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Keywords:

  • epicardium;
  • epicardially-derived cells;
  • heart;
  • stem cells;
  • coronary vasculogenesis

Abstract

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

After its initial formation the epicardium forms the outermost cell layer of the heart. As a result of an epithelial-to-mesenchymal transformation (EMT) individual cells delaminate from this primitive epicardial epithelium and migrate into the subepicardial space (Pérez-Pomares et al., Dev Dyn 1997; 210:96–105; Histochem J 1998a;30:627–634). Several studies have demonstrated that these epicardially derived cells (EPDCs) subsequently invade myocardial and valvuloseptal tissues (Mikawa and Fischman, Proc Natl Acad Sci USA 1992;89:9504–9508; Mikawa and Gourdie, Dev Biol 1996;174:221–232; Dettman et al., Dev Biol 1998;193:169–181; Gittenberger de Groot et al., Circ Res 1998;82:1043–1052; Manner, Anat Rec 1999;255:212–226; Pérez-Pomares et al., Dev. Biol. 2002b;247:307–326). A subset of EPDCs continue to differentiate in a variety of different cell types (including coronary endothelium, coronary smooth muscle cells (CoSMCs), interstitial fibroblasts, and atrioventricular cushion mesenchymal cells), whereas other EPDCs remain in a more or less undifferentiated state. Based on its specific characteristics, we consider the EPDC as the ultimate ‘cardiac stem cell’. In this review we briefly summarize what is known about events that relate to EPDC development and differentiation while at the same time identifying some of the directions where EPDC-related research might lead us in the near future. Anat Rec Part A 276A:43–57, 2004. © 2004 Wiley-Liss, Inc.

Heart morphogenesis is a complex developmental event in which a variety of cell types play a role. The initial step in cardiac development involves the formation of two, bilaterally located, heart fields of precardiac mesoderm. Within these heart fields, which are derived from the coelomic splanchnopleura, two precardiac precursor cell populations can be distinguished, i.e., the myocardial and the endocardial progenitor cells. Fusion of the heart fields, and subsequent rearrangement of the myocardial and endocardial progenitor cells, results in the formation of a tubular heart in which the two concentric epithelial layers of endocardium and myocardium (Peng et al., 1990; Markwald et al., 1996; reviewed in Wessels and Markwald, 2000) are separated by an acellular, extracellular matrix (ECM)-rich space, generally referred to as the cardiac jelly.

As the heart tube begins to loop, the cardiac jelly becomes unevenly distributed over the length of the heart tube. The jelly accumulates in the atrioventricular (AV) junction and in the outflow tract (OFT) and slowly disappears from the ventricular and atrial compartments (Fig. 1A). Subsequently, a subset of the endocardial cells that line the AV and OFT endocardial cushion tissues undergo an epithelial-to-mesenchymal transformation (EMT). This EMT involves the detachment of the endocardial cells from their neighboring cells and is accompanied by a drastic change in the structure of the cytoskeleton structure, a degradation of the basal lamina, and an active migration of endocardially derived mesenchymal cells into the cardiac jelly resulting in the formation of the cushion mesenchyme (reviewed in Mjaatvedt et al., 1999). The onset of EMT in the AV and OFT cushions occurs at approximately Hamburger-Hamilton stage (H/H) 15–16. The result of these events is that around stage H/H 16–17 in the avian heart, and 9.25 embryonic days (EDs) in the mouse, we can basically distinguish three cell types in the heart: cardiomyocytes, endocardial epithelium, and subendocardial mesenchyme.

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Figure 1. Origin and fate of the proepicardium. A:Very simplistic cartoon illustrating the extracardiac (or better, nonprecardiac) cell populations that contribute to the development of the heart. In the OFT, neural crest cells play an important role in the formation of the aortico pulmonary septum (APS), and in the atrial segment (Atr), the dorsal mesenchymal protrusion (DMP), a cell population that is continous with the mesenchyme of the dorsal mesocardium (DM), plays a part in the separation of the left vs. the right atrium. On the inferior aspect of the sinus venosus (SV) the epicardial precusrsor tissue, or proepicardium (PE), is recognizable. B: Detail of a H/H 17 chick embryo demonstrating the relatively large proepicardium at the margin of sinus venosus and liver and caudal to the developing atrium. C: A cross section of a H/H 17 proepicardium nicely shows the anatomy of this transient structure. The black arrows point to the mesothelial epithelium; the white arrows indicate the mesenchymal cells within the proepicardium; the asterisks illustrate the spaces within the proepicardium filled with ECM. D: A more advanced stage of epicardial development in a H/H 24 chick heart. The ventricular epicardium (arrows) is generating EPDCs by epicardial EMT that migrate into the subepicardium.

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At this point of development a new layer of epithelial cells starts to populate the myocardial surface of the heart (Ho and Shimada, 1978; Virágh and Challice, 1981; Kuhn and Liebherr, 1988; Hiruma and Hirakow, 1989; Fransen and Lemanski, 1990; Hirakow, 1992; Männer, 1992, 1993; Muñoz-Chápuli et al., 1997). This cell layer is known as the epicardium. Like the precardiac mesoderm, the epicardium also originates from the coelomic splanchnopleura. However, its differentiation takes place when the primitive cardiac tube is already formed and looped. Thus, the epicardial progenitor cells, found in the so-called proepicardium (Figs. 1B and C and 2A), are not derivatives of the precardiac embryonic fields. Therefore, the proepicardium is often, together with the cardiac neural crest, considered as one of the extra cardiac cell populations (see Fig. 1A). As all these cell populations do significantly contribute to the formation of the heart, it would actually be more appropriate to describe these cell populations as being non-precardiac-derived or non-heart-field-derived tissues.

