Hypoxia-induced Endothelial Barrier Dysfunction

描述（由申请人提供）：非心源性肺水肿是肺部医学中常见的疾病，由内皮屏障功能障碍引起。现在认识到，这种功能障碍可能是由程序性细胞信号传导引发的，而不是血管屏障的非特异性破坏。由于已知缺氧会改变内皮屏障，导致肺水肿，我们利用大鼠肺动脉微血管内皮细胞（EC）单层培养模型来研究缺氧对EC屏障功能的影响。初步研究提出了这样的假设：缺氧暴露通过p38 MAP激酶途径启动细胞信号传导，涉及MK2和HSP27活化，导致肌动蛋白细胞骨架和hic-5重新分布。这些信号事件与生物力学“栓系”和“刚度”的改变以及EC单层通透性的增强有关。“栓系”可能受p38 MAP激酶途径调控，而“僵硬”则受Rho/Rho激酶和肌球蛋白轻链磷酸化调控。我们计划在大鼠肺动脉微血管EC单层中更彻底地研究这些反应，并在人肺微血管的类似细胞中进行验证性研究。此外，我们将使用药物抑制剂在小鼠体内研究这些信号通路，以及p38通路中关键激酶MK2表达被“敲除”的小鼠。具体来说，我们计划：目标1：确定在缺氧暴露的内皮细胞单层中介导细胞骨架变化的重要信号通路；目的2：确定生物力学终点“僵硬”和“栓系”与内皮屏障功能生理决定因素的相关性，这些终点随着缺氧暴露而增加；目的3：评估体内缺氧时导致肺内皮通透性增加的信号事件。这项研究的结果有望进一步加深我们对内皮屏障功能障碍发展的机制的理解，并为肺水肿的治疗提供靶点。

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.