Abstract 5444: Plasminogen Activator Inhibitor-1 Inhibits Angiogenic Signaling By Blocking Cross-talk Between αVβ3 Integrin And Vascular Endothelial Cell Growth Factor Receptor-2
Background. Cross-talk between the vitronectin receptor, αVβ3 integrin, and vascular endothelial cell growth factor (VEGF) receptor-2 (VEGFR-2) plays a key role in controlling angiogenesis. Plasminogen activator inhibitor type-1 (PAI-1) competes with αVβ3 integrin for vitronectin (VN) binding. We tested the hypothesis that PAI-1 blocks angiogenesis by inhibiting VEGFR-2 activation.
Methods and Results. We studied the effects of PAI-1 on VEGF-induced adhesion, migration and tubule formation of human umbilical vein endothelial cells (HUVEC) grown in culture. PAI-1 (10 μg/mL) inhibited HUVEC adhesion, migration, and tubule formation on VN matrix. PAI-1 also inhibited VEGF-induced angiogenesis in ex vivo cultured aortic explant model and in vivo Matrigel implant angiogenesis model. To identify mechanisms underlying the anti-angiogenic properties of PAI-1, we examined the effect of PAI-1 on VEGF-induced phosphorylation of VEGFR-2 and ERK1/2. PAI-1 blocked VEGFR-2 phosphorylation in cells cultured on VN, but not in cells cultured on collagen or fibronectin. PAI-1 also blocked phosphorylation of ERK1/2 in a VN-dependent manner. PAI-1-R (a mutant that binds VN but does not inhibit proteases) and PAI-1-AK (a mutant that inhibits proteases but does not bind VN) each inhibited VEGFR-2 phosphorylation, suggesting that physical interaction between PAI-1 and VN was sufficient, but not required for PAI-1 to block VEGF signaling. Co-immunoprecipitation analyses demonstrated that PAI-1 blocked binding of β3 integrin to VEGFR-2, suggesting that PAI-1 blocks αVβ3 integrin-VEGFR-2 cross-talk.
Conclusion. Our data suggest that αVβ3 must engage VN to cross-talk with VEGFR-2 and support its activation by VEGF. PAI-1 inhibits angiogenic signaling by blocking αVβ3 integrin-VEGFR-2 cross-talk and preventing phosphorylation of VEGFR-2 and ERK1/2. The anti-angiogenic effect of PAI-1 appears to be mediated by binding to VN and blockade of αVβ3 integrin-VN interaction, and by pathways that do not require PAI-1-VN binding, suggesting that PAI-1-induced internalization of αVβ3 integrin may also inhibit VEGFR-2 activation. As a whole, these data suggest key roles for VN and PAI-1 in VEGF signaling that have important mechanistic and therapeutic implications.