal transformation (EMT) and migrates into the myocardium. These epicardial derived cells (EDC) differentiate into epithelial and smooth muscle cells of the coronary vessels. Signals required for this process are speculated to originate from the myocardium and endocardium, but there is no direct evidence for this activity. We have developed a novel in vitro system to directly address the question of “where do the signals required for early coronary vessel development originate?” In this system we co‐culture primary epicardial monolayers with embryonic myocytes. We detect an increase in epicardial EMT during co‐culture conditions compared to naïve epicardial cultures. We have identified several growth factors, including VEGF and TGF‐β2, which are produced by embryonic myocytes in vitro. Thus, our system allows for the identification of mediators responsible for epicardial EMT and, ultimately, EDC differentiation into the coronary vasculature. Supported by HL077493 and T32 HL07249.

Differential growth and multicellular villi direct proepicardial translocation to the developing mouse heart

In the mammalian system the proepicardium (PE) arises from mesothelium of the septum transversum before translocation to the heart where it forms the epicardium and progenitor cells of the coronary vessels. Despite its importance, the process in which PE cells translocate to the myocardium in mammals is not well defined. The current paradigm states that cellular cysts of PE float across the pericardial space and contact the outer surface of the myocardium. This mechanism does not provide a satisfactory explanation for the directionality or localization of PE migration. To better define PE migration, we performed a detailed study of mouse PE development. We provide thorough documentation that redefines the size of the PE migratory field and the mechanism of migration. Our new model incorporates differential growth and direct contact between multicellular PE villi and the myocardium as mechanisms in formation of the epicardium.

Epicardium-derived progenitor cells require beta-catenin for coronary artery formation.

We have previously identified several members of the Wnt/beta-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of beta-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of beta-catenin in coronary arteriogenesis, we have generated conditional beta-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of beta-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial beta-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-beta-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial beta-catenin pathway in coronary arteriogenesis.

Induction of proepicardial marker gene expression by the liver bud

Cells of the coronary vessels arise from a unique extracardiac mesothelial cell population, the proepicardium, which develops posterior to the sinoatrial region of the looping-stage heart. Although contribution of the proepicardial cells to cardiac development has been studied extensively, it remains unresolved how the proepicardium is induced and specified in the mesoderm during embryogenesis. It is known, however, that the proepicardium develops from the mesothelium that overlays the liver bud. Here, we show that the expression of proepicardial marker genes - Wt1, capsulin (epicardin, pod1, Tcf21) and Tbx18, can be induced in naïve mesothelial cells by the liver bud, both in vitro and in vivo. Lateral embryonic explants, when co-cultured with the liver bud, were induced to express these proepicardial marker genes. The same induction of the marker genes was detected in vivo when a quail liver bud was implanted in the posterior-lateral regions of a chick embryo. This ectopic induction of marker gene expression was not evident when other endodermal tissues, such as the lung bud or stomach, were implanted. This inductive response to the liver bud was not detectable in host embryos before stage 12 (16-somite stage). These results suggest that, after a specific developmental stage, a large area of the mesothelium becomes competent to express proepicardial marker genes in response to localized liver-derived signal(s). The developmentally regulated competency of mesothelium and a localized inductive signal might play a role in restricting the induction of the proepicardial marker gene expression to a specific region of the mesothelium. The data might also provide a foundation for future engineering of a coronary vascular progenitor population.