230 VEGFR2 and αvβ3 Recruitment and Cross-Talk at the Basal Aspect of HIV-1 Tat-Adherent Endothelial Cells Drives pp60src/ERK1/2 Activation, Cytoskeleton Organization and Pro-angiogenic Activation

Abstract

HIV-1 Tat protein released by infected cells accumulates in the extracellular matrix. In this substrate-immobilized form it promotes endothelial cell (EC) adhesion and consequent FAK/RhoA/pp60src/NF-kB activation and pro-angiogenic activation. All these effects, except pp60src activation, are dependent on αvβ3 engagement and activation by Tat. Here we demonstrate that EC adhesion to substrate-immobilized Tat induces the recruitment in the ventral plasma membrane (VPM) of αvβ3, paxillin, focal adhesion kinase (FAK), pp60src and the vascular endothelial growth factor receptor-2 (VEGFR2). As expected, paxillin and αvβ3 co-localize in the focal adhesion plaques of ECs adherent to the αvβ3-ligands Tat, vitronectin (VN) and fibrinogen (FG), but not to the α5β1-ligand collagen. At variance, VEGFR2 recruited at the VPM of Tat- (but not FG-) adherent ECs localizes in lipid rafts where it undergoes phosphorylation. In turn, VEGFR2 activation triggers pp60src and ERK1/2 phosphorylation. The VEGFR2/pp60src/ERK1/2 signaling pathway eventually leads to a proper cytoskeleton organization and pro-angiogenic activation of Tat-adherent ECs. In conclusion, VEGFR2 and αvβ3 are recruited in VPM of Tat-adherent ECs, in lipid rafts and focal adhesion plaques, respectively. VEGFR2 undergoes phosphorylation and triggers a signal transduction pathway (including pp60src and ERK1/2) that is distinct from the pathway activated by αvβ3. And is required for cytoskeleton re-organization and pro-angiogenic activation of ECs. These results provide new biochemical and biological insights in the cross-talk between integrin, angiogenic growth factors and tyrosine kinase receptors during angiogenesis.