Vascular Barrier Leakage in Inflammation

项目概要/摘要 血管屏障功能障碍导致血液成分异常转运到血管壁或周围 组织中的炎症是创伤、脓毒症、动脉粥样硬化、糖尿病和中风引起的炎性损伤的标志。 目前，没有直接靶向渗漏屏障的有效疗法，因为药物开发一直在进行。 在将细胞/动物数据转化为人类病理生理学方面存在知识差距和困难。我们 该计划通过比较分析内皮屏障的结构和功能来解决这些挑战， 炎症损伤的人和动物模型。我们在血液、血管、 界面和内皮屏障结构，旨在1）识别引起屏障的关键循环因子 渗漏及其细胞特异性产生和作用机制; 2）表征内皮表面 受体和细胞内信号，阐明它们的作用;和3）阐明细胞间的分子事件 连接，细胞骨架和糖萼，最终导致屏障开放。我们的工作一直是 在NHLBI的支持下，为开发新技术和变革性理论做出了贡献， 血管通透性我们是第一个表征内皮连接中nmMLCK信号传导的人之一。 动力学和细胞旁通透性。最近，我们报道了一种 dhhc 21介导的蛋白棕榈酰化在微血管渗漏中的新的翻译后修饰途径 以及感染和无菌损伤后白细胞-内皮细胞相互作用。基于这些令人兴奋的发现， 通过探索具有机械见解的新型诊断/治疗靶点， 将改变炎症的模式。目前的努力是针对中性粒细胞的表征， 细胞外陷阱，组蛋白和微泡，侧重于其细胞特异性的产生机制， 在微循环中发挥作用。研究正在测试棕榈酰化在囊泡生物发生中的作用， 货物组成和与内皮细胞的相互作用。这些因素的屏障破坏作用将是 揭示了内皮糖萼受体，细胞内信号转导， 和连接结构的翻译后修饰（棕榈酰化）。我们采用多方面的方法， 将创新的分子生物学和成像技术（许多是在我们的实验室开发的）结合到功能中， 临床相关条件下的血管渗透性分析。体外、离体和体内互补 体内实验设计测试药理学激活剂和抑制剂，分子操作， 在细胞-组织-机体水平上的遗传/嵌合改变。我们计划的一个独特方面在于翻译 通过对完整的功能可行的人体器官的研究所取得的影响。

PROJECT SUMMARY/ABSTRACT Vascular barrier dysfunction causes aberrant transport of blood components into the vessel wall or surrounding tissues, a hallmark of inflammatory injury in response to trauma, sepsis, atherosclerosis, diabetes, and stroke. Currently, there are no effective therapies that directly target the leaky barrier, as drug development has been hampered by knowledge gaps and difficulties in translating cell/animal data to human pathophysiology. Our program addresses these challenges via comparative analyses of endothelial barrier structure and function in human and animal models of inflammatory injury. We conduct three series of studies in the blood, blood-vessel interface and endothelial barrier structure, aimed at 1) identifying key circulating factors that cause barrier leakage and their cell-specific mechanisms of production and action; 2) characterizing endothelial surface receptors and intracellular signals that transduce their effects; and 3) elucidating molecular events in cell-cell junctions, cytoskeleton, and glycocalyx that ultimately lead to barrier opening. Our work has continuously been supported by the NHLBI contributing to the development of novel techniques and transformative theories in vascular permeability. We were among the first to characterize the nmMLCK signaling in endothelial junction dynamics and paracellular permeability during leukocyte activation. Recently, we reported the discovery of a new post-translational modification pathway, dhhc21-mediated protein palmitoylation, in microvascular leakage and leukocyte-endothelium interactions following infection and sterile injury. Built on these exciting findings, our program continues to advance by exploring novel diagnostic/therapeutic targets with mechanistic insights that will transform the paradigm of inflammation. Current efforts are directed to the characterization of neutrophil extracellular traps, histones and microvesicles, focusing on their cell-specific mechanisms of generation and function in the microcirculation. Studies are on-going to test the roles of palmitoylation in vesicle biogenesis, cargo composition and interaction with endothelial cells. The barrier-disrupting effects of these factors will be uncovered with in-depth molecular details on endothelial glycocalyx receptors, intracellular signal transduction, and post-translational modification (palmitoylation) of junction structures. We use a multifaceted approach that incorporates innovative molecular biology and imaging techniques (many developed in our lab) into functional analyses of vascular permeability under clinically relevant conditions. Complementary in vitro, ex vivo, and in vivo experiments are designed testing pharmacological activators and inhibitors, molecular manipulations, and genetic/chimeric alterations at cell-tissue-body levels. A unique aspect of our program lies in the translational impact achieved through the studies with intact functionally viable human organs.