The role of VEGF and eNOS during Early Embryonic Vascular Development.

摘要

Previous studies from our laboratory have demonstrated the important role played by VEGF during mouse embryonic blood vessel development. The emerging roles of eNOS in the regulation of VEGF-mediated effects in adult endothelial cells (ECs) suggested that eNOS may also regulate the development of blood vessels. To investigate the role of VEGF and eNOS during early vascular development, we used both the planar and the three-dimensional tissue spheroid cultures generated from murine allantoides. We show that spheroids generated in hanging drops contain an inner microcapillary network and that, upon treatment with VEGF, these blood vessels recapitulated the in vivo process of VEGF-mediated vascular hyperfusion. Moreover, VEGF-treated spheroids present an inner continuous EC layer coated with smooth muscle cells and surrounding an inner lumen. Lumenized spheroids exhibited properties similar to in vivo blood vessels in that they mimicked the contractile response to KCI and relaxed when exposed to acetylcholine. HDL-mediated relaxation was absent in lumenized spheroids generated using allantoides isolated from S1P3 nulls, highlighting the utility of lumenized spheroids as a tool for analyzing null mutations in mice. Finally, using lumenized spheroids we were able to recapitulate the fusion of paired dorsal aortae in a model that would explain dorsal aorta formation. Using the planar allantois culture system we next sought to investigate the role(s) of eNOS in regulating the VEGF-mediated proliferation in angioblasts and embryonic ECs (EECs). As was the case in vivo, utilizing the in vitro planar culture, we demonstrated for the first time that VEGF-mediated proliferation in angioblasts and EECs is dependent on eNOS phosphorylation of S1177 (peNOS) and NO synthesis. The VEGF/eNOS proliferative response was abolished in both in vivo and in vitro studies using Akt-1 nulls. Similarly, overexpression of a mutated eNOS on S1177 showed that the proliferation of human umbilical vein ECs (HUVECs) was also dependent on eNOS phosphorylation. Further, our studies in HUVECs provided novel findings that VEGF-mediated S-nitrosylation of cyclins in mitotic ECs is dependent on HSP90 and signalosome formation. Taken together, these studies demonstrate novel roles for VEGF and eNOS during embryonic vascular development relevant to adult EC biology and physiology.