Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

Inhibition of VE-PTP counters vascular leakage in inflammation via TIE-2, even in the absence of VE-cadherin.

No abstract

Key Points• The endothelial leukocyte receptor VCAM-1 triggers opening of endothelial junctions via dissociation of VE-PTP from VE-cadherin.• VCAM-1 and VEGF signaling use a similar signaling pathway to trigger the dissociation of VE-PTP from VE-cadherin.The vascular endothelial (VE) receptor protein tyrosine phosphatase (VE-PTP) associates with VE-cadherin and supports endothelial cell contact integrity. This complex is rapidly dissociated by adhesion of leukocytes to endothelial cells or by vascular endothelial growth factor. We have shown recently that this dissociation is indeed required for the opening of endothelial cell contacts during leukocyte extravasation in vivo. The leukocyte receptor and signaling mechanism that stimulates VE-cadherin/VE-PTP dissociation are unknown. Here, we identify vascular cell adhesion molecule 1 as the relevant receptor for lymphocytes in this process. As signaling steps downstream of this receptor, we determined the activation of Rac1, the generation of reactive oxygen species by nicotinamide adenine dinucleotide phosphate oxidase and the activation of the redox-sensitive tyrosine kinase Pyk2 as essential for VE-cadherin/VE-PTP dissociation. These signaling steps are also required for the dissociation induced by VE growth factor. Searching for the molecular mechanism of complex dissociation, we found that a model substrate of VE-PTP represented by a tyrosine-phosphorylated peptide of Tie-2 dissociates VE-PTP from VE-cadherin when introduced with the help of a Tat peptide. We suggest that lymphocyte binding to vascular cell adhesion molecule 1 triggers a signaling process that enables a VE-PTP substrate to dissociate VE-PTP from VEcadherin, thereby facilitating efficient transmigration. (Blood. 2013;122(14):2512-2522) IntroductionDuring inflammation, leukocytes extravasating from the blood into tissues have to migrate through the blood vessel wall. This is achieved by first binding to various cytokine-induced adhesion receptors on endothelial cells followed by inducing openings in the endothelial barrier.1 Although these openings can be transcellular, the vast majority of leukocytes use a paracellular route through endothelial junctions, as was documented in vitro 2 and more recently in vivo. 3,4 Vascular endothelial (VE)-cadherin is a junctional adhesion molecule that is of vital importance for the integrity of endothelial cell junctions. 5,6 Blocking of VE-cadherin adhesion by antibodies is sufficient to destabilize endothelial junctions in vivo and to increase leukocyte extravasation. 7,8 Furthermore, replacing the VE-cadherinb-catenin-a-catenin complex by a VE-cadherin-a-catenin fusion protein in knock-in mice stabilizes endothelial junctions efficiently and strongly inhibits leukocyte extravasation in various tissues. 4 This highlights the importance of regulating VE-cadherin function during leukocyte extravasation. It has been shown that tyrosine phosphorylation of VE-cadherin correlates with destabilization of endothelial junctions 9-11 and participates in vitro in the...

The endothelial layer of blood vessels controls the passage of cells and solutes from the blood into the surrounding tissue. Crucial for this regulation is the integrity of endothelial cell-cell junctions. Various molecular mechanisms control junctional integrity of the endothelial layer including GTPases, modulation of the actomyosin cytoskeleton and phosphorylation and dephosphorylation of junctional proteins. Several kinases and phosphatases have been identified that are good candidates for the regulation of the endothelial barrier function. For some of them, in vivo evidence has recently been presented that highlights their importance in either the regulation of vascular permeability or leukocyte extravasation. This review will summarize current knowledge about the regulation of endothelial junctions by kinases and phosphatases. In particular, the role of the endothelial specific phosphatase VE-PTP in the context of endothelial cell contact stability will be highlighted.