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      The Integrin Antagonist Cilengitide Activates αVβ3, Disrupts VE-Cadherin Localization at Cell Junctions and Enhances Permeability in Endothelial Cells

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          Cilengitide is a high-affinity cyclic pentapeptdic αV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through αVβ3/αVβ5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the β1 family, and little is known on the effect of cilengitide on endothelial cells expressing αVβ3 but adhering through β1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on αVβ3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the β1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface αVβ3, stimulated phosphorylation of FAK (Y 397 and Y 576/577), Src (S 418) and VE-cadherin (Y 658 and Y 731), redistributed αVβ3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density β1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of αVβ3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

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          The role of adherens junctions and VE-cadherin in the control of vascular permeability.

          Endothelial cells control the passage of plasma constituents and circulating cells from blood to the underlying tissues. This specialized function is lost or impaired in several pathological conditions - including inflammation, sepsis, ischemia and diabetes - which leads to severe, and sometimes fatal, organ dysfunction. Endothelial permeability is regulated in part by the dynamic opening and closure of cell-cell adherens junctions (AJs). In endothelial cells, AJs are largely composed of vascular endothelial cadherin (VE-cadherin), an endothelium-specific member of the cadherin family of adhesion proteins that binds, via its cytoplasmic domain, to several protein partners, including p120, beta-catenin and plakoglobin. Endogenous pathways that increase vascular permeability affect the function and organization of VE-cadherin and other proteins at AJs in diverse ways. For instance, several factors, including vascular endothelial growth factor (VEGF), induce the tyrosine phosphorylation of VE-cadherin, which accompanies an increase in vascular permeability and leukocyte diapedesis; in addition, the internalization and cleavage of VE-cadherin can cause AJs to be dismantled. From the knowledge of how AJ organization can be modulated, it is possible to formulate several pharmacological strategies to control the barrier function of the endothelium. We discuss the possible use of inhibitors of SRC and other kinases, of agents that increase cAMP levels, and of inhibitors of lytic enzymes as pharmacological tools for decreasing endothelial permeability.
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            Endothelial cell-cell junctions: happy together.

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              Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

              Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

                Author and article information

                Role: Editor
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                12 February 2009
                : 4
                : 2
                : e4449
                [1]Division of Experimental Oncology, Centre Pluridisciplinaire d'Oncologie (CePO), Faculty of Biology and Medicine, University of Lausanne, and NCCR Molecular Oncology, ISREC, Epalinges, Switzerland
                University of Birmingham, United Kingdom
                Author notes

                Conceived and designed the experiments: GCA CR. Performed the experiments: GCA LP. Analyzed the data: GCA LP CR. Wrote the paper: GCA CR.

                Alghisi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                : 21 September 2008
                : 2 January 2009
                Page count
                Pages: 14
                Research Article
                Cell Biology/Cell Adhesion
                Cell Biology/Cell Signaling
                Cardiovascular Disorders/Vascular Biology



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