Endotoxin TLR4 Signaling Regulates Lung Endothelial Paracellular Pathway

描述（由申请方提供）：革兰氏阴性细菌血流感染及其伴随的内毒素血症与血管渗漏综合征相关，包括危及生命的急性呼吸窘迫综合征（ARDS）。Toll样受体（TLR）-4是内毒素或细菌脂多糖（LPS）的受体之一，由内皮细胞（EC）表达。虽然对TLR 4信号传导有很多了解，但它如何与肺微血管内皮细胞旁通路的开放相结合尚不清楚。我们已经证明，LPS增加EC蛋白的酪氨酸磷酸化富集到EC-EC边界，并诱导肌动蛋白重组。先前的PTK抑制和F-肌动蛋白稳定各自保护免受LPS诱导的屏障破坏。我们现在提供的数据确定的粘附小带（ZA）蛋白，VE-钙粘蛋白，3-连环蛋白，和p120 ctn，作为底物的LPS诱导的酪氨酸磷酸化，和激活一个或多个src家族PTK（SFK）作为一个先决条件的LPS诱导的开放的细胞旁途径。我们提出以下具体目标：1。鉴定TLR 4应答性、MyD 88依赖性信号传导/接头分子，其将LPS刺激与SFK激活偶联，并打开人肺微血管内皮细胞（HMVEC-Ls）中的细胞旁途径。2.确定LPS诱导的ZA多蛋白复合物内蛋白质-蛋白质相互作用的变化，ZA-肌动蛋白细胞骨架连接，以及相对的VE-钙粘蛋白胞外域之间的变化。3.确定SFK，LPS通过该SFK增加连接和/或肌动蛋白细胞骨架蛋白的酪氨酸磷酸化，诱导ZA重组，并打开内皮细胞旁途径。4.为了确定PTP是否响应于LPS刺激，反调节SFK活化、ZA蛋白的酪氨酸磷酸化增加、ZA多蛋白复合物的重组和/或细胞旁途径的打开。了解LPS调节EC-EC嗜同性粘附和细胞旁途径的机制不仅对脓毒症相关的急性肺损伤和血管内液体和大分子进入组织的运动有意义，而且对中性粒细胞外渗也有意义。公共卫生相关性：许多细菌会导致危及人类生命的感染，释放出一种称为内毒素的物质，这种物质可以与血管细胞上的受体结合。当内毒素与这种受体结合时，它可以触发这些细胞之间的空间的打开和体液泄漏到组织中，包括肺的空气空间。了解导致这种高死亡率的生化事件，血管渗漏综合征应提供潜在的治疗干预目标。

DESCRIPTION (provided by applicant): Gram-negative bacterial bloodstream infections and their attendant endotoxemia are associated with vascular leak syndromes, including the life-threatening, Acute Respiratory Distress Syndrome (ARDS). One receptor for endotoxin or bacterial lipopolysaccharide (LPS), Toll-like receptor (TLR)-4, is expressed by endothelial cell (EC)s. Although much is known about TLR4 signaling, how it is coupled to opening of the pulmonary microvascular endothelial paracellular pathway is unknown. We have demonstrated that LPS increases tyrosine phosphorylation of EC proteins enriched to EC-EC boundaries and induces actin reorganization. Prior PTK inhibition and F-actin stabilization each protects against LPS-induced barrier disruption. We now present data identifying the zonula adherens (ZA) proteins, VE-cadherin, 3-catenin, and p120ctn, as substrates for LPS- induced tyrosine phosphorylation, and activation of one or more src family PTK (SFK)s as a prerequisite for LPS-induced opening of the paracellular pathway. We propose the following SPECIFIC AIMS: 1. To identify the TLR4-responsive, MyD88-dependent signaling/adapter molecule(s) that couple the LPS stimulus to SFK activation and opening of the paracellular pathway in human lung microvascular endothelia (HMVEC-Ls). 2. To define LPS-induced changes in protein-protein interactions within the ZA multiprotein complex, the ZA-actin cytoskeletal linkage, and between opposing VE-cadherin ectodomains. 3. To identify the SFK(s) through which LPS increases tyrosine phosphorylation of junctional and/or actin cytoskeletal proteins, induces ZA reorganization, and opens the endothelial paracellular pathway. 4. To establish whether PTP¿ counter-regulates SFK activation, increased tyrosine phosphorylation of ZA proteins, reorganization of the ZA multiprotein complex, and/or opening of the paracellular pathway, in response to the LPS stimulus. Understanding the mechanism(s) through which LPS regulates EC-EC homophilic adhesion and the paracellular pathway has implications not only for sepsis-associated acute lung injury and the movement of intravascular fluid and macromolecules into tissues but for neutrophil extravasation as well. PUBLIC HEALTH RELEVANCE: Numerous bacteria that cause life-threatening infections in humans, release a substance called endotoxin, which can bind to a receptor on the cell that line blood vessels. When endotoxin engages this receptor, it can trigger opening of the spaces between these cells and leakiness of body fluids into the tissues, including the air-spaces of the lung. Understanding the biochemical events that lead to this high-mortality, vascular leak syndrome should offer potential targets for therapeutic interventions.