Microvascular Barrier Dysfunction in Thermal Injury

描述（由申请人提供）：微血管屏障损伤是与创伤和炎症性疾病相关的重要问题。在严重烧伤的患者中，水肿不仅发生在局部，而且发生在远离伤口的组织中，导致低血容量性休克和包括肠道和肺在内的多个器官的功能障碍。系统性炎症的终末器官效应尚未在细胞或分子水平上得到很好的表征。这个长期项目的目标是阐明烧伤后微血管通透性增高的内皮特异性机制。我们在上一个资助期的工作导致了独特的实验模型和成像技术的发展，使定量分析热损伤啮齿动物模型中的微血管功能。这些研究还揭示了烧伤引起的信号传导和内皮屏障的结构变化，其特征在于细胞骨架收缩和细胞-细胞连接开放。在这一竞争性的更新申请中，我们计划将调查扩展到对内皮屏障分子的更深入的研究，重点关注我们新发现的棕榈酰化介导细胞旁通透性的途径。提出了三个具体目标：1）建立棕榈酰化在烧伤诱导的微血管损伤中的病理生理学重要性和功能作用; 2）表征棕榈酰化酶（DHHC PAT）介导的体内和体外渗透性反应的分子基础; 3）确定调节微血管渗透性的内皮细胞-细胞粘附连接屏障中棕榈酰化的分子靶点。待检验的中心假设是，烧伤后激活的内皮棕榈酰化通过促进β-连环蛋白从细胞-细胞连接处隔离而促成微血管屏障衰竭的发病机制，β-连环蛋白是通过棕榈酰化的药理学或分子抑制而减弱的终点细胞反应。这一新的和机制的途径将在一系列的互补研究中进行评估，结合在体内微循环分析与孤立的微血管实验和培养的内皮细胞测定。将开发和测试创新的实验模型和分子工具。来自该项目的数据可能会导致创伤微血管功能的病理生理调节的新理论。该研究也有可能为炎症性疾病确定新的治疗或诊断靶点。

DESCRIPTION (provided by applicant): Microvascular barrier injury represents a significant problem associated with trauma and inflammatory disease. In patients with major burns, edema occurs not only locally but also in tissues remote from the wound, leading to hypovolemic shock and dysfunction of multiple organs including the gut and lungs. The end-organ effects of systemic inflammation have not been well characterized at the cellular or molecular level. The goal of this long- standing project is to elucidate the endothelial-specific mechanisms of microvascular hyperpermeability following burns. Our work during the previous funding period has led to the development of unique experimental models and imaging techniques that enable quantitative analyses of microvascular function in rodent models of thermal injury. The studies also revealed burn-elicited signaling and structural changes in the endothelial barrier characterized by cytoskeleton contraction and cell-cell junction opening. In this competing renewal application, we plan to extend the investigation to a more in-depth examination of endothelial barrier molecules, focusing on our newly discovered pathway of palmitoylation in mediating paracellular permeability. Three specific aims are proposed: 1) to establish the pathophysiological importance and functional role of palmitoylation in burn-induced microvascular injury; 2) to characterize the molecular basis of palmitoylating enzyme (DHHC PAT)-mediated permeability responses in vivo and in vitro; and 3) to identify molecular targets of palmitoylation in endothelial cell-cell adherens junction barrier that regulate microvascular permeability. The central hypothesis to be tested is that endothelial palmitoylation activated following burn injury contributes to the pathogenesis of microvascular barrier failure by promoting ß-catenin sequestration from cell-cell junctions, an end-point cellular response attenuated by pharmacological or molecular inhibition of palmitoylation. This novel and mechanistic pathway will be evaluated in a series of complementary studies integrating in vivo microcirculation analyses with isolated microvessel experiments and cultured endothelial cell assays. Innovative experimental models and molecular tools will be developed and tested. Data derived from this project may lead to a new theory about the pathophysiological regulation of microvascular function in trauma. The study also has the potential to identify novel therapeutic or diagnostic targets for inflammatory diseases.