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Figure 2. Epicardium and EPDC types.A:A BrdU/cytokeratin (CK)-labeled proepicardium from a H/H 17 chick embryo is shown. The proepicardium has a cauliflower appearance with multiple finger-like protrusions (proepicardial villi, arrows). Mesothelial cells in the surface of the proepicardium, as well as some mesenchymal cells (double arrowheads) in the proepicardial matrix (asterisks), are CK-positive (green staining). CK/BrdU-positive cells appear as light blue dots (arrowheads). B: The transplantation of quail propepicardium into H/H 16–17 chick host embryo results in the formation of an epicardial mesothelium (Ep) of quail origin (green nuclear staining, QCPN positive) that expresses epicardial-specific markers like CK (red cytoplasmic staining). Some EPDCs can be seen invading the underlying myocardium as shown in a H/H 32 chimera (Myo, arrowheads). C: In quail-to-chick proepicardial chimeras EPDCs form coronary vessels. In this H/H 36 chimera a transversal section of a coronary artery illustrates the concentric arrangement of EPDCs in the layers of the vessels. Endothelial cells (QCPN positive, arrowheads) are found in the innermost intima layer, whereas the smooth muscle cells (caldesmon positive, red staining) pointed with arrows constitute the tunica media. Caldesmon-negative mesenchymal cells (likely fibroblasts, double arrowheads) cover the outermost surface of the vessel, giving rise to the adventitia. D: Development of blood islands associated with epicardially derived coronaries is a frequently observed event. An H&E (hematoxylin-eosin) staining of a H/H 25 chick embryo shows a blood island structure (arrow) in the inner curvature of the heart. See a detail in the insert. E: A subpopulation of quail-derived (QCPN-positive) EPDCs invades the AV cushions (AVCs) of proepicardial chimeras accumulating in the subendocardial areas (H/H 36 chimera, arrowheads). F: Proepicardial cells do not differentiate into myocardium in vivo, but they do so in vitro as shown in this picture. After 24 hr on a collagen gel culture, some avian proepicardial cells differentiate into myosin-expressing cells (MF20 positive, green staining, arrow). Small groups of cells with a faint MF20 expression are often found in the periphery of the culture (arrowheads). Nuclei are counterstained with propidium iodide.

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The myocardium and epicardium are initially separated by a virtually acellular gelatinous space, commonly referred to as the subepicardial space or subepicardium. This subepicardium is rich in ECM components and resembles in many respects the cardiac jelly. During and after the formation of the epicardial sheet, a subset of epicardial cells detach from their epithelial context (Fig. 1D) and generate in the subepicardium a population of epicardially derived cells (EPDCs) through an epicardial EMT (Muñoz-Chápuli et al., 1996; Pérez-Pomares et al., 1997, 1998a; Dettman et al., 1998; Gittenberger-de Groot et al., 1998). The epicardial EMT is observed at the AV junction, in the ventricular epicardium, and in the epicardium at the junction between the ventricles and the OFT, but not in the atrial epicardium. As a result of the two ongoing EMTs in the heart, two different populations of mesenchymal cells are generated at either side of the myocardium. Whereas the fate of the mesenchymal cells in the endocardial cushion seems to be restricted to become mesenchyme in the developing valves, a series of recent studies have demonstrated that following epicardial EMT, a subset of EPDCs initially migrate into the myocardium (see Fig. 2B) and subsequently differentiate (in vitro as well as in vivo) into a variety of cell types, including undifferentiated subepicardial mesenchyme, interstitial fibroblast, coronary endothelium, coronary smooth muscle cells (CoSMCs), and blood progenitors. (For a schematic representation illustrating the fate of EPDCs as well as the molecular mechanisms that might be involved in their differentiation, see Fig. 3). In addition, under certain in vitro conditions the proepicardial cells can also differentiate into cardiomyocytes. The clonal differentiation abilities of the EPDCs to give rise to multiple cardiac cell types closely resemble those described for other stem cell populations. In this review we will present and discuss data that suggest that the EPDC is a true cardiac stem cell. In addition, we will discuss some aspects of the role of EPDCs in cardiac morphogenesis.

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Figure 3. Differentiation pathways of EPDCs. This cartoon, which is partly based on observations discussed in this review, and complemented by results of others as well as unpublished results from ongoing studies from our own labs, serves as our current work model for the studies on epicardial development. Thus we propose that BMPs and TGFβs are involved in the induction/regulation of epicardial (pink) EMT. Furthermore, there is a growing body of evidence that VEGF, FGF, PDGF, and TGFβ play a role in the regulation of EPDC differentiation (isolated pink cell). Specifically, VEGF and FGF are responsible for the differentiation of EPDCs into angioblasts (orange) and hemangioblasts (blue). A subset of clustered hemangioblasts can, dependent on their exposure to VEGF, further differentiate into endothelial cells, whereas others will transdifferentiate into hemopoietic cells (yellow). These processes are crucial for the initial phases of coronary vasculogenesis/angiogenesis. The growth factors PDGF and TGFβ1 are thought to be responsible for the differentiation of EPDCs into a fibroblast (yellow)/smooth muscle (red) pathway. The cartoon also shows where in the embryonic heart EPDCs can be found.

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WHAT IS THE EPICARDIUM?

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The epicardium is the outermost cardiac epithelium that covers the surface of the heart. The space that is sandwiched between the myocardium and epicardium is known as the subepicardium or subepicardial space. Within the subepicardial space, subepicardial mesenchymal cells can be found that are derived from the epicardium as a result of epicardial EMT (Pérez-Pomares et al., 1997, 1998a; Dettman et al., 1998; reviewed in Männer et al., 2001). These cells are therefore generally referred to as EPDCs (Gittenberger-de Groot et al., 1998). It has been suggested that sources other than the epicardial mesothelium could also contribute to the subepicardial mesenchyme (Langford et al., 1990; Van den Eijnde et al., 1995). Our observations (Pérez-Pomares, unpublished results) indicate that the relative contributions from nonepicardial tissues to the subepicardial mesenchyme may vary between vertebrate species and are possibly more important in avians than in mammals.

THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

Until a decade ago, little was known ago about the origin, function, and fate of the epicardium. It was generally considered to be an inert layer of cells, its basic role being to protect the myocardium from external factors. It was believed for many years that the epicardium was a derivative of the myocardium, hence the term myoepicardium (Mollier, 1906), which was frequently used in studies on cardiac embryology (Streeter, 1945; De Haan, 1965; Patten, 1968). Interestingly, however, an alternative, extracardiac source for the epicardium had been proposed as early as 1909 (Kurkiewicz, 1909). The established concept of the myoepicardium was not significantly challenged, however, until Manasek (1968, 1969) published his seminal studies on embryonic heart morphology. After publication of these papers, other studies followed that supported the nonmyocardial origin for the epicardial tissue. Thus it was shown that the epicardium derives from a proliferation of coelomic cells located in the septum transversum in mammals and analogous regions in other vertebrates (i.e., between liver and sinus venosus). The epicardium is actually a synapomorphy of the vertebrate subphyllum and is not found in other chordates such as the urochordates and cephalochordates (Hirakow, 1985). Its evolutionary relevance seems to be related to the ability of the epicardium to generate a mesenchymal cell population, a process that is characteristic of the entire embryonic splanchnopleura (Pérez-Pomares and Muñoz-Chápuli, 2002).

PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

As indicated above, the epicardium originates from the proepicardium. The proepicardium is continuous with the splanchnopleural coelomic epithelium and is a proliferative tissue as shown by simple bromodeoxyuridine (BrdU) uptake experiments (Fig. 2A). Interestingly, after proepicardial ablation the coelomic cells of the area keep dividing and form a secondary cluster of cells that provide the heart with an epicardial-like compensatory tissue (Pérez-Pomares et al., 2002b).

The proepicardium is composed of an external mesothelium (i.e., a mesodermal epithelium), which is continuous with the coelomic/splanchnopleural epithelium. This epithelium develops multiple, finger-like protrusions (a.k.a. proepicardial villi in the avians) into the pericardial cavity pointing to the ventricular surface of the heart. The form and extent of these protrusions differ in length between species (Männer et al., 2001). The proepicardial epithelium covers an ECM (see also Nahirney et al., 2003). The extent of this ECM varies between different vertebrate groups. Whereas in the avian embryo the initially acellular proepicardial ECM is rather extensive, in other species there is relatively little ECM and the entire proepicardium is rather compact. As a result of proepicardial EMT, the ECM becomes populated with mesenchymal cells (Pérez-Pomares et al., 1997, 1998a, 1998b, 2002a, 2002b) (see Fig. 1C). It can, however, not be ruled out that some of the mesenchymal cells within the proepicardium are derived from other tissues.

In the fast-growing number of papers on epicardial development, the terms proepicardium, proepicardial organ, and proepicardial serosa (with a variety of abbreviations for all these terms) are inconsistently used to describe the precursor tissues of the epicardium. Most authors do not distinguish between the extracardiac isolated cauliflower-like protrusions of the pericardial coelom (called proepicardium in Viragh et al., 1993) and the transient bridging structure (in the avian, recognizable for approximately 12–15 hr) that is seen when the proepicardium becomes attached to (and spreads over) the myocardium of the heart. It is important to note that when first introduced (Viragh et al., 1993), the term proepicardial organ was specifically used to describe this transient structure and to discriminate it from the less advanced proepicardium stage. Therefore, the term proepicardial organ indicates a stage in proepicardial to epicardial transition observed in the avian heart. This bridging structure is, however, never seen in the murine (and other mammalian) heart, and the use of this term in these species is therefore incorrect. Because of this confusing situation, it is our suggestion that proepicardial organ should be avoided in any stage of proepicardial development.

FROM PROEPICARDIUM TO EPICARDIUM

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The process of attachment of proepicardial cells to the myocardial surface is an intriguing event. In avians, the proepicardium connects through attachment of the proepicardial villi to the myocardial surface in the right-sided AV junction (inner curvature). This results in the formation of a transient tissue bridge that has been named proepicardial organ (Viragh et al., 1993) (see discussion above) but has also been described as secondary mesocardium (Männer, 1992), which is an equally imprecise choice of nomenclature. A recent study nicely illustrates the involvement of the ECM in this process (Nahirney et al., 2003). As the embryonic cephalic and cervical flexures develop, the right AV region becomes a perfect anchoring point for the proepicardial protrusions because it is the closest area of the myocardial surface to the proepicardial villi. The mechanisms that control the spatiotemporal migration pattern of the proepicardially derived cells have not yet been elucidated but are under investigation in several laboratories. The spatiotemporal migration pattern of the epicardium over the surface of the heart is basically the same in all vertebrates studied thus far. These first areas to become covered are the AV and cono-ventricular (CV) grooves resulting in the formation of a characteristic set of epicardial cuffs in these intersegmental areas. Subsequently, the epicardium spreads over the rest of the myocardial surface (Vrancken Peeters et al., 1995; Moss et al., 1998; Xavier-Neto et al., 2000).

In the mammalian embryo, the primary mechanism through which the proepicardial cells connect to the cardiac surface is via the release of groups, or vesicles, of cells from the proepicardial protrusions into the pericardial cavity (Van den Eijnde et al., 1995; Pérez-Pomares et al., 1997; Männer et al., 2001). These cells attach to the myocardial surface of the heart in more or less the same areas in which the proepicardial protrusions attach in the avian heart. How the isolated vesicles are directed to these specific areas of the developing heart is not known. It is likely that this process is partly controlled by spatiotemporal regulation of the expression of adhesion molecules and ECM components on the myocardial surface.

THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The subepicardium is a harboring space that will eventually host a population of mesenchyme. This subepicardium is a hydrated ECM, rich in proteins, including collagens I, IV, V, and VI and fibronectin (Tidball, 1992; Hurlé et al., 1994; Kálmán et al., 1995; Bouchey et al., 1996; Kim et al., 1999), flectin (Tsuda et al., 1998), fibulin-2 (Tsuda et al., 2001), GP68 (Morita et al., 1998), laminin and proteoglycans (Kálmán et al., 1995), vitronectin, fibrillin-2, elastin (Bouchey et al., 1996), and tenascin-X (Burch et al., 1995). It has been shown that both the epicardium and myocardium contribute proteins to this matrix (Bouchey et al., 1996). Actually, the myocardial synthesis of fibronectin seems to precede the arrival of the epicardial cells to the myocardium (Kálmán et al., 1995) and may play a role in directing epicardial migration. The subepicardium also accumulates growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) (Tomanek et al., 1998, 1999, 2001, 2002; Zheng et al., 2001) and is likely to retain other myocardially secreted growth factors like hepatocyte growth factor (HGF) (Rappolee et al., 1996; Song et al., 1999), transforming growth factors (TGFβs), and bone morphogenetic proteins (BMPs) (Yamagishi et al., 1999; Nakajima et al., 2000). The release of these factors appears to be partially controlled by the interactions with the matrix.

The dynamics of the EPDCs (see below) are in part determined by the dimension and molecular milieu of the subepicardial region where they are generated. The intersegmental grooves (e.g., AV groove) accumulate more subepicardial matrix than other cardiac regions. As a result, the subepicardial space is wider in these areas. Also, it is in these areas that relatively high numbers of EPDCs are generated that are involved in the formation of the very prominent epicardial sulcus tissues (Wessels et al., 1996) and the assembly of large coronary vessels by coronary vasculogenesis (see Fig. 4). It is important to point out that the largest coronary vessels develop in the areas where considerable amounts of subepicardial matrix have accumulated and where the density of EPDCs is the highest (Pérez-Pomares et al., 1997, 1998a, 1998b). To date it has not been elucidated what drives this local accumulation of the subepicardial matrix and the EPDCs. However, it is noteworthy that in most areas of epicardial matrix/EPDC accumulation (with the exception of the interventricular groove), the luminal side of the myocardium is covered by endocardial cushion tissues in which more or less the same processes (accumulation of ECM and mesenchymal cells) take place as well (see below). Hence, it is tempting to speculate that the (growth) factors that are involved in the development of the endocardial cushions also play a role in the regulation of the epicardial events. However, to date there is no convincing evidence that this is indeed the case (Pérez-Pomares et al., 1998a; Morabito et al., 2001).

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Figure 4. Coronary vasculogenesis. This plate demonstrates the formation of the coronary vasculature. All panels of this plate show staining of coronary endothelial cells using the QH1 antibody. A: Cross section from a H/H 30 quail heart immunohistochemically stained with QH1 showing the formation of subepicardial (arrow) and intramyocardial (arrowhead) coronary vessels. D: A section of a proepicardial quail-to-chick chimera at stage H/H 31 in which the proepicardially derived endothelial cells are immunofluorescently detected using QH1 (nuclei (red) were counterstained using propidium iodide). These staining protocols are particularly useful when studying aspects of the formation of coronary vessels in relation to the respective cardiac cell layers. In addition, they are helpful when studying the relationship of the developing vascular components in relation to other cell types. However, for a detailed spatial description of coronary vasculogenesis (i.e., the formation of vessels by assembly of angioblasts) they lack three-dimensional information. B, C, E, and F: Demonstrate the usefulness of whole-mount immunofluorescent microscopy for elucidating coronary patterning. Quail hearts at different stages of development (B = H/H 26; C = H/H 31; E and F = H/H 30) were fixed in Amsterdam's Fixative and immunolabeled with QH1. Low-magnification scanning laser confocal imaging allows the study of spatiotemporal progression of coronary pattern formation (cf. B and C, dorsal aspects of hearts), while high-magnification imaging very clearly demonstrates how coronary vessels develop and grow by assembly and addition of isolated angioblasts (arrows in E and F). Note the typical mesenchymal appearance of the angioblasts. It is interesting to point out that at this stage the coronary vessels are already filled with blood cells, whereas the connection of the coronary network to the aorta reportedly does not take place until stage H/H 32.

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CARDIAC EMT AND GENERATION OF EPDCs

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The first EMT (for a general review on EMT, see Hay, 1995) described in the heart was that of the EMT taking place in the endocardial cushion tissues in the AV junction and OFT (Markwald et al., 1975, 1977). It was demonstrated that this localized EMT gives rise to the valvuloseptal mesenchyme of the cardiac valvular primordia. Almost 20 years later, we showed that this process also takes place in the epicardium where it results in the formation of subepicardial mesenchyme (Pérez-Pomares et al., 1997). The mesenchymal cells that migrate into the subepicardial space and that are derived from the proepicardially derived primitive epicardium (Pérez-Pomares et al., 1997, 1998a, 1998b; Dettman et al., 1998) are generally referred to as EPDCs (see above).

Although the transformation events in the respective areas of the heart are now quite well characterized from a morphological point of view, information on the molecular mechanisms that regulate these events is only slowly emerging. For the endocardial EMT it has been suggested that several growth factors of the TGFβ superfamily play a role in eliciting and sustaining this process. These factors include TGFβs 2 and 3 and BMPs 2 and 4 (Nakajima et al., 1998, 2000; Romano and Runyan, 1999, 2000), as well as other proteins (ES130) (Rezaee et al., 1993, Krug et al., 1995). In recent studies it has been suggested that FGF isoforms (1, 2, and 7) as well as VEGF and EGF might be playing a role in stimulating epicardial EMT, while TGFβ might inhibit the transformation process (Morabito et al., 2001). Thus it appears that the molecular mechanisms controlling EMT in different areas of the heart might not be the same. It is important to emphasize that despite the controversy about the factors that induce EMT, it has also been reported that some transcription factors (i.e., ets-1, slug) are found in both endocardial and epicardial transforming cells (Carmona et al., 2000; Macias et al., 1998; Romano and Runyan, 1999). The spatiotemporal expression of ets-1, slug, WT1, and RALDH2 in avian cardiac development is summarized in Figure 5.

