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：
10457247
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/10457247
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 Lead 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 Pharmacology Phosphorylation Plasma Platelet Activation Play Proteins Pulmonary Edema Pulmonary Inflammation Respiratory Failure Role S-nitro-N-acetylpenicillamine SNAP receptor Sepsis Severities Signal Transduction Testing The Sun Thrombosis Time Up-Regulation Vascular Diseases cadherin 5 design endothelial dysfunction enhancer-binding protein AP-2 gain of function hypercholesterolemia in vivo innovation insight interest interstitial knock-down lung injury microbial mortality mouse model novel novel strategies novel therapeutic intervention organ injury 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.

项目概要急性呼吸窘迫综合征（ARDS）是危重患者呼吸衰竭的常见原因，每年约有 75000 人死亡和 360 万住院天数。现有的范式是 ARDS 结果肺内皮广泛功能障碍，导致微血管血栓形成、间质性血栓形成水肿、肺泡充盈和呼吸衰竭，尽管这些机制导致内皮功能障碍仍然未知。该提议源于将 Latexin 的上调与内皮细胞联系起来的新观察肺功能障碍。我们发现健康的肺内皮细胞表达低水平的乳胶蛋白，而对湍流或细菌 LPS 的反应显着增加。此外，我们发现内皮屏障通过减少 LPS 暴露的肺内皮细胞中 Latexin 的表达来增强功能，并且总体而言小鼠乳胶素缺乏可减少脂多糖引起的肺水肿、肺部炎症和死亡率。从机制上讲，我们发现 Latexin 通过复杂的机制介导其作用，包括与酶 Src 激酶并促进其膜易位和粘着蛋白 VE-钙粘蛋白的磷酸化路口 (AJ)。此外，质谱分析显示 Latexin 与其他几种物质存在物理相互作用。蛋白质，包括网格蛋白和 AP2，表明在内吞运输中发挥作用，以及 Syntaxin-3 和 SNAP3，表明在胞吐作用中发挥作用。与后一种机制一致，我们发现 Latexin 缺乏减少了肺内皮分泌冯维伦布兰德因子。综上所述，这些观察结果使我们提出以下关于 Latexin 在肺内皮生物学中的作用和发病机制的中心假设 ARDS 中的内皮功能障碍。我们假设 Latexin 在驱动激动剂中发挥致病作用诱导肺内皮功能障碍，抑制其与其他关键蛋白质的相互作用将减轻肺水肿、肺部炎症和微血管血栓并发症的严重程度在实验诱发的 ARDS 中。为了检验这个假设，我们提出了 3 个独立但机械的假设链接的具体目标。在目标 1 中，我们将确定内皮特异性删除 Latexin 可增强内皮屏障保护并减少小鼠微血管血栓形成和 LPS 死亡率。在目标 2 中，我们将描述 Latexin 调节 VE-钙粘蛋白膜生物利用度的分子机制通过对相关基因进行各种体外和体内功能丧失和获得功能研究来研究内皮 AJ 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。在目标 3 中，我们将描述 Latexin 介导内皮细胞分泌 vWF 的分子机制，重点关注其在 SNARE 复合体形成中的作用。总的来说，该提案将建立 Latexin 的机制调节肺内皮的关键病理行为，并为开发新的治疗方法提供方向治疗肺部炎症性血管疾病（例如 ARDS）的方法。