Vascular Barrier Leakage in Inflammation

项目摘要/摘要 血管屏障功能障碍导致血液成分异常地输送到血管壁或周围 组织，这是创伤、败血症、动脉粥样硬化、糖尿病和中风时炎性损伤的标志。 目前，还没有直接针对渗漏屏障的有效疗法，就像药物开发那样 受制于知识空白和将细胞/动物数据转换为人类病理生理学的困难。我们的 计划通过对比分析血管内皮细胞屏障的结构和功能来应对这些挑战 炎症性损伤的人和动物模型。我们对血液、血管进行了三系列研究 界面和内皮屏障结构，旨在1)识别导致屏障的关键循环因素 渗漏及其细胞特有的产生和作用机制；2)内皮细胞表面的特征 转导其作用的受体和细胞内信号；3)阐明细胞-细胞内的分子事件 最终导致屏障开放的连接、细胞骨架和糖基化。我们的工作一直是 在NHLBI的支持下，促进了新技术和变革性理论的发展 血管通透性。我们是首批研究内皮细胞连接中nmMLCK信号的人之一。 白细胞激活过程中的动力学和细胞旁通透性。最近，我们报道发现了一种 微血管渗漏中新的翻译后修饰途径--dhc21介导的蛋白质棕榈酰化 感染和无菌损伤后白细胞与内皮细胞的相互作用。基于这些令人兴奋的发现，我们的 计划继续推进，探索具有机械洞察力的新诊断/治疗靶点 将改变炎症的模式。目前的研究方向是中性粒细胞的特性。 细胞外陷阱、组蛋白和微囊泡，重点研究它们特定的细胞产生和 在微循环中发挥作用。正在进行的研究正在测试棕榈酰化在囊泡生物发生中的作用， 货物组成及其与内皮细胞的相互作用。这些因素的障碍破坏效应将是 揭示了内皮细胞糖基化受体、细胞内信号转导、 和连接结构的翻译后修饰(棕榈酰化)。我们使用多方面的方法， 将创新的分子生物学和成像技术(许多是我们实验室开发的)融入到功能 临床相关条件下的血管通透性分析。体外、体外和体内互补 活体实验旨在测试药理激活剂和抑制剂、分子操作和 细胞-组织-身体水平上的遗传/嵌合改变。我们节目的一个独特之处在于翻译 通过对具有完整功能的人体器官进行研究而取得的影响。

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.