Mechanotransduction via endothelial adhesion molecule CD31 initiates transmigration and reveals a role for VEGFR2 in diapedesis.

Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.

Time-Variant SRC Kinase Activation Determines Endothelial Permeability Response.

In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.

Targeted Recycling of the Lateral Border Recycling Compartment Precedes Adherens Junction Dissociation during Transendothelial Migration.

Leukocyte transendothelial migration (TEM) requires two major events: local dissociation of adherens junctions manifested as gaps in vascular endothelial (VE)-cadherin staining at the site of TEM and targeted trafficking of the lateral border recycling compartment (LBRC) to the site of TEM. However, the association between LBRC recycling and VE-cadherin gaps remains unknown. We found that when targeting of the LBRC is selectively inhibited using established methods, such as a function blocking anti-platelet endothelial cell adhesion molecule 1 antibody, depolymerizing microtubules, or microinjection of an antibody that inhibits kinesin, VE-cadherin gaps do not form around the blocked leukocyte. This is the first time that the LBRC has been implicated in this process. We obtained similar results for neutrophils and monocytes and in studies using live cell imaging microscopy conducted under fluid shear conditions. Depolymerizing microtubules did not affect the ability of leukocytes to induce tyrosine phosphorylation of VE-cadherin. A VE-cadherin double mutant (Y658F, Y731F) expressed in endothelial cells acted as a dominant negative and inhibited VE-cadherin gap formation and TEM, yet targeting of the LBRC still occurred. These data suggest that targeting of the LBRC to the site of TEM precedes VE-cadherin clearance. Recruitment of the LBRC may play a role in clearing VE-cadherin from the site of TEM.

Transdominant regulation of integrin function: mechanisms of crosstalk.

Transdominant inhibition of integrins or integrin-integrin crosstalk is an important regulator of integrin ligand binding and subsequent signaling events that control a variety of cell functions in many tissues. Here we discuss examples of integrin crosstalk and detail our current understanding of the molecular mechanisms that are involved in this receptor phenomenon. The cytoskeleton associated protein talin is a key regulator of integrin crosstalk. We describe how the interaction of talin and the cytoplasmic tail of beta integrin is controlled and how competitive inhibitors of this binding play a role in integrin crosstalk. We conclude with a discussion of how integrin crosstalk impacts the interpretation of integrin inhibitor and knockdown studies in both the laboratory and clinical setting.

Recruitment of vimentin to the cell surface by beta3 integrin and plectin mediates adhesion strength.

Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires beta3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack beta3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing beta3 integrin (CHOwtbeta3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the beta3 integrin cytoplasmic tail. Moreover, CHOwtbeta3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated beta3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.

Integrin cross-talk in endothelial cells is regulated by protein kinase A and protein phosphatase 1.

In endothelial cells (ECs) beta1 integrin function-blocking antibodies inhibit alphavbeta3 integrin-mediated adhesion to a recombinant alpha4-laminin fragment (ralpha4LN fragment). beta1 integrin sequestration of talin is not the mechanism by which beta1 integrin modulates alphavbeta3 integrin ligand binding. Rather, treatment of the ECs with beta1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta1 integrin function-blocking antibodies on beta3 integrin serine phosphorylation and EC-ralpha4LN fragment binding. beta3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alphavbeta3 integrin-ralpha4LN fragment binding, we mutated it to alanine (beta3S752A) or aspartic acid (beta3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta3S752A integrin attach robustly to ligand. CHO cells expressing beta3S752D integrin do not. Because the beta3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta3 integrin. Instead, we have tested an hypothesis that PKA regulates beta3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alphavbeta3-mediated cell adhesion to the ralpha4LN fragment when both PKA and beta1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta1 integrin negatively modulates alphavbeta3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo.

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G(919-1207) to support endothelial cell adhesion. In particular, endothelial cell adhesion to G(919-1207) is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the alphavbeta3 integrin in angiogenesis.