Novel Mechanisms Controlling Endothelial Junctions and Vascular Permeability

项目摘要 血管内皮在血流和血管内皮细胞之间形成高度调节的渗透性屏障。 间质组织过度的血管渗漏导致许多炎症相关的疾病过程， 包括水肿、败血症、急性呼吸窘迫综合征（ARDS）、缺血性中风和出血。到 为了防止炎症期间由血管渗漏引起的组织损伤，需要能够 增强内皮屏障功能。细胞间连接控制内皮细胞旁的变化 血管内皮钙粘蛋白（VE-钙粘蛋白，CDH 5）是内皮细胞通透性的主要调节因子， 连接和细胞旁通透性。我们将探索一种新的假设和方法来理解其 基于我们对细胞内其他钙粘蛋白变构调节的发现， 表面响应信号事件。要检验的主要假设是，VE的变构调节- 钙粘蛋白与肌动蛋白细胞骨架的活动相协调，是它调节细胞内钙粘蛋白表达的关键机制。 内皮细胞的通透性对一系列血管因子的反应。我们将使用两个新的激活 抗VE-钙粘蛋白的单克隆抗体（mAb），其可防止由 凝血酶、VEGF和TNF α作为主要工具，在体外和体内检验这一假设，并研究 调控机制。具体目标是：A.确定VE-钙粘蛋白细胞表面调节是否是一种 一系列内皮生理过程的共同机制。体外实验将用于 确定它是否受到其他关键因素的类似调节，包括组胺，血管生成素，Tie 2 受体和S1 P。将检查来自不同微血管床的内皮细胞。协同 将进行实验以确定流动和剪切力影响mAb诱导的屏障功能。我们还将 研究活化mAb是否抑制白细胞渗出或影响内皮形态发生， 血管生成B。阐明VE-钙粘蛋白细胞表面的细胞和生化机制 调控我们将研究单克隆抗体激活其的结构和生物物理基础，并测试其作用。 VE-钙粘蛋白和相关连环蛋白的磷酸化激活。我们还将探讨 在细胞表面调节和内吞作用以及改变细胞骨架功能的屏障之间。 C.研究在小鼠体内，通过mAb激活VE-钙粘蛋白是否能增强渗漏性肠梗阻中的屏障功能。 血管疾病或炎症。我们将测试激活mAb对诱导的急性诱导的 血管渗漏和白细胞浸润我们还将研究它们对小鼠炎症模型的影响。 涉及脉管系统的疾病过程，包括败血症和炎症性肠病。这些研究 将帮助我们了解内皮渗透性的调节，并开发新的方法来增强 内皮屏障功能，以减少炎症过程和疾病。

Project Summary The vascular endothelium forms a highly regulated permeability barrier between the blood stream and interstitial tissues. Excessive vascular leakiness contributes to many inflammation related disease processes, including edema, sepsis, acute respiratory distress syndrome (ARDS), ischemic stroke, and hemorrhage. To prevent tissue damage resulting from vascular leakiness during inflammation, there is a need to be able to enhance endothelial barrier function. Intercellular junctions control changes in endothelial paracellular permeability, and Vascular-endothelial cadherin (VE-cadherin, CDH5) is a major regulator of endothelial junctions and paracellular permeability. We will explore a novel hypothesis and approach to understanding its role in permeability regulation, based on our findings of allosteric regulation of other cadherins at the cell surface in response to signaling events. The main hypothesis to be examined is that allosteric regulation of VE- cadherin, in coordination with activities of the actin cytoskeleton, is a key mechanism by which it regulates endothelial permeability in response to a range of vascular factors. We will use two novel activating monoclonal antibodies (mAbs) to VE-cadherin that prevent the increase in endothelial permeability induced by thrombin, VEGF, and TNFa, as major tools to test this hypothesis both in vitro and in vivo and to study the mechanism of regulation. The specific aims are: A. Determine whether VE-cadherin cell surface regulation is a common mechanism for a range of endothelial physiological processes. In vitro experiments will be used to determine whether it is similarly regulated by other key factors, including histamine, angiopoietins, Tie2 receptors, and S1P. Endothelial cells from different microvascular beds will be examined. Collaborative experiments will be done to determine flow and shear forces affect mAb induced barrier function. We will also investigate whether activating mAbs inhibit leukocyte diapedesis or affect endothelial morphogenesis and angiogenesis. B. Elucidate the cellular and biochemical mechanisms underlying VE-cadherin cell surface regulation. We will investigate the structural and biophysical basis of its activation by mAbs and test the roles of the phosphorylation of VE-cadherin and associated catenins in activation. We’ll also explore the relationship between cell surface regulation and endocytosis as well as barrier altering cytoskeletal functions. C. Investigate whether activation of VE-cadherin by mAbs enhances barrier function in vivo in mice in leaky vascular conditions or inflammation. We’ll test the effects of activating mAbs on induced acute induced vascular leak, and leukocyte infiltration. We will also examine their effects on mouse models of inflammatory disease processes involving the vasculature, including sepsis and Inflammatory Bowel Disease. These studies will help us understand regulation of endothelial permeability and develop novel approaches to enhance endothelial barrier function to reduce inflammatory processes and diseases.