Analysis of coronary vascular development in an animal model of congenital heart disease - in vivo and in vitro studies to detect a role of proepicardial organ derived cells

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We examined distributional patterns of quail-derived proepicardial organ (PEO) cells in the hearts of chick-quail chimeric embryos produced following the procedure described by Manner. Quail PEO cells formed epicardium at day 5 after incubation, beneath which epithelial-mesenchymal transformation (EMT) of PEO cells were observed. Quail PEO cells also were detected in the myocardium and around the future ostia of coronary arteries at day 6 and at day 9 after incubation, respectively. In the mouse embryos at the coincident developmental stage with chick, tenascin-C was expressed in PEO but was suppressed when PEO cells moved to the cardiac surface, migrating into the myocardium. Tenascin-C expression was upregulated in EMT. Mouse PEO cells cultured on the collagen gels in the presence of fetal calf serum formed tube structures. RT-PCR analyses of RNAs extracted from cultured PEO demonstrated expression of various vascular growth factors including bFGF, HGF, VEGF, Flt-1, Flk- 1, Ang-1, Ang-2 and Tie-2, however, the expression of these growth factors were downregulated when bis-diamine was added into the culture medium. In rat embryos, vascular plexus was formed between the epicardium and myocardium at 14.5 embryonic day (ED) and vascular structure was recognized in the myocardium at ED 15.5. The vascular plexus was dispersed and vascular structures were not formed in the embryonic hearts when bis-diamine was given to mother rats at ED 10.5. Immunohistological studies showed delayed expression of the vascular growth factors in those rat embryos treated with bis-diamine.These results suggested that bis-diamine caused the abnormal development of coronary vasculature by making a direct effect on growth of PEO cells and by disturbing EMT and expression of vascular growth factors.