Retinoic acid and VEGF delay smooth muscle relative to endothelial differentiation to coordinate inner and outer coronary vessel wall morphogenesis.

RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.

Epicardial development in the axolotl, Ambystoma mexicanum.

Recent studies on avian and mammalian embryos have established that the epicardium is derived, not from the early heart tube, but from mesothelial tissue overlying the sinus venosus. We tested the validity of this concept for Amphibia by examining normal and cardiac lethal (c/c) mutant axolotl embryos (stages 35-43) by electron microscopy. In axolotl embryos, the myocardial surface of the heart remains exposed to the pericardial fluid through stage 39. At this stage the transverse septum releases into the pericardial cavity mesothelial cells that subsequently flatten over the adjacent ventricular myocardium. However, mesothelial cells observed on the developing epicardium always appear rounded and may extend a filopodium up to 75 microns. This apparent "substrate-dependent" difference in mesothelial cell shape may promote the extension of the epicardium over the rest of the myocardium. The initial site of epicardial formation persists in the adult as the ventricular pericardial stalk that connects the epicardium to the peritoneal lining of the transverse septum. Cardiac lethal (c/c) mutant embryos, despite the non-contractility of their myocardia, form their epicardia in the same way. This suggests that the c/c mutation does not impair those properties of the myocardium that render it a suitable substrate for epicardial spreading. The abnormal pattern of epicardial coverage of the edematous stage 41 c/c mutant heart could be the result of its abnormally large myocardial surface area, the abnormal proximity of the atrium to the transverse septum, and/or the absence of heart contractions which could aid the dispersion of mesothelial cells within the pericardial cavity. Despite species differences, epicardial development in the axolotl is similar to the general pattern described for higher vertebrate embryos.