Therapeutic Mechanisms of RhoA Inhibition In Acute Lung Injury

项目总结（见说明）; 被诊断患有ARDS的受试者表现出约40%的死亡率，因此需要新的方法来管理这种严重的病症。本项目将研究G+和G-细菌诱导人内皮细胞和ALI/ARDS中内皮屏障功能障碍的全新机制。它遵循项目1中提出的关于RhoA/Rac 1失衡在内皮屏障功能障碍和ALI/ARSD中的关键作用的研究。在这里，我们专注于激活内皮RhoA，产生内皮细胞高通透性培养和增加肺毛细血管通透性，在体内。我们提出了一种新的机制，调节RhoA活性，可能是有用的管理ALI和ARDS。 来自项目1和已发表研究的初步数据表明，pp 60 src是RhoA激活的关键步骤，其导致小热休克蛋白27（hsp 27）的磷酸化，这是F-肌动蛋白应力纤维形成和内皮屏障功能障碍的主要原因。激酶，ppGOsrc是一个众所周知的热休克蛋白90（hsp 90）的客户端蛋白，我们最近发表的热休克蛋白27与热休克蛋白90免疫共沉淀。 初步数据表明，hsp 90抑制剂，17-AG降低LPS诱导的ppSOsrc激活和hsp 27磷酸化，以及LPS诱导的RhoA激活，在内皮细胞。此外，我们最近已经证明，hsp 90抑制预防和逆转LPS诱导的内皮屏障功能障碍，在文化，并减少毛细血管通透性过高，炎症，肺功能障碍和死亡率在LPS诱导的ALI小鼠模型。尽管如此，hsp 90介导的ppBOsrc、RhoA和hsp 27活化调节对G+和G-诱导的内皮屏障功能障碍，特别是人内皮细胞屏障功能的影响，以及在体内ALI的管理中的影响仍然未知。本项目将测试的假设，热休克蛋白90是一个重要的调节人内皮细胞高通透性，在体外，和急性肺损伤，在体内。我们将进一步测试的假设，热休克蛋白90发挥这些作用，部分，通过控制的命运的两个关键蛋白质（ppSOsrc，热休克蛋白27）参与RhoA的激活和信号转导。 这些研究为ALI/ARDS的治疗提供了令人兴奋的新可能性。

PROJECT SUMMARY (See instructions); Subjects diagnosed with ARDS exhibit ~40% mortality, thus demanding fresh approaches to the management of this serious condition. This Project will investigate a totally new mechanism of both G+ and G- bacteria-induced endothelial barrier dysfunction in human endothelial cells and in ALI/ARDS. It follows the studies on the crucial role RhoA/Rac1 imbalance in endothelial barrier dysfunction and ALI/ARSD, proposed in Project 1. Here we focus on the activation of endothelial RhoA that produces endothelial hyperpermeability in culture and increased pulmonary capillary permeability, in vivo. We propose a novel mechanism of regulating RhoA activity that could be useful in the management of ALI and ARDS. Preliminary data from Project 1 and published studies suggest that pp60src is a key step in RhoA activation, which leads to the phosphorylation of the small heat shock protein 27 (hsp27), a major cause of F-actin stress fiber formation and endothelial barrier dysfunction. The kinase, ppGOsrc is a well-known heat shock protein 90 (hsp90) client protein and we recently published that hsp27 co-immunoprecipitates with hsp90. Preliminary data suggest that the hsp90 inhibitor, 17-/\AG reduces both LPS-induced ppSOsrc activation and hsp27 phosphorylation, as well as LPS-induced RhoA activation, in endothelial cells. Furthermore, we have recently demonstrated that hsp90 inhibition prevents and reverses LPS-induced endothelial barrier dysfunction, in culture, and reduces capillary hyper-permeability, inflammation, lung dysfunction and mortality in a mouse model of LPS-induced ALI. Still, the effects of hsp90-mediated regulation of ppBOsrc, RhoA and hsp27 activation on G+ and G- induced endothelial barrier dysfunction, especially human endothelial cell barrier function, and in the management of ALI, in vivo, remain unknown. This project will test the hypothesis that hsp90 is an important regulator of human endothelial cell hyper-permeability, in vitro, and of ALI, in vivo. We will further test the hypothesis that hsp90 exerts these actions, in part, by controlling the fate of two key proteins (ppSOsrc, hsp27) that are involved in RhoA activation and signal transduction. These studies represent an exciting new possibility in the management of ALI/ARDS.