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

描述(申请人提供)：内皮作为血管内外空间之间的屏障，其通透性调节营养因子和炎症细胞进入损伤或炎症部位。因此，探讨血管通透性增加的基本机制对于了解肺损伤和脓毒症中全身炎症的途径至关重要。整合素是一种与细胞外基质配体结合的异源二聚体细胞表面蛋白，对细胞的生长、发育、迁移和黏附至关重要。整合素将细胞外基质中的配体连接到肌动蛋白细胞骨架上，并已被证明影响肌动蛋白细胞酮重排，从而直接影响细胞与细胞之间的接触，从而影响内皮通透性。谢泼德实验室研究了(v(3整合素)在这方面：虽然(3整合素零突变纯合子小鼠被发现具有正常的生长发育，但在凝血酶或血管内皮生长因子等增加血管通透性的药物存在下，(3整合素缺失小鼠与野生型相比通透性增加。此外，(v(3)整合素被发现是皮层肌动蛋白形成所必需的，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，将GST融合到(3)整合素的细胞质尾部，从内皮细胞胞浆中亲和捕获潜在的结合伙伴。得到的复合体被提交给质谱学测序，并鉴定了已知的肌动蛋白调节蛋白Cortactin。从人肺血管内皮细胞分离的整合素复合体的Western印迹实验证实了(v(3)与皮质素之间的联系。在这个应用中，我提出了旨在评估Cortactin和(v(3)之间这种相互作用的功能意义的实验。具体地说，我想要探讨的一个关键机制问题是，(v(3)与Cortactin的结合是否是整合素增强内皮屏障作用所必需的。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渗漏的理解，并将为肺损伤和脓毒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.