Abstract 237: VEGF-Mediated Phosphorylation of eNOS Regulates Angioblast and Endothelial Cell Proliferation
To evaluate when nitric oxide (NO) is first expressed in the EC lineage, the expression pattern of eNOS, phosphorylated eNOS (P-eNOS), and key proteins that define the endothelial lineage (i.e., Flk-1, TAL-1, CD31) were assessed in 7.0-8.5dpc mouse embryos. Immunohistochemical analyses revealed that embryonic endothelial cells (Flk-1+/TAL-1+/CD31+) expressed eNOS prior to their investment by smooth muscle cells while isolated angioblasts (Flk-1+/TAL-1+/CD31-) did not express eNOS. Based on eNOS expression we identified a cell type, transitional angioblasts (eNOS+/FLK-1+/TAL-1+/CD31-), intermediate between embryonic endothelial cells (EECs) and angioblasts. Transitional angioblasts are further distinguished from angioblasts by their initiation of cell-cell contacts with other eNOS+ Flk-1+/TAL-1+/CD31- cells or with EECs. Analysis of P-eNOS and phospho-histone H3 expression in transitional angioblasts and EECs showed a tight correlation between P-eNOS expression and cell proliferation. This correlation was also observed in cultured human umbilical vein endothelial cells (HUVECs) treated with nocodazole to induce a G2/M cell cycle block. Given that VEGF mediates both EC division and NO production, we evaluated whether VEGF-mediated NO production is critical for neovascular processes (vasculogenesis/angiogenesis). We show using allantoic cultures that VEGF-mediated mitosis in Flk-1+ cells is decreased by the eNOS inhibitors L-NIO and 17-AAG, suggesting that VEGF-mediated eNOS phosphorylation is regulating cell proliferation. Supporting such a role, we found that the alterations in VEGF-mediated vascular patterning we observed in response to eNOS inhibitors were consistent with an overall reduction in the EEC numbers and that VEGF-induced NO production in HUVECs increased the S-nitrosylation of proteins important for the regulation of the G2/M transition, such as cyclin B. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation regulates transitional angioblast, EEC, and EC proliferation.
- © 2012 by American Heart Association, Inc.