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.
D001808:Blood Vessels / Q000033:anatomy & histology; D002196:Capillaries / Q000033:anatomy & histology / Q000378:metabolism / Q000502:physiology; D018920:Coculture Techniques; D016228:Endothelial Growth Factors / Q000502:physiology*; D004730:Endothelium, Vascular / Q000201:enzymology / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D009154:Mutation; D018919:Neovascularization, Physiologic; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A; D020741:rho GTP-Binding Proteins / Q000502:physiology
D016228:Endothelial Growth Factors; D039302:Integrin alphaVbeta3; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020741:rho GTP-Binding Proteins