KRIT1/Rap1 Regulation of Vascular Homeostasis

项目摘要。血管通透性是组织动态平衡的重要因素 炎症反应。在炎症过程中，内皮细胞的变化导致富含蛋白质的液体局部渗漏。 以及循环白细胞的募集和激活，伴随着细胞间连接的破裂和 屏障功能下降。这些事件也是许多疾病中可见的内皮功能障碍的特征 状态，包括关节炎、动脉粥样硬化、阿尔茨海默病和肿瘤血管生成。尽管有很多信号 这些通路有助于调节血管通透性，但我们仍不清楚这些通路是如何发挥作用的 在细胞和组织中协调。KRIT1是多个信号通路的中央调节者，参与了 血管通透性的调节，因此被定位为控制内皮细胞程度的变阻器。 对炎症刺激的激活和/或特异性反应。我们认为，对活性氧的调节 KRIT1的物种(ROS)是KRIT1的S对内皮细胞黏附连接稳定性的影响与KRIT1之间的关键连锁 血管通透性。此外，更多的研究表明，KRIT1在造血细胞中的表达是 KRIT1杂合子小鼠的通透性增加所需的，这表明KRIT1在 内皮细胞和白细胞都依赖于通透性调节。我们将在一系列测试中测试我们的总体假设 目标相辅相成。首先，我们将评估ROS是否是内皮细胞-细胞接触丧失所必需的 通过测量ROS依赖的KRIT1耗竭细胞和动物的完整性和增加血管通透性 体内外内皮屏障功能和微血管通透性的变化。第二，我们将确定 KRIT1限制内皮细胞产生活性氧的机制(S)，使用一系列 细胞培养实验旨在评估KRIT1通过限制ROS的产生来限制ROS产生这一新假设 RAP1与NADPH氧化酶的相互作用。最后，在目标3中，我们将探索造血的贡献 KRIT1对血管通透性的调节。我们建立了细胞培养和动物模型 有必要在分子水平上研究KRIT1在这一过程中的作用，并在 复杂的环境中完整的血管网络，因此我们处于独特的位置来定义相关的 并探索控制体内血管通透性调节协调的信号优先级。

Project Summary. Vascular permeability is an important element of tissue homeostasis and the normal inflammatory response. During inflammation, changes in the endothelium lead to localized leakage of protein-rich fluid and recruitment and activation of circulating leukocytes, accompanied by a breakdown of intercellular junctions and a decrease in barrier function. These events are also characteristic of the endothelial dysfunction seen in many disease states, including arthritis, atherosclerosis, Alzheimer's disease, and tumor angiogenesis. Though many signaling pathways contribute to the regulation of vascular permeability, we still do not understand how these pathways are coordinated in cells and tissues. KRIT1 is a central regulator of multiple signaling pathways involved in the regulation of vascular permeability, and as such, is positioned to act as a rheostat to control the degree of endothelial activation and/or specific responses to inflammatory stimuli. We propose that the regulation of reactive oxygen species (ROS) by KRIT1 is the key linkage between KRIT1's effects on endothelial adherens junction stability and vascular permeability. Moreover, additional studies have revealed that KRIT1 expression in hematopoietic cells is required for the increase in permeability in KRIT1 heterozygous mice, suggesting that KRIT1 plays a novel role in both endothelial- and leukocyte-dependent regulation of permeability. We will test our overall hypothesis in a series of complementary aims. First, we will assess whether ROS are required for the loss of endothelial cell-cell contact integrity and increased vessel permeability in KRIT1 depleted cells and animals by measuring ROS-dependent changes in endothelial barrier function and microvessel permeability in vitro and in vivo. Second, we will determine the mechanism(s) by which KRIT1 limits reactive oxygen species production in endothelial cells, using a series of cell culture experiments designed to evaluate the novel hypothesis that KRIT1 limits ROS production by restricting the interaction of Rap1 with NADPH oxidase. Finally, in Aim 3 we will explore the contribution of hematopoietic KRIT1 to the regulation of vascular permeability. We have established the cell-culture and animal models necessary to examine the role of KRIT1 in this process at the molecular level, and authenticate these findings in the complex environment of the intact vascular network, thus we are uniquely positioned to define the relevant pathways and explore signaling priorities that control the coordination of vascular permeability regulation in vivo.