Hypoxia-induced Endothelial Barrier Dysfunction

缺氧引起的内皮屏障功能障碍

• 批准号: 6858931
• 负责人: Usamah S Kayyali
• 金额: $ 32.6万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-07 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6858931
• 关键词: biological signal transduction; cytoskeleton; heat shock proteins; hypoxia; immunoprecipitation; laboratory mouse; microcirculation; mitogen activated protein kinase; myosins; phosphorylation; protein localization; protein structure function; pulmonary edema; serine threonine protein kinase; two dimensional gel electrophoresis; vascular endothelium

DESCRIPTION (provided by applicant): Non-cardiac pulmonary edema is a frequent affliction in pulmonary medicine and is caused by endothelial barrier dysfunction. It is now recognized that this dysfunction may be initiated by programmed cell signaling as opposed to non-specific disruption of the vascular barrier. Since the endothelial barrier is known to be altered by exposure to hypoxia leading to pulmonary edema, we have utilized a rat pulmonary artery microvascular endothelial cell (EC) monolayer culture model to study the effects of hypoxia on EC barrier function. Preliminary studies have led to the hypothesis that exposure to hypoxia initiates cell signaling through the p38 MAP kinase pathway involving MK2 and HSP27 activation that leads to actin cytoskeleton and hic-5 redistribution. These signaling events are associated with an alteration in biomechanical "tethering" and "stiffness" and enhanced permeability of the EC monolayer. "Tethering" may be regulated by the p38 MAP kinase pathway while "stiffness" is regulated by Rho/Rho kinase and myosin light chain phosphorylation. We plan in the present proposal to study these responses more thoroughly in the rat pulmonary artery microvascular EC monolayer and to perform confirmatory studies in similar cells from human pulmonary microvasculature. In addition, we will investigate these signaling pathways in mice in vivo using pharmacological inhibitors, as well as mice in which the expression of MK2, a key kinase in the p38 pathway, has been "knocked-out". Specifically, we plan to: Aim 1: Define signaling pathways important for mediating cytoskeletal changes in endothelial cell monolayers exposed to hypoxia; Aim 2: Determine the relevance of the biomechanical endpoints "stiffness" and "tethering", which increase with exposure to hypoxia, to physiological determinants of endothelial barrier function; and Aim 3: Evaluate the signaling events that lead to increased pulmonary endothelial permeability in hypoxia in vivo. The findings of this study are expected to further our understanding of mechanisms involved in the development of endothelial barrier dysfunction, in general, and to suggest targets for treatment of pulmonary edema.

描述(申请人提供)：非心性肺水肿是一种常见的肺部疾病，由内皮屏障功能障碍引起。现在人们认识到，这种功能障碍可能是由程序性细胞信号转导启动的，而不是血管屏障的非特异性破坏。我们利用大鼠肺微血管内皮细胞(EC)单层培养模型，研究了缺氧对EC屏障功能的影响。初步研究认为，缺氧通过p38 MAP激酶途径启动细胞信号转导，涉及MK2和HSP27的激活，导致肌动蛋白细胞骨架和HIC-5的重新分布。这些信号事件与生物力学“系绳”和“僵硬”的改变以及EC单层通透性的增强有关。“拴系”可能受p38 MAP激酶途径的调节，而“僵硬”则受Rho/Rho激酶和肌球蛋白轻链磷酸化的调节。在目前的方案中，我们计划在大鼠肺微血管内皮细胞单层中更深入地研究这些反应，并在来自人类肺微血管系统的类似细胞中进行验证性研究。此外，我们将在体内使用药物抑制剂研究这些信号通路，以及p38途径中的关键激酶MK2表达已被敲除的小鼠。具体地说，我们计划：目标1：确定介导缺氧下内皮细胞单层细胞骨架变化的重要信号通路；目标2：确定随着缺氧暴露而增加的生物力学终点“僵硬”和“系留”与内皮屏障功能的生理决定因素的相关性；以及目标3：评估体内缺氧时导致肺内皮细胞通透性增加的信号事件。这项研究的发现有望进一步加深我们对内皮屏障功能障碍发生机制的理解，并为肺水肿的治疗提供靶点。