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Figure 5. Molecular patterning of epicardial cells and EPDCs. Columns show the levels of expression of three transcription factors (ets-1, slug, and WT1) and the retinoic acid synthesizing enzyme RALDH2 in epicardial and EPDCs (red = high expression; purple = regular expression; pale pink = traces of expression; white = no expression; noninvestigated situations are indicated with a question mark). Subdivisions in the columns correspond to the expressions in three significant stages of embryonic epicardial development (H/H 26, H/H 29, and H/H 31). Rows correspond to different heart chambers, and the colors indicate heart layers from the outer epicardium to the inner endocardium. CT: cushion tissue; EN: endocardium; EP: epicardium; MYO: myocardium; SE: subepicardium; SEN: subendocardium.

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STEM CELLS IN THE EMBRYO

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

In the early embryo, embryonic stem (ES) cells have been widely described and have shown to exhibit pluripotential abilities that are not matched by any other stem cell type (Hadjantonakis and Papaioannou, 2001). The so-called hematopoietic stem cell (HSC) is an early derivative of the ES cell type and expresses a spectrum of markers that is characteristic of the phenotype of the stem cell (i.e., c-Kit, Sca-1, and CD34) (Bacigalupo et al., 2002; Jackson et al., 2001). After their migration from the para-aortic/aorta-gonad-mesonephros region, the HSCs populate various organs before settling into the bone marrow (reviewed in Dieterlen-Lievre et al., 2002). Recently the mesangioblast has been suggested to be the para-aortic stem cell as this cell has been demonstrated to differentiate in vitro as well as in vivo into multiple mesodermal tissues other than blood cells (Minasi et al., 2002). Clonal abilities of SCs are often tested in vitro by culturing the cells in a medium that is thought to mimic their natural environment. This approach, however, might obscure their true potential. It is commonly accepted that the adult organism contains organ-resident stem cells whose specific stem molecular profile seems to be modulated by the organ environment (Poulsom et al., 2002). It is likely that some of these cells contribute to the development of their host organ, but it is not clear to what extent they contribute to the heterogeneous population of adult bone marrow stem cells (Hadjantonakis and Papaioannou, 2001).

CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

Recent studies indicate a significant contribution of stem cells, likely of bone marrow origin, to the homeostasis and/or repair of the adult heart (Jackson et al., 2001; Orlic et al., 2001; Hughes, 2002; Laflamme et al., 2002; Quaini et al., 2002). In addition, the existence of a so-called myocardial stem cell population has been suggested (Hierlihy et al., 2002), but it is not clear how this population contributes to the respective established cardiac cell types in the heart, nor has the origin/phenotype of these cells been properly described.

IS THE EPDC A TRUE STEM CELL?

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

A series of studies has demonstrated that a subset of EPDCs can differentiate into multiple cell types, including cardiac fibroblasts, coronary endothelial and smooth muscle cells, and valvuloseptal mesenchymal cells (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996; Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Pérez-Pomares et al., 1998a, 1998b; 2002a, 2002b; Landerholm et al., 1999; Männer, 1999; Vrancken Peeters et al., 1999; Lu et al., 2001; Wada et al., 2003). This demonstrates that the EPDC is a multipotential cell and raises the question whether we can consider the EPDC as a true cardiac stem cell.

The definition of a stem cell is dependent on the context in which a cell is studied and the research interest of the researchers involved. Our working definition for a stem cell comes from the work of Potten (1998): “Stem cells can be defined as relatively undifferentiated, proliferative cells that maintain their numbers, while at the same time producing a range of differentiated progeny that may continue to divide” (see also Potten and Loeffler, 1990). We think that the native EPDC (i.e., the primary mesenchymal derivative of the epicardial epithelium after EMT) meets these criteria very well as outlined below. After EMT the EPDC is a relatively undifferentiated cell. This EPDC expresses genes characteristic for undifferentiated cells such as Slug (Carmona et al., 2000) and WT1 (Moore et al., 1999, Carmona et al., 2001, Pérez-Pomares et al., 2002b), and, in general, does not express any markers for advanced stages of differentiation (e.g., smooth muscle markers (actin, caldesmon) and endothelial markers (PECAM, Flk1, QH1)). Several in vitro studies demonstrate high proliferative activity of the epicardial epithelium and EPDCs. In addition, BrdU uptake experiments have shown high in vivo mitotic activities in these tissues as well (Pérez-Pomares and Wessels, unpublished observations). In conclusion, given the fact that the EPDC meets all stem cell criteria outlined above, we consider the EPDC a cardiac stem cell.

THE FATE OF THE EPDC

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

EPDCs and Coronary Endothelium

The origin of coronary endothelium has been a controversial topic. In a series of elegant retroviral labeling studies, Mikawa and colleagues (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996) clearly showed that the coronary system does not derive from the myocardium or from the endocardium, but that instead the proepicardium was the source of coronary endothelium. The retroviral studies did not, however, provide any data on the specific origin of the coronary progenitors found within the proepicardium. Recently, we have published a paper that specifically addresses this issue and in which we unequivocally demonstrate that it is indeed the proepicardial/epicardial mesothelium itself that differentiates into coronary endothelial cells (Pérez-Pomares et al., 2002a).

A possible mesothelial origin for coronary endothelium was first proposed in a primitive vertebrate, the dogfish (Scyliorhinus canicula), based on careful morphological and ultrastructural analyses (Muñoz-Chápuli et al., 1996). Subsequently, immunohistochemical studies, showing the presence of mesothelial markers in early vascular precursors (angioblasts), and new data from proepicardial quail-to-chick transplantations (Pérez-Pomares et al., 1997, 1998a, 1998b, 2002b; Pérez-Pomares and Muñoz-Chápuli, unpublished results) were found to strongly support this idea (Figs. 2C and 4D). Finally, a recent study, in which specific mesothelial retroviral tagging in a chick-to-chick chimerization process was combined with direct carboxyfluorescein (CCFSE; Molecular Probes) labeling, convincingly showed that proepicardial/epicardial mesothelial cells differentiate in vivo into coronary endothelium (Pérez-Pomares et al., 2002a). These results do not exclude, however, the possibility of additional contribution of other non-epicardially-derived angioblasts to the coronary vasculature.

