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: 10661225
• 负责人: Ross S Summer
• 金额: $ 12.6万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10661225
• 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

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)是危重病患者呼吸衰竭的常见原因， 每年造成约75000人死亡，360万天住院。现有的模式是ARDS导致 从广泛的肺内皮功能障碍，导致微血管血栓，间质 水肿、肺泡积水和呼吸衰竭，尽管导致内皮功能障碍的机制 仍然不为人所知。这一建议源于新的观察结果，将Latexin上调与内皮细胞联系起来 肺部功能障碍。我们发现，健康的肺内皮细胞表达低水平的Latexin，而 对湍流或细菌内毒素的反应显著增加。此外，我们发现内皮屏障 通过减少脂多糖暴露的肺内皮细胞中Latexin的表达而增强功能 小鼠缺乏乳胶蛋白可减少内毒素引起的肺水肿、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 Src酶及其促进粘附区VE-钙粘附素的膜转位和磷酸化 连接点(AJ)。此外，质谱学显示，Latexin与其他几个 蛋白质，包括笼蛋白和AP2，表明在细胞内转运中发挥作用，以及Synaxin-3和SNAP3， 表明它在胞吐作用中起了作用。与后一种机制一致，我们发现Latexin缺乏症减少了 肺内皮细胞分泌von Willenbrand因子。综上所述，这些观察结果让我们提出 关于Latexin在肺内皮细胞生物学中的作用及其发病机制的以下中心假说 急性呼吸窘迫综合征的内皮功能障碍。我们假设，Latexin在驱动激动剂- 诱导的肺内皮细胞功能障碍以及抑制其与其他关键蛋白的相互作用将 减轻肺水肿、肺部炎症和微血管血栓并发症的严重程度 在实验诱导的ARDS中。为了验证这一假设，我们提出了3个独立但机械的假设 相互关联的具体目标。在目标1中，我们将建立内皮特异性Latexin删除增强 保护内皮屏障，减少小鼠微血管血栓形成和对内毒素的死亡率。在目标2中，我们 将描述Latexin调节VE-cadherin膜生物利用度的分子机制 通过对相关基因进行不同的体外和体内功能丧失和获得研究来获得内皮AJ 对Src介导的VE-钙粘蛋白磷酸化和笼蛋白介导的内皮细胞内吞作用的影响。在《目标3》中，我们将 阐明Latexin介导内皮细胞分泌vWF的分子机制 关于它在诱捕复合体形成中的作用。总之，这项提案将建立Latexin 调节肺内皮细胞的关键病理行为，为开发新的治疗方法提供方向 肺部炎症性血管疾病的治疗方法，如ARDS。