VEGF‐A and α V β 3 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 α V β 3 integrin is critical in fibroblast‐mediated angiogenesis because enhancement of both VEGF production by fibroblasts and β 3 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‐Ras N17 ), 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 β 3 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 α V β 3 on fibroblast‐mediated microvascular network formation.