REGULATION OF ENDOTHELIAL CELL PERMEABILITY BY RHO/ROCK SIGNALING

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 微血管通透性过高是出血性休克相关炎症的标志。尽管酒精中毒导致三分之一的意外死亡，近50%的急诊室患者血液酒精浓度呈阳性，但尚未研究酒精中毒如何影响体内创伤或炎症引起的正常微血管交换或微血管渗漏。需要更好地了解微循环如何在功能上适应酒精中毒，以改善复苏和预防水肿，不受控制的炎症和感染的策略。我们实验室的研究表明，内皮细胞之间的细胞骨架和粘附连接在维持正常屏障功能和作为全身炎症过程中高通透性反应的效应器中起着重要作用。我们的初步数据表明，急性酒精暴露破坏内皮肌动蛋白细胞骨架和细胞间连接。我们还观察到，酒精给药加剧了体内对炎症刺激的高渗透性反应。这些事件似乎涉及全身和局部（内皮细胞）水平的氧化应激。因此，我们假设酒精通过引起内皮细胞氧化应激介导的细胞骨架重排，削弱连接和对炎症刺激产生过度反应，加剧失血性休克诱导的微血管渗漏。为了达到检验这一假设的具体目的，将：1）表征急性酒精中毒和失血性休克联合过程中的微血管屏障异常，这将提供目前不可用的体内数据，2）测试酒精诱导的氧化应激在酒精中毒和失血性休克联合过程中的作用，以及3）检测在酒精中毒和出血的复合过程中内皮细胞骨架破坏在微血管屏障功能障碍中的作用。这些中间目标将使用固定压力出血模型结合急性酒精给药来解决。一个综合的方法，利用在体内活体显微镜的肠系膜微循环，建立隔离灌注小静脉技术，和分子生物学方法将被用来成功地完成这项研究。从这项研究中获得的数据将提供新的见解休克时微血管渗漏的机制。这些研究的最终目的是促进发展更好的休克病人复苏方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microvascular hyperpermeability is a hallmark of inflammation associated with hemorrhagic shock. Despite the fact that alcohol intoxication contributes to one-third of accidental deaths and that nearly 50% of emergency room patients have positive blood alcohol concentrations, there have been no studies of how alcohol intoxication affects normal microvascular exchange or microvascular leak caused by trauma or inflammation in vivo. A better understanding of how the microcirculation functionally adapts to alcohol intoxication is needed to improve strategies for resuscitation and prevention of edema, uncontrolled inflammation, and infection. Studies from our laboratory have shown that the cytoskeleton and adherens junctions between endothelial cells play important roles in both the maintenance of normal barrier function and as effectors of the hyperpermeability response during systemic inflammation. Our preliminary data show that acute alcohol exposure disrupts the endothelial actin cytoskeleton and intercellular junctions. We have also observed that alcohol administration exacerbates the in vivo hyperpermeability reaction to inflammatory stimuli. These events appear to involve oxidative stress at both the systemic and local (endothelial cell) level. Thus, we hypothesize that alcohol exacerbates hemorrhagic shock-induced microvascular leakage by causing oxidative stress-mediated cytoskeletal rearrangement in endothelial cells, weakening junctions and creating an exaggerated response to inflammatory stimuli. To intermediate specific aims to test this hypothesis will be to: 1) characterize microvascular barrier abnormalities during combined acute alcohol intoxication and hemorrhagic shock, which will provide in vivo data not currently available, 2) test the role of alcohol-induced oxidative stress during combined alcohol intoxication and hemorrhagic shock, and 3) examine the role of endothelial cytoskeletal disruption in microvascular barrier dysfunction during combined alcohol intoxication and hemorrhage. These intermediate aims will be addressed using a fixed pressure hemorrhage model combined with acute alcohol administration. An integrated approach utilizing in vivo intravital microscopy of the mesenteric microcirculation, an established isolated perfused venule technique, and molecular biology methods will be used to successfully complete this study. The data obtained from this study will provide new insights into the mechanisms underlying microvascular leakage during shock. The ultimate goal of these studies is to promote development of better resuscitation methods for shock patients.