VEGFR2 signals with VE‐cadherin and PECAM to facilitate leukocyte transendothelial migration

The migration of leukocytes out of the blood and into tissue is a critical component of the inflammatory process. To gain access to the target tissue and exert their beneficial or deleterious effects, leukocytes undergo a complex series of adhesive interactions with endothelial cells, which line the vessel wall. In the putative committed step in this process, the leukocyte must find the junction between adjacent endothelial cells and crawl through. This process, commonly referred to as transendothelial migration (TEM), has long been known to require homophilic interaction between PECAM on leukocyte and the endothelial cells. Recently our group showed that the process also involves calcium signaling originating through the cation channel TRPC6. We hypothesized that PECAM may be functioning in TEM using some of the interactions recently shown to be involved in its role in the mechanotransduction of fluid sheer stress. Perhaps engagement of leukocyte PECAM with endothelial cell PECAM at the cell border provides the sheer stress in this case. Here we report the unexpected role of a macromolecular PECAM complex that facilitates signaling to TRPC6 in TEM. Specifically, we show that endothelial cell PECAM binds to VEGFR2 and VE-cadherin via the transmembrane domain of VE-cadherin. Swapping the VE-cadherin transmembrane domain with that of N-cadherin abrogated TEM. Likewise, deletion of the cytoplasmic c-terminal tail of PECAM also abolished this signaling cascade. Stimulation of this complex with antibodies against PECAM induces the phosphorylation of VEGFR2 on tyrosine Y1175. Mutation of that critical residue to phenylalanine (Y1175F) prevented VEGFR2 phosphorylation and blocked TEM. Furthermore we show that this complex signals specifically to PLC1γ (and not PLCβ3) leading to its phosphorylation. Blocking its function with the inhibitor U73122 also blocked TEM. Diacylglycerol liberated by PLCγ1 activates TRPC6. We also confirmed the role of VEGFR2 in vivo using to separate models of inflammation. First, we examined the recruitment of neutrophils to inflamed skin using the croton oil dermatitis model. In control animals, neutrophils are robustly recruited to and migrate across post-capillary venules. In VEGFR2 knockout (KO) mice, most neutrophils are found inside the vessel with a corresponding decrease in those found out in tissue. Similarly, live animal imaging (intravital microscopy) of the inflamed cremaster muscle showed no defect in neutrophil rolling or adhesion in the VEGFR2 KO animals, but a profound disruption in their ability to undergo TEM compared to control wild type mice. Taken together, these data confirm the role of a VEGFR2/PECAM/VE-cadherin mechanotransduction complex that signals through PLC1γ to facilitate leukocyte TEM upstream of the action of TRPC6. Uncovering this signaling pathway opens up several new targets for potential therapeutic intervention.