Targeting Latexin Signaling for Endothelial Barrier Dysfunction in Inflammatory Lung Injury

靶向乳胶蛋白信号传导治疗炎症性肺损伤中的内皮屏障功能障碍

基本信息

批准号：
10676165
负责人：
Ross S Summer
金额：
$ 64.89万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10676165
关键词：
Accounting Acute Lung Injury Acute Respiratory Distress Syndrome Adherens Junction Agonist Alveolar Attenuated Automobile Driving Bacterial Pneumonia Behavior Binding Biological Availability Biology Blood Platelets Blood Vessels Blood flow Cells Cessation of life Clathrin Coagulation Process Complex Critical Illness Data Data Set Development Diabetes Mellitus Edema Endocytosis Endothelial Cells Endothelium Enzymes Exocytosis Experimental Models Exposure to Extravasation Floods Fostering Functional disorder Genes Goals Grant Human In Situ In Vitro Inflammation Inflammatory Injury Investigation Knockout Mice Knowledge Laboratories Length of Stay Link Lipopolysaccharides Lung Mass Spectrum Analysis Mediating Membrane Modeling Molecular Morbidity - disease rate Mus Obesity Organ PC3.1 antigen Pathogenesis Pathogenicity Pathologic Phosphorylation Plasma Platelet Activation Play Proteins Pulmonary Edema Pulmonary Inflammation Respiratory Failure Role SNAP receptor SNAP23 gene Sepsis Severities Signal Transduction Testing Thrombosis Up-Regulation Vascular Diseases aspirate cadherin 5 design endothelial dysfunction enhancer-binding protein AP-2 gain of function hypercholesterolemia in vivo innovation insight interest interstitial knock-down loss of function lung injury microbial mortality mouse model novel novel strategies novel therapeutic intervention organ injury pharmacologic prevent pulmonary vascular disorder respiratory response shear stress src-Family Kinases syntaxin 3 thrombotic complications tool trafficking transcriptome sequencing translational progress von Willebrand Factor

项目摘要

Project Summary The Acute Respiratory Distress Syndrome (ARDS) is a common cause of respiratory failure in the critically ill, accounting for ~75000 deaths/year and 3.6 million hospital days. The existing paradigm is that the ARDS results from widespread dysfunction of the pulmonary endothelium, leading to microvascular thrombosis, interstitial edema, alveolar flooding and respiratory failure, although the mechanisms leading to endothelial dysfunction remain unknown. This proposal arises from novel observations linking the upregulation of Latexin to endothelial dysfunction in the lung. We found that healthy lung endothelium expresses low levels of Latexin whereas levels markedly increase in response to turbulent flow or bacterial LPS. Further, we found that endothelial barrier function was enhanced by reducing Latexin expression in LPS-exposed lung endothelial cells and that global deficiency of Latexin in mice reduced LPS-induced pulmonary edema, lung inflammation and mortality. Mechanistically, we found that Latexin mediated its effects through complex mechanisms, including binding to the enzyme Src kinase and facilitating its membrane translocation and phosphorylation of VE-cadherin at adherens junctions (AJ). Additionally, mass spectroscopy revealed that Latexin physically interacts with several other proteins, including clathrin and AP2, suggesting a role in endocytic trafficking, and syntaxin-3 and SNAP3, suggesting a role in exocytosis. Consistent with this latter mechanism, we found that Latexin deficiency reduced von Willenbrand factor secretion from lung endothelium. Taken together, these observations lead us to propose the following central hypothesis regarding the role of Latexin in lung endothelial biology and the pathogenesis of endothelial dysfunction in ARDS. We hypothesize that Latexin plays a pathogenic role in driving agonist- induced endothelial dysfunction in the lung and that inhibiting its interactions with other key proteins will reduce the severity of pulmonary edema, lung inflammation and microvascular thrombotic complications in experimentally-induced ARDS. To test this hypothesis, we propose 3 independent but mechanistically linked Specific Aims. In Aim 1, we will establish that endothelial-specific deletion of Latexin enhances endothelial barrier protection and reduces microvascular thrombosis and mortality to LPS in mice. In Aim 2, we will delineate the molecular mechanisms by which Latexin regulates VE-cadherin membrane bioavailability at endothelial AJs by performing various in vitro and in vivo loss- and gain-of-function studies for genes linked to Src-mediated VE-cadherin phosphorylation and clathrin-mediated endocytosis in ECs. In Aim 3, we will delineate the molecular mechanisms by which Latexin mediates vWF secretion from endothelial cells, focusing on its role in SNARE complex formation. In toto, this proposal will establish the mechanisms by which Latexin regulates key pathological behaviors in lung endothelium and provide direction for developing novel therapeutic approaches for inflammatory vascular diseases of the lung, such as the ARDS.

项目摘要急性呼吸窘迫综合征（ARDS）是重病呼吸衰竭的常见原因，约75000人/年死亡和360万医院的核算。现有的范式是ARDS结果肺部内皮的广泛功能障碍，导致微血管血栓形成，间隙水肿，肺泡洪水和呼吸衰竭，尽管导致内皮功能障碍的机制仍然未知。该提议源于将乳胶蛋白上调与内皮上调联系起来的新颖观察结果肺部功能障碍。我们发现健康的肺内皮表达低水平的乳胶素，而水平响应湍流或细菌LP的响应显着增加。此外，我们发现内皮障碍通过降低暴露于LPS的肺内皮细胞中的乳胶素表达来增强功能小鼠乳胶蛋白的缺乏降低了LPS诱导的肺水肿，肺部炎症和死亡率。从机械上讲，我们发现乳胶素通过复杂机制介导了其作用，包括与酶SRC激酶并促进其膜的膜易位和在粘附蛋白上的磷酸化和磷酸化连接（AJ）。此外，质谱法表明乳胶素在物理上与其他几种相互作用蛋白质，包括网状蛋白和AP2，表明在内吞运输中起作用，以及义务素3和SNAP3，提示在胞吐作用中起作用。与后一种机制一致，我们发现乳胶缺乏率降低了 von Willenbrand因子因肺内皮的分泌。综上所述，这些观察结果使我们提出了关于乳胶素在肺内皮生物学中的作用以及以下中心假设 ARDS中的内皮功能障碍。我们假设乳胶素在驱动激动剂中起致病作用肺部诱导的内皮功能障碍并抑制其与其他关键蛋白的相互作用减少肺水肿，肺部炎症和微血管血栓形成并发症的严重程度在实验诱导的ARD中。为了检验该假设，我们独立提出了3个，但机械机械地提出了链接的特定目标。在AIM 1中，我们将确定乳胶的特定于内皮的缺失增强内皮屏障保护并降低了小鼠的微血管血栓形成和死亡率。在AIM 2中，我们将描述乳胶素调节VE-钙粘蛋白膜生物利用度的分子机制内皮AJS通过对连接的基因进行各种体外和体内损失和功能性研究研究在EC中，SRC介导的VE-钙粘蛋白磷酸化和网格蛋白介导的内吞作用。在AIM 3中，我们将描述乳胶介导从内皮细胞中介导的VWF分泌的分子机制，聚焦关于其在军鼓复合物形成中的作用。在Toto中，该提案将确定乳胶蛋白的机制调节肺内皮中的关键病理行为，并为发展新型治疗提供方向肺部炎症血管疾病的方法，例如ARDS。