Objective Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 down-regulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin (VN)-dependent cooperative binding interaction between VEGFR-2 and αVβ3. Approach and Results We studied PAI-1's effects on VEGF signaling in human umbilical vein endothelial cells (HUVECs). PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in HUVECs grown on VN, but not on fibronectin or collagen. PAI-1 inhibited binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to VN and another requiring binding to very-low-density-lipoprotein receptor (VLDLR). PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. Conclusions PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a VN-dependent, pro-angiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, VN, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.

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BackgroundCross-talk between integrins and cadherins regulates cell function. We tested the hypothesis that vitronectin (VN), a multi-functional adhesion molecule present in the extracellular matrix and plasma, regulates vascular permeability via effects on VE-cadherin, a critical regulator of endothelial cell (EC) adhesion.Methodology/Principal FindingsAddition of multimeric VN (mult VN) significantly increased VE-cadherin internalization in human umbilical vein EC (HUVEC) monolayers. This effect was blocked by the anti-αVβ3 antibody, pharmacological inhibition and knockdown of Src kinase. In contrast to mult VN, monomeric VN did not trigger VE-cadherin internalization. In a modified Miles assay, VN deficiency impaired vascular endothelial growth factor-induced permeability. Furthermore, ischemia-induced enhancement of vascular permeability, expressed as the ratio of FITC-dextran leakage from the circulation into the ischemic and non-ischemic hindlimb muscle, was significantly greater in the WT mice than in the Vn −/− mice. Similarly, ischemia-mediated macrophage infiltration was significantly reduced in the Vn −/− mice vs. the WT controls. We evaluated changes in the multimerization of VN in ischemic tissue in a mouse hindlimb ischemia model. VN plays a previously unrecognized role in regulating endothelial permeability via conformational- and integrin-dependent effects on VE-cadherin trafficking.Conclusion/SignificanceThese results have important implications for the regulation of endothelial function and angiogenesis by VN under normal and pathological conditions.