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.

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The contribution of epicardially‐derived and their progenitors, proepicardial (PE) cells, to the development of the atrioventricular (AV) valves have been recently described in mouse linage tracing experiments. Results presented here indicate that PE cell epithelial to mesenchymal transformation (EMT) is dependent on the presence of AV cushions. Once in the AV cushions, PE cells appear to regulate the expression and deposition of fibrous extracellular matrix (ECM) proteins. Using a tubular, 3D culturing system, we show that AV cushions induce PE cells to undergo an EMT and migrate into AV cushions where they stimulate the expression and deposition of ECM proteins including tenascin C, periostin and collagen I, which are critical for proper valve function. It was tested if members of the TGFβ family of proteins were sufficient to stimulate PE EMT and ECM expression. Only the combination of TGFβ 1, 2 and 3, was found to regulate these processes. When PE cells were cultured on tube scaffolds containing TGFβ 1, 2 and 3 together, PE cell EMT increased compared to cultures with only a single TGFβ isoform. qRT‐PCR and confocal analysis found increased expression and localization of ECM molecules in these transformed cells. These results indicate all three isoforms of TGFβ are required for epicardial EMT, which appear to be necessary the critical expression and deposition of fibrous ECM proteins during valve development.

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