Recent studies from our labs also show the differentiation of proepicardial/epicardial cells into angioblasts/endothelium in in vitro culture systems using either Matrigel™ or collagen type I gels (Pérez-Pomares et al., 2002a; Wessels et al., unpublished). The commitment of EPDCs into the angioblastic lineage seems to be under the control of the myocardial production of VEGF and bFGF (Tomanek et al., 1998, 1999, 2001, 2002; Wessels et al., unpublished). VEGF is generally considered to regulate vascular differentiation and morphogenesis via VEGFR-2, although it is not clear if it is indispensable for angioblastic specification and differentiation (Vokes and Krieg, 2002). This endothelial differentiation process is mediated by the transcription factor SCL/TAL (expressed in the subepicardium) (Pérez-Pomares et al., unpublished results), which is known to be essential in the angioblastic splanchnopleural differentiation (Drake et al., 1997; Drake and Flemming, 2000) as well as in the specification of hematopoietic cells (Robb et al., 1995; Begley and Green 1999).

EPDCs and Blood Cells

Whereas vasculogenesis of the extraembryonic vasculature involves the formation of blood islands (vesicles with an outer mantle of differentiated endothelial cells and inner mass consisting of blood cells) (Risau and Flamme, 1995), early intraembryonic vascularization does not require their formation (Drake and Fleming, 2000). Interestingly, during early stages of cardiac development, blood islands are seen in the subepicardium (Hutchins et al., 1988; Viragh et al., 1990; Hirakow, 1992; Pérez-Pomares et al., 1997, 1998a, 1998b). The larger ones are typically found in the intersegmental junctions in which high numbers of EPDCs accumulate (see above) and where the largest coronary vessels eventually develop (Fig. 2D). Despite these correlations, virtually nothing is known to date on the role and/or significance of these structures within the context of coronary vasculogenesis. In relation to this process two different subepicardial cell types need consideration. First, the regular angioblast, which has a mesenchymal appearance, expresses endothelial markers and is committed to the endothelial lineage (Fig. 4E and F). The subepicardium also contains big round cells that express hemangioblastic cell markers (Vrancken Peeters et al., 1997; Pérez-Pomares and Wessels, unpublished results). This cell morphologically resembles the early intraembryonic hemopoietic stem cells of the para-aortic region (Jaffredo et al., 1998). The use of quail-to-chick proepicardial chimeras to study proepicardial derivatives has provided some information concerning the lineage relationship between endothelial and blood cells in the subepicardial blood islands. The blood islands are often lined by quail-derived (QH1-positive) endothelium, a result that is consistent with the idea that both endothelium and blood cells derive from a common hemangioblastic progenitor (Risau and Flamme, 1995; Eichmann et al., 1997).

EPDCs and CoSMCs

The differentiation of proepicardial/epicardial cells into CoSMCs was first reported in a series of studies based on a retroviral in vivo labeling (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996). Subsequently, a number of studies using quail-to-chick proepicardial transplantations confirmed and expanded these observations (Fig. 2C) (Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Männer, 1999; Vrancken Peeters et al., 1999; Pérez-Pomares et al., 2002a, 2002b). In vitro studies indicate that in the presence of serum, EPDCs can differentiate into CoSMC (Landerholm et al., 1999). Furthermore, CoSMC is regulated by a rho-A-mediated reorganization of α-actin filaments associated with p160 rho-kinase activity (Lu et al., 2001).

The relationship between coronary endothelial and smooth muscle cell differentiation of EPDCs is intriguing. An important issue that needs to be addressed is the sequence of events related to the in vivo differentiation of EPDCs into the endothelial/smooth muscle cell lineage. In vitro studies have demonstrated that endothelial cells can induce differentiation of CoSMC precursors into mature CoSMCs (Landerholm et al., 1999; Lu et al., 2001; Wada et al., 2003). A particular question that needs to be resolved, however, is whether in vivo endothelial cells are required for differentiation of CoSMC precursors into mature CoSMCs. Recent studies (Yamashita et al., 2000) have provided evidence for SMC differentiation after PDGF-BB induction from an early embryonic bipotential ancestor characterized as a flk-1/VEGFR-2-positive cell type. In the absence of PDGF-BB these cells would differentiate into endothelium after VEFG induction. The elucidation of this and related growth factor-mediated differentiation mechanisms involved in EPDC development forms part of the research efforts in our laboratories.

EPDCs and Cardiac Fibroblasts

Virtually all the data available on epicardial differentiation into cardiac fibroblasts are derived from quail-to-chick chimera studies. Because of the limited availability of appropriate tools to determine fibroblast phenotype in the epicardial lineage within one tissue section, the conclusion that EPDCs differentiate into fibroblasts is largely based on histological/topological analysis of the distribution of EPDCs (Gittenberger-de Groot et al., 1998; Pérez-Pomares et al., 2002a, 2002b) (see Fig. 2C). The epicardially derived population of fibroblasts seems to be closely related to the CoSMCs. In fact, all the quail-to-chick proepicardial chimeric transplantation studies show a gradient in the expression of SMC markers from the media to the non-SMC adventitia of the coronaries. The close ontogenetic relationship between SMCs and fibroblasts has been discussed previously (Hungerford and Little, 1999). Finally, it has been demonstrated that non-epicardially-derived fibroblasts can transdifferentiate and give rise to other cells types (Kon and Fujiwara, 1994). Thus, it is feasible that at later developmental stages epicardially derived fibroblasts can transdifferentiate and contribute to other cardiac tissues.

