Development of the epicardium is critical to proper heart formation. It provides all of the precursor cells that form the coronary system and supplies signals that stimulate cardiac myocyte proliferation. The epicardium forms from mesothelial cells associated with the septum transversum and is referred to as the proepicardium (PE). Two different methods by which these PE cells colonize the developing heart have been described. In avians, PE cells form a bridge to the heart over which PE cells migrate onto the heart. In fish and mammals, PE cells form vesicles of cells that detach from the mesothelium, float through the pericardial cavity, and attach to the heart. A previous study of rat PE development investigated this process at the histological level. Protein markers have been developed since this study. Thus, we investigated this important developmental process coupled with these new markers using other visualization techniques such as scanning electron microscopy (SEM) and confocal microscopy. Finally, a novel, three-dimensional (3-D) culture system was used to confirm the identity of the PE cells. In this study, we found convincing evidence that the rat PE cells directly attach to the heart in a manner similar to that observed in avians.

Valvular defects are among the most common and deleterious of all cardiac malformations. The early cardiac cushions are located in the atrioventricular (AV) canal of the embryonic heart. These cushions contain cells that are the primordia of the cardiac valves and membranous septa. Significant progress has been made in delineating the molecular mechanisms that regulate the early steps of cushion formation; however, little is known about how these cushions differentiate into valve leaflets. Here, a new threedimensional collagen tube culturing system was tested for its ability to sustain the development and maturation of the AV cushion anlagen. We report that AV cushion tissues grown within the collagen tube scaffold recapitulate aspects of AV valve development both at the molecular and morphological levels.

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, VEGF164, 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.

Coronary vascular disease is one of the leading causes of mortality and morbidity in the United States. Therefore, a mechanistic understanding of coronary vessel morphogenesis would aid in the innovation of new therapies targeting vascular disorders. Moreover, a functionally equivalent in vitro model system allows for the delineation of the molecular mechanisms that regulate coronary vessel development. In this study, we present a novel in vitro model system. This three-dimensional (3-D) model system consists of a tubular scaffold, which is engineered from type-I collagen and has been optimized to support the growth of embryonic cardiac tissues. In this report, proepicardial (PE) cells, the developmental precursors of coronary vessels, have been isolated from several model species and cultured on this scaffold. In this model system, the PE cells were able to recapitulate several aspects of coronary vessel morphogenesis including epicardial formation, the epicardial to mesenchymal transformation, and de novo coronary vessel development or vasculogenesis. The differentiation of PE cells was characterized using a variety of specific protein markers. The potential uses of this novel coronary developmental model are discussed.

Cardiac malformations are the most common birth defect and cardiac disease is the leading cause of death in the industrialized world. In this study a new tissue engineering technique was developed that enables the construction of a three-dimensional (3-D) model system in which aspects of cardiac valve and vessel formation are recapitulated. The main component of this system is an engineered collagen tube substrate, on which developing cardiac tissues were allowed to differentiate. Valve anlagen, and cardiac vessel primordia (proepicardial tissues) were cultured on the tube scaffold with and without coculturing with embryonic cardiac myocytes so that the role cell/cell interactions could be determined. First, to investigate whether the 3-D collagen tube scaffold would be effective in sustaining atrioventricular canal (AV) valve development, stage 22 (day 5) chicken AV cushions were cultured within the lumen of collagen tubes with and without cardiac myocytes. Morphological and immunohistochemical analysis was carried out using electron and light microscopic techniques. AV canal explants grown within the collagen tubes retained their in vivo configuration and grew into leaflet-like structures (Fig.1) During normal valve development, the expression of periostin and fibrillin-2 are useful markers of valve differentiation. Both of these proteins are believed to be produced from the mesenchymal cells within the cushion. Staining of the cushion inside the tube showed the presence of both periostin and fibrillin-2. Periostin was observed throughout the cushion explant with the most intense staining at the periphery. This pattern of expression is analogous to that seen in vivo at closely approximated stages of development 1 . Experiments are ongoing to determine the role that myocytes play in this growth and development. Additionally, studies to determine if these cushions will remodel to become more leaflet in shape have begun (Fig. 2). The ability of proepicardial organ cells (PEO) to grow on the collagen tube scaffold was also investigated. Stage 17 (day 3) chicken and quail PEOs were isolated and incubated on the surface of collagen tubes seeded with or without rat embryonic myocytes. The PEO cells were processed for scanning electron microscopy and immunohistochemical analysis at days 3, 7, 14 and 21 of culture. These migrating PEO cells express matrix proteins such as fibronectin (FN), heparin sulphate proteoglycan (HSPG) and alpha cytokeratin. Collagen tubes containing PEO cells recapitulated the staining for cytokeratin and fibronectin. Interestingly, the staining for FN and cytokeratin were rarely co-localized (Fig.3). However both FN and cytokeratin were seen only where PEO cells had migrated over the surface of the tube. Scanning electron microscopy demonstrated that the ultrastructural makeup of these migrating PEO cells is also identical to that observed in vivo. Cultured PEO cells showed the ability to migrate over the tube in a sheet like fashion and maintained the typical apical microvilli appearance (Fig...