Beta 3 Integrin Phosphorylation and VEGF Signaling.

The process of angiogenesis is critical for tumor survival as well as for ischemic remodeling of vasculature after thrombotic occlusion. This process is a result of finely tuned co-operation between angiogenic growth factors, primarily from the VEGF family, and their receptors and cell receptors for extracellular matrix, the integrins. The role of alpha v beta 3 integrin in neovasculature development appears to be controversial asthe blockade of this receptor with antibodies inhibited angiogenesis while the genetic ablation of the beta3 subunit resulted in increased angiogenesis due to the over-expression of VEGFR2 on endothelial cells (EC). In order to understand the role of beta 3 integrin in VEGF-induced responses, we utilized knock-in mice with a mutation within the beta 3 subunit (Y747/759 to F, DiYF) that results in beta 3 that is unable to undergo tyrosine phosphorylation, an event critical for integrin outside-in signaling. Using EC isolated from WT and DiYF mice, we have found that defective tyrosine phosphorylation resulted in impaired EC adhesion and spreading on vitronectin but not on fibronectin, laminin and collagen. Migration in response to VEGF was reduced in DIYF EC by 3 fold compared to WT when vitronectin and fibrinogen but not fibronectin and collagen were used as substrates. In a wound healing assay on vitronectin, DiYF but not WT EC failed to close the wound. Video microscopy of migrating EC revealed that WT EC form forward protrusions and then retract the back of the cell, which results in successful directed migration. DiYF EC also formed protrusions, but could not form stable adhesion and retract the tail of the cell. As a result, DiYF EC were moving forward and then back again, failing to migrate in the direction of the wound. Importantly, DiYF but not WT EC were not able to form complete capillaries on Matrigel in response to VEGF and the average length of EC tubes was 3.5 fold lower compared to WT. Interestingly, the DiYF mutation resulted in impaired integrin activation (inside-out signaling) in response to VEGF as well as PMA based on soluble fibrinogen and WOW-1 binding. We have found that VEGF treatment of WT EC resulted in beta 3 integrin phosphorylation, which occurred 5 minutes after stimulation and reached the maximum at 30 min. In DiYF EC, this response was absent. Most importantly, immediately after VEGF stimulation, VEGFR2 formed a complex with beta 3 integrin. This complex was not observed in DiYF EC. Moreover, phosphorylation of VEGFR2 (but not the total protein level) was significantly reduced in DiYF EC compared to WT. As a result, major intracellular signaling events triggered by VEGF stimulation, such as phoshorylation of Akt, ERK1/2 and p38 MAPK, were severely impaired in DiYF EC. The finding that beta 3 phosphorylation regulates an activation of VEGFR2 and subsequent signaling events in EC provides novel insights on EC biology and mechanisms of VEGF-induced angiogenesis.