EPDCs and Valvuloseptal Mesenchyme

The quail-to-chick proepicardial chimera studies have shown that a subset of EPDCs populates the AV cushions (Gittenberger-de Groot et al., 1998; Männer, 1999; Pérez-Pomares et al., 2002b) (Fig. 2E). Little is known about the relationship of these cells with other epicardially derived lineages, nor has their role in cushion morphogenesis been elucidated. In a recent study, in which we combined the quail-to-chick proepicardial chimerization with extensive molecular characterization of the donor-derived cells (i.e., quail EPDCs) (Pérez-Pomares et al., 2002b), we showed that two molecules, i.e., the Wilms' tumor transcription factor (WT1) and the retinoic acid converting enzyme RALDH2, are characteristically expressed in early stages of EPDC differentiation. We demonstrated that as the EPDCs differentiate into the endothelial and CoSMC lineages, both WT1 and RALDH2 are downregulated. The EPDCs located in the AV cushions are also WT1/RALDH2-negative and express caldesmon, a smooth muscle marker that is also expressed in CoSMCs from EPDC origin. Although there are no true SMCs in the adult vertebrate AV valves, around 10% of the cells in the valves have been reported to show an intermediate phenotype between fibroblasts and vascular SMCs (Filip et al., 1986). Inhibition of normal epicardial development resulted in a series of cardiac defects, including abnormalities in the AV valve apparatus (Pérez-Pomares et al., 2002b). Thus, although the role of smooth muscle-like EPDCs in valve development remains to be elucidated, the above results suggest an involvement of these cells in valve morphogenesis, likely through an interaction with endocardial and/or endocardially derived cells.

EPDCs and Myocardium

The old hypothesis about a material contribution of epicardial cells to the developing myocardium (Morris, 1976) has recently been refuted by in vivo studies (Männer, 1999). Recent results from our labs (Fig. 2F) as well as those from others (Kruithof et al., in preparation), however, indicate that under certain in vitro conditions proepicardially derived cells can differentiate into cardiomyocytes. These studies also indicate that this differentiation process can be manipulated by the addition of stimulating (e.g., BMP2) and inhibiting (e.g., FGF2 and VEGF) growth hormones. It is important to realize that the in vitro conditions do not reflect the normal extracellular in vivo environment. The question therefore is whether the in vitro myocardial differentiation of proepicardially derived cells reflects some kind of in vitro artifact or whether this phenomenon indicates that in the heart regulatory mechanisms are in place that specifically prevent the differentiation of EPDCs into cardiomyocytes. Such a mechanism could be important to prevent uncontrolled myocardialization of epicardially derived tissues (e.g., in the AV sulcus) during normal embryonic development. In this respect it is tempting to speculate that accessory AV bundles as seen in patients with Wolff-Parkinson-White syndrome could be the result of uncontrolled epicardial-to-myocardial differentiation, rather than a result of myocardial invasion of the mesenchyme as a result of ingrowth of existing myocardium (see e.g. Van den Hoff et al., 1999).

Undifferentiated EPDCs

While many EPDCs are differentiating into the respective cell lineages as described above, a contingent of relatively undifferentiated EPDCs will remain within the subepicardial space and myocardial tissues. The majority of the cells within the subepicardium express RALDH2 as well as WT1, whereas the EPDCs that migrate into the myocardium gradually loose their RALDH2 expression while retaining the expression of WT1. It is likely that this persistence of WT1 expression is related to the maintenance of the undifferentiated stem cell state in these EPDCs (Carmona et al., 2001; Pérez-Pomares et al., 2002b; Wagner et al., 2002), a mechanism also suggested for hematopoietic progenitors (Baird and Simmons, 1997).

TO DIFFERENTIATE OR NOT TO DIFFERENTIATE

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

Little is known about the spatiotemporal regulation of EPDC differentiation. We recently proposed WT1 as a candidate for the regulation of the state of differentiation of the EPDC as downregulation of WT1 expression correlates with the differentiation of EPDCs into specific cell types as shown by the upregulation of lineage markers (Pérez-Pomares et al., 2002b). Multiple targets have been suggested for this transcription factor, including some growth factors (IGFII, PDGF-A, TGFβ1) and receptors for growth factors/morphogens (RARα, EFGR, IGFIR) (reviewed in Little et al., 1999).

WT1 is involved in the development of the more common pediatric solid tumor (Brown et al., 1992; Haber and Housman, 1992) as well as in certain forms of leukemia (King-Underwood et al., 1996; Bergmann et al., 1997). WT1 expression has been reported to tightly control the proliferation/differentiation status of ES cells through regulation of cyclins (Wagner et al., 2001). In addition, it has been reported that stem cells respond to retinoids by upregulating WT1 expression (Scharnhorst et al., 1997). This is important since retinoic acid is known to be essential in cardiac development (Dyson et al., 1995; Gruber et al., 1996; Kastner et al., 1997; Niederreither et al., 2000; Chen et al., 2002; Stuckmann et al., 2003). Furthermore, it has been demonstrated that retinoic acid is actively synthesized by RALDH2 in the epicardiums of both mammals and avians (Moss et al., 1998; Xavier-Neto et al., 2000; Pérez-Pomares et al., 2002b). Thus the interaction between WT1 and RALDH2 likely plays a critical role in the differentiation of the EPDC.

EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The role of the epicardium and EPDCs in regulating myocardial development has recently gained a lot of attention (Gittenberger-de Groot et al., 2000; Chen et al., 2002; Pérez-Pomares et al., 2002b; Stuckmann et al., 2003). Although experimental or genetic alterations of epicardial development were known to cause severe defects in the development of the compact myocardium (see Table 1), it was only after the description of EPDCs' immigration into the myocardial layers (Mikawa and Gourdie, 1996; Gittenberger-de Groot et al., 1998; Männer, 1999; Perez-Pomares et al., 2002b) that most of the pieces of the puzzle started to fall into place. These studies prompted a renewed interest in a study that initially gained little attention (Eid et al., 1992). In this study, Eid et al. (1992) showed alterations in the myosin synthesis program in myocardiocytes in the absence/presence of epicardium and concluded that epicardial cells did secrete factors that could influence myocardial development. It is noteworthy that these results were published before the publications frequently cited in this review that describe the fate of proepicardially derived cells, which probably explains why the significance of the results of the myocardial-epicardial coculture experiments has only recently been fully appreciated.

Table 1. Animal Models for Altered Epicardial Development and/or Thin Myocardial Syndrome*
Genes altered (or experimentally-induced effects)Expression/cells affectedCardiac phenotypeReference
  • *

    See text for a detailed explanation.

