ENDOTHELIAL LATERAL JUNCTIONS AND LEUKOCYTE TRAFFICKING

内皮横向连接和白细胞运输

• 批准号: 6602438
• 负责人: FRANCIS W. LUSCINSKAS
• 金额: $ 6.87万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6602438
• 关键词: SCID mouse; cadherins; cell adhesion; cell adhesion molecules; cell cell interaction; cell migration; hemodynamics; human tissue; immunofluorescence technique; inflammation; laboratory mouse; molecular cloning; neutrophil; tissue /cell culture; vascular endothelium

The inflammatory response is a fundamental component in host defense. The firm adhesion and transmigration of leukocytes across the vascular endothelium during inflammation occurs at the level of post-capillary and collecting venules. Although much attention has been paid to leukocyte- endothelial adhesive interactions, only recently have the molecular mechanisms involved in leukocyte passage through the endothelial cell lateral junctions become a focus of study. Recently, we have shown that neutrophil adhesion triggers disruption of adherens junctions at endothelial lateral borders, prior to the transmigration event (1). Specifically, the VE-cadherin complex, consisting of VE-cadherin, alpha- catenin, beta-catenin, gamma-catenin (also termed plakoglobin), and p120/p100 non-covalently linked, was disrupted within 3 minutes of initial neutrophil binding, and results in loss of this complex from lateral junctions and cleavage of both VE-cadherin and beta-catenin. Washed membranes (protease inhibitor-treated) prepared from neutrophils were able to mediate these changes, suggesting that the stimulus for disruption of the endothelial VE-cadherin complex resides in neutrophil surface and that granule proteolytic components do not appear to be required. The focus of this project is to characterize more fully the endothelial dependent-mechanisms involved in regulation of lateral junctions during leukocyte transmigration using immunological, cell biological and molecular biological strategies in in vitro and in vivo models. The proposed studies will test critically whether the observed changes in the composition of lateral junctions are necessary for leukocyte transmigration. Specific Aim I will determine the step in cell adhesion that trigger dissociation of the VE-cadherin complex during leukocyte transmigration, using live time immunofluorescence assays to monitor changes in VE-cadherin localization during leukocyte adhesion in a in vitro flow model, and secondly, test the hypothesis that dissociation and cleavage of the VE-cadherin complex is necessary for transmigration. Specific Aim II will attempt to identify other, as yet uncharacterized, endothelial molecules localized to cell-to-cell junctions that are involved in leukocyte adhesion-transmigration, using a monoclonal antibody screening approach. The experiments proposed in Specific Aim II will determine the intracellular signaling pathways involved in leukocyte- induced VE-cadherin dissociation using pharmacological approaches and the live time immunofluorescence assays developed in Specific Aim I. Specific Aim IV will utilize in vivo models of acute or chronic inflammation to test the potential pathophysiologic relevance of leukocyte-induced alterations in adherens junctions. The proposed studies are relevant to the overall them of the program and will work in concert with the other project to gain a better understanding of the role of the vascular endothelial lining in inflammation and immune reactions.

炎症反应是宿主防御中的基本组成部分。这 白细胞的坚固粘附和跨血管的迁移 炎症期间内皮发生在毛细血管后的水平和 收集静脉。尽管已经对白细胞引起了很多关注 - 内皮粘合剂相互作用，直到最近才具有分子 白细胞通过内皮细胞涉及的机制 横向连接成为研究的重点。最近，我们已经证明 中性粒细胞粘附触发了粘附连接处的破坏 内皮外侧边界，移民事件发生之前（1）。 具体而言，由VE-钙粘蛋白，α-组成的VE-钙粘蛋白复合物 catenin，beta-catenin，伽马 - 钙蛋白（也称为plakoglobin）和 P120/P100非共价链接，在初始后3分钟内被破坏 嗜中性粒细胞结合，并导致该复合物因侧向丧失 VE-钙粘蛋白和β-catenin的结合和裂解。洗了 由中性粒细胞制备的膜（蛋白酶抑制剂治疗）能够 调解这些变化，表明刺激的破坏 内皮VE-钙粘蛋白复合物位于中性粒细胞表面， 似乎不需要颗粒蛋白水解成分。 该项目的重点是更充分地表征内皮 在调节横向连接的调节期间涉及的依赖机制 白细胞使用免疫，细胞生物学和 体外和体内模型中的分子生物学策略。这 拟议的研究将批判性测试观察到的 侧结的组成是白细胞所需的 轮回。具体目的我将确定细胞粘附的步骤 这会触发白细胞期间VE-钙粘蛋白复合物的解离 移民，使用活时间免疫荧光测定法进行监测 白细胞粘附过程中VE-钙粘蛋白定位的变化 体外流程模型，其次，检验了解离和 VE-钙粘蛋白复合物的裂解对于移民是必需的。 特定的目标II将尝试识别其他尚未表征的其他AIM II 内皮分子位于细胞到细胞连接处的内皮分子 使用单克隆抗体参与白细胞粘附转移术 筛选方法。特定目标中提出的实验II将 确定与白细胞相关的细胞内信号通路 使用药理学方法诱导VE-钙粘蛋白解离并 在特定目标I中开发的活时间免疫荧光测定法。 AIM IV将利用急性或慢性炎症的体内模型 测试白细胞诱导的潜在病理生理相关性 粘附连接的改变。 拟议的研究与该计划的总体相关， 将与另一个项目协同合作，以获得更好的理解 血管内皮衬里在炎症和免疫中的作用 反应。