VEGF-A and alphaVbeta3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells.

We recently showed that normal fibroblasts mediate capillary-like differentiation of human microvascular endothelial cells (HMVEC) in a 3-D angiogenesis model. Here, we show that a collaborative effect of VEGF-A and alphaVbeta3 integrin is critical in fibroblast-mediated angiogenesis because enhancement of both VEGF production by fibroblasts and beta3 integrin expression in HMVEC can rescue capillary-like endothelial differentiation under reduced serum conditions. To investigate the downstream signaling mechanisms, we compared N-Ras and Rho/Rac/Cdc42, as well as phosphatidylinositol 3-kinase (PI3-K) and Akt, for their involvement in the capillary-like network formation. The dominant-negative mutant of N-Ras (N-RasN17), but not the mutants of Rho/Rac/Cdc42, suppressed network formation. Overexpression of a constitutively active form of PI3-K rescued the network formation, which was inhibited by a dominant-negative >beta3 integrin; however, an active form of Akt failed to rescue the inhibition but induced a phenotypic change in HMVEC. Moreover, PI3-K is a downstream target of N-Ras because it could be co-immunoprecipitated with N-Ras, and its active form could rescue the inhibitory effect of N-Ras N17. Thus, our data indicate the existence of N-Ras- and PI3-K-dependent but Rho/Rac/Cdc42- and Akt-independent signaling mechanisms for the synergistic effect of VEGF-A and alphaVbeta3 on fibroblast-mediated microvascular network formation.