N-Myc k.o.Myocardial cells. Not described in epicardiumHypoplastic ventricular myocardium; epicardial/coronary alterations not describedMoens et al. (1993)
TEF-1 k.o.Myocardial cells. Not described in epicardiumHypoplastic ventricular myocardium; epicardial/coronary alterations not describedChen et al. (1994)
Neuropilin overexpressionEndocardium and cardiac endotheliumHypoplastic ventricular myocardium; aberrant coronary vasculatureKitsukawa et al. (1995)
VCAM k.o.MyocardiumHypoplastic ventricular myocardium; poor epicardial integrity, and abnormal coronary developmentKwee et al. (1995)
α-4 integrin k.o.EpicardiumPoor epicardial integrity, and abnormal coronary developmentYang et al. (1995)
RXRα k.o.Myocardium, and epicardiumHypoplastic ventricular myocardium; epicardial/coronary alterations not describedDyson et al. (1995); Gruber et al. (1996); Chen et al. (2002)
βARK k.o.Myocardial cells. Not described in epicardiumHypoplastic ventricular myocardium; epicardial/coronary alterations not describedJaber et al. (1996)
GP130 k.o.Ubiquitous in mouse embryonic cellsHypoplastic ventricular myocardium; epicardial/coronary alterations not describedYoshida et al. (1996)
Erythropoietin receptor k.o.Pericardium, epicardium, and endocardiumHypoplastic ventricular myocardium; epicardial detachment and underdeveloped subepicardium; abnormal coronary morphogenesisWu et al. (1999)
WT1 k.o.EpicardiumHypoplastic ventricular myocardium; poor epicardial integrity reduced EPDCs, and abnormal coronary morphogenesisMoore et al. (1999)
FOG-2 k.o.MyocardiumHypoplastic ventricular myocardium, and abnormal coronary morphogenesisTevosian et al. (2000)
Proepicardial manual ablation or experimental delay of epicardial growthEpicardiumPerturbation of EPDCs production, abnormal coronaries and ventricular hypoplasiaMänner (1993); Gittenberger-de Groot et al. (2000; Pérez-Pomares et al. (2002b)
C3, rhoGD1 (inhibitors of rhoA signaling)Epicardium (others not tested)Alteration of EPDC differentiation into CoSMCLu et al. (2001)
Connexin 43 k.o.Myocardium, and epicardiumAbnormal coronary patterning and presence of conal “pouches”Li et al. (2002)
Antisense ETS-1, 2 targetingEpicardium, myocardium, and endocardiumAlteration of and myocardial coronary developmentMacías et al. (1998); Lie-Venema et al. (2003)

The combined results of a series of papers on the relationship between the localization of EPDCs and myocardial development indicate that the presence of EPDCs in the myocardial layers determines the thickening of the myocardial walls of the respective cardiac segments. Myocardial walls without EPDCs remain very thin (i.e., OFT myocardium and atrial myocardium), whereas the myocardial wall of the ventricles in which many EPDCs are found become relatively thick. When proepicardial development is perturbed and EPDC migration into the ventricular myocardium is inhibited, the ventricular wall thickness does not increase to its normal size (Pérez-Pomares et al., 2002b). In addition, the altered epicardial migration and production of EPDCs also leads to dysplastic cushion tissues and perturbation of the coronary vascular network with associated arterioventricular connections (pouches/fistulae) (Gittenberger-de Groot et al., 2001; Pérez-Pomares et al., 2002b).

SUMMARY

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

Epicardial biology is a relatively new field in cardiovascular developmental biology. The pluripotent stem cell character of the epicardial cell, as well as its recently revealed overall importance in cardiac development, has resulted in a prominent place for the EPDC in numerous research projects within an increasing numbers of labs worldwide. Many of these studies are focused on the analysis of the mechanisms that control the conversion of the epicardial epithelial (mesothelial) cells into a population of pluripotent EPDCs that contributes to many cardiac tissues. Others are looking at different aspects of EPDC development, including the regulatory role of EPDCs in myocardial proliferation and the involvement of EPDCs in endocardial cushion morphogenesis. Elucidating the pathways that control epicardial differentiation will not only help us to further understand normal heart morphogenesis, but will also provide new insights in the mechanisms that underlie the etiology of congenital heart disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED

The authors thank Dr. Muñoz-Chápuli for sharing with us some results from his lab as well as for all past and present discussions on epicardial issues. Special thanks also to Tanya Rittman for help in the artistic design of some of the illustrations. Finally, we also thank the members of our labs who have contributed in one way or another to the results included and discussed in this review: Dr. D. Macías, Dr. C. García-Garrido, Dr. R. Carmona, M. González-Iriarte, A. Phelps, M. Sedmerova, B. Riley, I. Moralez, H. Tripp, G. McCoy, and C. Maharaj. This work was supported by funds from the NIH (NIH PO1 HL52813 and NIH PO1 HD39946) and AHA (grant AHA-GIA 005099U) to Dr. Wessels and Dr. J.M. Pérez-Pomares.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. WHAT IS THE EPICARDIUM?
  4. THE ORIGIN OF THE EPICARDIUM: A HISTORICAL PERSPECTIVE
  5. PROEPICARDIUM VS. PROEPICARDIAL ORGAN: WHAT'S IN A NAME?
  6. FROM PROEPICARDIUM TO EPICARDIUM
  7. THE SUBEPICARDIAL EXTRACELLULAR ENVIRONMENT
  8. CARDIAC EMT AND GENERATION OF EPDCs
  9. STEM CELLS IN THE EMBRYO
  10. CONTRIBUTIONS OF STEM CELLS TO THE ADULT HEART
  11. IS THE EPDC A TRUE STEM CELL?
  12. THE FATE OF THE EPDC
  13. TO DIFFERENTIATE OR NOT TO DIFFERENTIATE
  14. EPDCs AND HEART MORPHOGENESIS: A MODEL ON EPICARDIAL-MYOCARDIAL INTERACTIONS
  15. SUMMARY
  16. Acknowledgements
  17. LITERATURE CITED
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