␣v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with ArgGly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some ␣v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of ␣v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for ␣v␤3 and ␣v␤5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. IntroductionThe formation of new blood vessels from existing vasculature, or angiogenesis, is essential for successful tumor growth and for the development of metastases. Previous work has suggested that certain endothelial cell integrins, including ␣v␤3 and ␣v␤5, actively contribute to the angiogenic process. 1,2 More recently, endogenous inhibitors of angiogenesis have been shown to target these integrins. 3 Integrins are transmembrane receptors for extracellular matrix (ECM) and basement membrane proteins that are composed of 2 noncovalently associated subunits, ␣ and ␤. 4 To date, 18 ␣ and 8 ␤ subunits have been identified in mammals, and their association in various combinations leads to the formation of at least 24 receptors with distinct ligand specificity. Besides mediating stable adhesion, integrins transmit signals that regulate cell survival, growth, motility, and remodeling of their extracellular environment. [4][5][6] The integrins ␣v␤3 and ␣v␤5 bind to ECM molecules through an Arg-Gly-Asp (RGD)-binding site. Based on the concept that they are proangiogenic receptors, specific inhibitors, including blocking monoclonal antibodies, RGD peptides, and RGD peptidomimetics, have been developed and evaluated in vivo. 7 Indeed, pharmacologic agents targeted to ␣v␤3, ␣v␤5, or both, have been reported to block tumor and retinal angiogenesis. [8][9][10][11][12][13][14] Some of these angiogenesis inhibitors, including a humanized monoclonal anti␣v␤3 (Vitaxin; MedImmune, Gaithersburg, MD) and an ␣v␤3/ ␣v␤5-selective RGD-based cyclic peptide (cilengitide), have entered clinical trials. 15,16 Early experiments show that a monoclonal antibody directed against ␣v␤3 inhibits angiogenesis by inducing the apoptosis of angiogenic blood vessels. 17...