Regulation of Endothelial Permeability by Integrin alpha-v beta-3

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DESCRIPTION (provided by applicant): The endothelium serves as a barrier between the intra and extravascular spaces, and its permeability regulates access of trophic factors and inflammatory cells to sites of injury or inflammation. Probing basic mechanisms of increased vascular permeabilty is thus crucial to understanding pathways of systemic inflammation in lung injury and sepsis. Integrins are heterodimeric cell surface proteins that attach to extracellular matrix ligands and are vital to cellular growth, development, migration, and adhesion. Integrins link ligands in extracellular matrix to the actin cytoskeleton and have been shown to affect actin cytosketal rearrangement, which directly impacts cell-cell contacts and hence endothelial permeability. The Sheppard lab has studied (v(3 integrin in this regard: while mice homozygous for a null mutation of (3 integrin were found to have normal growth and development, in the presence of agents that increase vascular permeability like thrombin or VEGF, (3 null mice had increased permeability compared to wild type. Moreover, (v(3 integrin was found to be necessary for the formation of cortical actin in response to the barrier enhancing agonist sphingosine 1 phosphate. Since joining the lab this year, I have used glutathione S-transferase (GST) fusion proteins consisting of GST fused to the cytoplasmic tail of (3 integrin for affinity capture of potential binding partners from endothelial cytosol. The resulting complexes were submitted for mass spectrometric sequencing, with identification of the protein cortactin, a known actin regulatory protein. The association between (v(3 and cortactin was verified by Western blot of integrin complexes isolated from human pulmonary artery endothelial cells. In this application, I propose experiments designed to assess the functional significance of this interaction between cortactin and (v(3. Specifically, a crucial mechanistic question that I wish to probe is whether binding of cortactin by (v(3 is necessary for the integrin's endothelial barrier enhancing effect. Elucidating how the interaction between integrins and actin binding proteins modulates barrier function will deepen our understanding of vascular leak and will provide avenues for targeted therapy of lung injury and sepsis.