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

整合素 alpha-v beta-3 调节内皮通透性

• 批准号: 7996943
• 负责人: Mallar Bhattacharya
• 金额: $ 5.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996943
• 关键词: Actin-Binding Protein; Actins; Adhesions; Affect; Affinity; Agonist; Binding; Biology; Blood Vessels; Cell Line; Cell Surface Proteins; Cells; Chimeric Proteins; Complex; Cytoplasmic Tail; Cytoskeleton; Cytosol; DNA Sequence Rearrangement; Endothelial Cells; Endothelium; Extracellular Matrix; Glutathione S-Transferase; Growth and Development function; Human; Immune system; Inflammation; Inflammatory; Inflammatory Response; Injury; Integrin alphaVbeta3; Integrins; Knockout Mice; Lead; Life; Ligands; Link; Liquid substance; Mus; Pathway interactions; Permeability; Proteins; Regulation; Research; Sepsis; Site; Thrombin; Trauma; Vascular Endothelial Growth Factors; Vascular Permeabilities; Western Blotting; cell growth; design; genetic regulatory protein; human EMS1 protein; insight; lung injury; migration; null mutation; public health relevance; pulmonary artery endothelial cell; research study; response; sphingosine 1-phosphate

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.

描述（由申请方提供）：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性细胞进入损伤或炎症部位。因此，探索血管通透性增加的基本机制对于理解肺损伤和脓毒症中全身性炎症的途径至关重要。整合素是异源二聚体细胞表面蛋白，其附着于细胞外基质配体，并且对细胞生长、发育、迁移和粘附至关重要。整合素将细胞外基质中的配体连接到肌动蛋白细胞骨架，并且已显示影响肌动蛋白细胞骨架重排，其直接影响细胞-细胞接触，从而影响内皮通透性。Sheppard实验室已经在这方面研究了β β整联蛋白：虽然发现β整联蛋白无效突变的纯合子小鼠在增加血管通透性的试剂如凝血酶或VEGF存在下具有正常的生长和发育，但β无效小鼠与野生型相比具有增加的通透性。此外，发现β 3整合素对于响应屏障增强激动剂1磷酸鞘氨醇的皮质肌动蛋白的形成是必需的。自从今年加入实验室以来，我已经使用了谷胱甘肽S-转移酶（GST）融合蛋白，该融合蛋白由GST融合到β整合素的胞质尾部组成，用于从内皮细胞胞质溶胶中亲和捕获潜在的结合伴侣。将所得复合物提交用于质谱测序，鉴定蛋白质corneum，一种已知的肌动蛋白调节蛋白。通过从人肺动脉内皮细胞分离的整合素复合物的Western印迹证实了β 3和corneum之间的关联。在这个应用中，我提出了实验设计，以评估这种相互作用之间的功能意义coronin和（v）（3。具体地说，我希望探讨的一个关键的机制问题是，整合素的内皮屏障增强作用是否需要β-cornein与β-cornein的结合。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。