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

描述（由申请人提供）：内皮作为血管内和血管外空间之间的屏障，其渗透性调节营养因子和炎症细胞进入损伤或炎症部位。因此，探究血管通透性增加的基本机制对于理解肺损伤和败血症中全身性炎症的途径至关重要。整合素是一种异二聚体细胞表面蛋白，它附着在细胞外基质配体上，对细胞生长、发育、迁移和粘附至关重要。整合素将细胞外基质中的配体与肌动蛋白细胞骨架连接起来，并已被证明影响肌动蛋白细胞骨架重排，这直接影响细胞间接触，从而影响内皮细胞的通透性。Sheppard实验室在这方面对(v（3整合素）进行了研究：虽然发现(3整合素零突变的纯合小鼠具有正常的生长发育，但在凝血酶或VEGF等增加血管通透性的药物存在下，(3)零小鼠的通透性比野生型增加。此外，(v(3)整合素被发现是形成皮质肌动蛋白所必需的，以响应屏障增强激动剂鞘氨醇1磷酸。自今年加入实验室以来，我一直使用谷胱甘肽s -转移酶（GST）融合蛋白，该融合蛋白由GST融合到(3整合素的细胞质尾部组成，用于从内皮细胞溶胶中亲和力捕获潜在的结合伙伴。所得到的复合物被提交进行质谱测序，鉴定了一种已知的肌动蛋白调节蛋白。从人肺动脉内皮细胞中分离的整合素复合物Western blot证实了(v(3)与内联蛋白之间的关系。在本应用中，我提出了旨在评估接触和(v(3)之间这种相互作用的功能意义的实验。具体来说，我希望探讨的一个关键机制问题是，整合素增强内皮屏障的作用是否需要（v(3)）结合接触。阐明整合素和肌动蛋白结合蛋白之间的相互作用如何调节屏障功能将加深我们对血管泄漏的理解，并将为肺损伤和败血症的靶向治疗提供途径。

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.