Nox 4 in Endothelial Cell ROS Production, Signaling and Motility

描述（由申请人提供）：血管中活性氧（ROS）的增加与炎症、血管渗漏、动脉粥样硬化和其他心血管疾病有关。在血管内皮中，NADPH氧化酶已被确定为ROS的主要来源，ROS调节内皮细胞生长、迁移、细胞骨架组织和屏障功能的信号通路。内皮细胞NADPH氧化酶与吞噬氧化酶类似，由胞质（p47phox、p67phox和Rac1）和膜结合（gp91phox和p22phox）组分组成。近年来，已经发现了几种Nox 2 （gp91phox）的同源物，即Nox1、Nox3、Nox4和Nox5。我们和其他人发现，与Nox2相比，Nox4 mRNA在不同血管床的内皮细胞中的表达水平要高得多；然而，Nox4在ROS生成和内皮细胞功能中的作用尚不清楚。在初步实验中，我们发现Nox4不仅参与高氧和tnf α诱导的超氧化物/活性氧的产生，还参与调节运动和毛细血管形成的内皮细胞信号转导。该提案的总体假设是“Nox4在调节参与超氧化物/活性氧产生、迁移和毛细血管形成的内皮信号通路中起关键作用”。这一假设将在原代人肺内皮细胞和转基因Nox4和Nox2 （gp91phox）小鼠中进行验证，使用高氧和tnf - α作为NADPH氧化酶的调节剂。特异性目的1：表征Nox 4在HPAECs和小鼠肺ECs中的表达，并确定其在ROS生成中的作用；特异性目的2：探讨高氧诱导人肺内皮细胞Nox4表达和活化的分子机制；特异性目的3：表征高氧条件下调节Nox4依赖性内皮细胞迁移和毛细血管形成的信号通路；特异性目的4：探讨Nox4在小鼠肺损伤模型中ROS生成和血管渗漏中的作用。这些实验将为Nox4作为NADPH氧化酶的一个组成部分在调节内皮细胞ROS产生和功能中的作用提供新的见解，这可能会导致开发最小化肺损伤的治疗策略。

DESCRIPTION (provided by applicant): Increased generation of reactive oxygen species (ROS) in the vasculature has been linked to inflammation, vascular leakiness, atherosclerosis and other cardiovascular disorders. In the vascular endothelium, NADPH oxidase has been identified as a major source of ROS, which regulates signaling pathways involved in endothelial cell growth, migration, cytoskeletal organization and barrier function. The endothelial NADPH oxidase, similar to phagocytic oxidase, consists of cytosolic (p47phox, p67phox and Rac1) and membranebound (gp91phox and p22phox) components. Recently, several homologues of Nox 2 (gp91phox) namely Nox1, Nox3, Nox4 and Nox5 have been identified. We and others have found that Nox4 mRNA is expressed at much higher levels, compared to Nox2, in the endothelial cells from different vascular beds; however, the role of Nox4 in ROS production and endothelial cell functions is not well understood. In preliminary experiments, we have identified that Nox4 is not only involved in hyperoxia- and TNF-alpha-induced superoxide/ROS production but also in endothelial cell signal transduction regulating motility and capillary tube formation. The overall hypothesis of this proposal is that "Nox4 plays a key role in regulating endothelial signaling pathways involved in superoxide/ROS production, migration and capillary tube formation". This hypothesis will be tested in primary human lung endothelial cells and in genetically modified Nox4 and Nox2 (gp91phox) mice using hyperoxia- and TNF-alpha as modulators of NADPH oxidase. Specific Aim 1: To characterize expression of Nox 4 in HPAECs and mouse lung ECs and determine its role in ROS production; Specific Aim 2: To investigate molecular mechanisms of increased expression and activation of Nox4 by hyperoxia in human lung endothelial cells ; Specific Aim 3: To characterize signaling pathways that regulate Nox4 dependent endothelial cell migration and capillary tube formation in response to hyperoxia; Specific Aim 4: To investigate the role of Nox4 in ROS generation and vascular leakiness in an in vivo murine model of lung injury. These experiments will provide novel and new insights into the role of Nox4, as a component of NADPH oxidase, in regulating endothelial cell ROS production and function that may lead to development of therapeutic strategies minimizing lung injury.