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

• 批准号: 10677334
• 负责人: Ross S Summer
• 金额: $ 10.61万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10677334
• 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

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）是危重病人呼吸衰竭的常见原因， 约75000例死亡/年和360万个住院日。现有的范式是， 肺内皮广泛功能障碍，导致微血管血栓形成，间质性 水肿、肺泡灌流和呼吸衰竭，尽管导致内皮功能障碍的机制 仍然未知。这一建议源于新的观察结果，将Latexin的上调与内皮细胞的凋亡联系起来。 肺功能障碍我们发现，健康的肺内皮细胞表达低水平的Latexin，而 对湍流或细菌LPS的响应显著增加。此外，我们发现内皮屏障 通过降低LPS暴露的肺内皮细胞中Latexin的表达， 在小鼠中缺乏Latexin可降低LPS诱导的肺水肿、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激酶和促进其膜转位和粘附分子上VE-钙粘蛋白的磷酸化 连接（AJ）。此外，质谱显示Latexin与几种其他的 蛋白质，包括网格蛋白和AP 2，表明在内吞运输中的作用，以及突触融合蛋白3和SNAP 3， 表明在胞吐作用中起作用。与后一种机制一致，我们发现Latexin缺乏减少了 从肺内皮分泌von Willenbrand因子。综合起来，这些观察使我们提出 以下关于乳胶蛋白在肺内皮生物学中的作用和肺动脉高压的发病机制的中心假设， 内皮功能障碍。我们假设Latexin在驱动激动剂中起致病作用- 诱导的肺内皮功能障碍，抑制其与其他关键蛋白的相互作用， 减轻肺水肿、肺部炎症和微血管血栓并发症的严重程度 实验性诱导的ARDS为了检验这一假设，我们提出了3个独立的，但机械 联系具体目标。在目的1中，我们将建立内皮特异性Latexin缺失增强内皮细胞的细胞凋亡。 内皮屏障保护和减少微血管血栓形成和死亡率的LPS小鼠。在目标2中， 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研究， Src介导的VE-钙粘蛋白磷酸化和网格蛋白介导的内皮细胞内吞作用。在目标3中，我们 描述Latexin介导内皮细胞分泌vWF的分子机制，重点关注 在SNARE复合物形成中的作用。总的来说，这项提案将建立一种机制， 调节肺内皮细胞的关键病理行为，为开发新的治疗药物提供方向。 肺的炎症性血管疾病，如ARDS的方法。