Regulatory Mechanisms for Resolution of Inflammatory Microvascular Leakage

描述(由申请人提供)：微血管高通透性是93%重症监护患者出现的全身性炎症反应综合征(SIRS)的特征。全身炎症反应综合征使创伤患者的复苏严重复杂化，并使临床结果恶化，常常进展为多器官衰竭。据估计，美国每年与SIRS相关的医疗成本负担为167亿美元。尽管对炎症诱导的微血管高通透性的了解取得了进展，但炎症刺激后微血管通透性恢复到正常水平的机制尚不清楚。此外，一旦炎症级联反应已经启动，现有的临床治疗方法对于恢复微血管屏障的完整性并不有效。我们提出了一种新的信号机制范例，负责创伤引起的炎症后微血管完整性的恢复。我们的初步数据显示，在失血性休克的大鼠模型中，给予一种多胺包被的、细胞可穿透的RND3，一种Rho家族小GTP酶，可以降低微血管的高通透性。我们还用内皮细胞的活细胞成像观察到，片状脂膜的形成和更新代表了一种以前未被描述的细胞行为，对正常的内皮屏障完整性很重要。我们假设RND3通过抑制RhoA/ROCK介导的细胞收缩和激活rac1介导的增强细胞间连接完整性来促进炎症攻击后内皮屏障的恢复。我们的具体目标是：1)验证RND3的丝氨酸磷酸化和膜靶向RND3是降低微血管高通透性所必需的预测；2)测试RND3促进岩石介导的细胞收缩的负反馈抑制，从而增强内皮屏障完整性的预测；3)测试RND3促进rac1介导的片状脂膜的形成和VE-钙粘素在连接处的稳定，促进内皮屏障完整性的预测。提出的新的综合方法利用改进的和相关的失血性休克大鼠模型，结合活体肠系膜微循环的活体显微镜和分离的小静脉方法来评估微血管的通透性。我们将通过使用一种创新的方法将RND3蛋白传递到肠系膜微循环来探索RND3的核心作用。培养的内皮细胞单层通透性模型、活体内皮细胞内GFP-肌动蛋白和GFP-VE-钙粘蛋白的动态成像，以及生化研究将支持体内和分离小静脉的研究。这项研究的结果将使我们能够发展一种新的理论，即炎症期间内皮屏障功能如何恢复，这将为新的治疗方法奠定基础。发现可用于解决微血管高通透性的靶点将使创伤患者的治疗发生革命性变化，并将为治疗与多种疾病相关的浮肿创造新的机会。

DESCRIPTION (provided by applicant): Microvascular hyperpermeability is a hallmark of the systemic inflammatory response syndrome (SIRS) present in 93% of intensive care patients. SIRS severely complicates resuscitation of trauma victims and worsens clinical outcomes, frequently progressing to multiple organ failure. The estimated annual US health care cost burden associated with SIRS is $16.7 billion. Despite advances in understanding of inflammation- induced microvascular hyperpermeability, the mechanisms that restore microvascular permeability to normal following an inflammatory challenge are unknown. Moreover, existing clinical treatments are not effective for restoration of microvascular barrier integrity once the inflammatory cascade has been initiated. We propose a novel paradigm of signaling mechanisms responsible for restoration of microvascular integrity following inflammation caused by traumatic injury. Our preliminary data show that administration of a polyamine-coated, cell permeable form of Rnd3, a Rho family small GTPase, reduces microvascular hyperpermeability in a rat model of hemorrhagic shock. We also have observed with live cell imaging of endothelial cells that lamellipodia formation and turnover represent a previously uncharacterized cell behavior important for normal endothelial barrier integrity. We hypothesize that Rnd3 promotes endothelial barrier restoration after inflammatory challenges by inhibiting RhoA/ROCK-mediated cell contraction and activating Rac1-mediated enhancement of intercellular junction integrity. Our specific aims are to: 1) Test the prediction that Ser phosphorylation and membrane targeting of Rnd3 are required for Rnd3 to reduce microvascular hyperpermeability; 2) To test the prediction that Rnd3 promotes negative feedback inhibition of ROCK-mediated cell contraction, which enhances endothelial barrier integrity; 3) To test the prediction that Rnd3 enhances Rac1-mediated lamellipodia formation and stabilization of VE-cadherin at junctions, promoting endothelial barrier integrity. The proposed novel, integrated approach capitalizes on a refined and relevant rat model of hemorrhagic shock combined with intravital microscopy of the in vivo mesenteric microcirculation and isolated venule methods to assess microvascular permeability. We will explore the central role of Rnd3 by employing an innovative method to deliver cell permeable Rnd3 protein to the mesenteric microcirculation. Cultured endothelial cell monolayer permeability models, imaging of GFP-actin and GFP-VE-cadherin dynamics in live endothelial cells, and biochemical studies will support the in vivo and isolated venule studies. The results of this study will enable us to develop a new theory of how endothelial barrier function can be restored during inflammation, which will serve as the foundation for novel therapies. Discovery of targets that can be used to resolve microvascular hyperpermeability will revolutionize the treatment of trauma patients, and will also create new opportunities to treat edema associated with a wide range of diseases.