Caveolin-1 and NO Regulate PMN-mediated Increases in Vascular Permeability

Caveolin-1 和 NO 调节 PMN 介导的血管通透性增加

• 批准号: 7370182
• 负责人: RICHARD D MINSHALL
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-10 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7370182
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acute Lung Injury; Address; Adhesions; Albumins; Biochemical; Blood Vessels; Caveolae; Complement; Cultured Cells; Cysteine; Data; Disease; Dynamin 2; Edema; Endothelial Cells; Extravasation; Gene Deletion; Generations; ITGB2 gene; Image Analysis; Inflammation; Inflammatory; Injury; Intercellular adhesion molecule 1; Knockout Mice; Life; Lung; Mediating; Microvascular Permeability; Molecular Target; Mus; Neutrophil Activation; Oxidants; PH Domain; Pathway interactions; Permeability; Peroxonitrite; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphorylation; Plasma Proteins; Production; Proteins; Pulmonary Edema; Reaction; Regulation; Reperfusion Injury; Role; Sepsis; Signal Pathway; Signal Transduction; Small Interfering RNA; Superoxides; Testing; Vascular Permeabilities; base; caveolin 1; design; lung injury; monolayer; neutrophil; nitration; novel; protein expression; src-Family Kinases; trafficking; transcytosis; uptake

DESCRIPTION (provided by applicant): Increased lung vascular endothelial permeability induced by activation of polymorphonuclear leukocytes (PMNs) adherent to endothelial cells via ICAM-1 leads to protein-rich pulmonary edema formation and acute lung injury. However, the mechanisms responsible for PMN-induced increased endothelial permeability are incompletely understood. Our supporting data demonstrate that increased caveolae- mediated transcellular transport of albumin may contribute to increased endothelial permeability, and thus may be a factor leading to leaky lung microvessels. In this renewal application, we will address the role of PMNs in inducing the activation of caveolae-dependent transcytosis of albumin and promoting edema formation. The studies will test the hypotheses that (i) fMLP activation of PMNs stimulates caveolae- mediated albumin transcytosis in endothelial cells and mediates the increase in endothelial permeability and lung injury, and (ii) PMN activation of Src kinase in endothelial cells regulates eNOS-mediated NO production via PI3-kinase and Akt signaling and that the NO derived from PMN-endothelial interaction regulates transcellular and junctional permeability pathways to increase lung microvessel permeability. We will delineate the signaling pathways responsible for the activation of endothelial permeability and its consequences in the mechanism of lung edema formation. The approaches to be used include knockout mice, siRNA-induced suppression of protein expression, endothelial permeability assessment of transcellular and paracellular pathways, imaging analysis of caveolae mediated trafficking in live endothelial cells, and biochemical assessments of relevant signaling pathways. Studies in intact mouse lungs will be complemented by studies utilizing mouse lung endothelial cells to provide a more detailed and mechanistic understanding of the signaling basis of PMN-activated increase in endothelial permeability. These studies will provide a novel perspective into the mechanism of increased transendothelial permeability in lungs induced by PMN activation, with the hope of identifying novel molecular targets and better designing strategies directed at treating protein-rich pulmonary edemagenesis and acute lung injury. PROJECT NARRATIVE: Vascular injury and protein-rich pulmonary edema formation are life-threatening complications associated with inflammatory diseases such as ischemia/reperfusion injury and sepsis. We hypothesize that increased transendothelial albumin transport via a caveolar mechanism contributes to neutrophil-induced endothelial hyperpermeability during inflammation. This project therefore addresses the role of caveolae-mediated albumin uptake and transcytosis as compared to paracellular leakage of plasma protein in neutrophil-mediated increase in endothelial permeability and lung injury. We will assess the role of neutrophil activation of Src kinase in endothelial cells in regulating caveolin-1, dynamin-2, and eNOS function, and determine the consequences of increased albumin transport and NO production in regulating the transcellular and junctional permeability pathways.

描述（由申请方提供）：通过ICAM-1活化粘附于内皮细胞的多形核白细胞（PMN）诱导肺血管内皮通透性增加，导致富蛋白肺水肿形成和急性肺损伤。然而，负责PMN诱导的内皮细胞通透性增加的机制还不完全清楚。我们的支持性数据表明，小窝介导的白蛋白跨细胞转运增加可能导致内皮通透性增加，因此可能是导致肺微血管渗漏的一个因素。在这个更新申请中，我们将讨论中性粒细胞在诱导白蛋白小窝依赖性胞吞转运激活和促进水肿形成中的作用。这些研究将检验以下假设：（i）PMNs的fMLP激活刺激内皮细胞中小窝介导的白蛋白转胞吞作用并介导内皮渗透性和肺损伤的增加，和（ii）内皮细胞中Src激酶的PMN激活通过PI 3-激酶和Akt信号传导调节eNOS介导的NO产生，并且来自PMN-内皮相互作用调节跨细胞和交界渗透性途径以增加肺微血管渗透性。我们将描述负责激活内皮通透性的信号通路及其在肺水肿形成机制中的后果。待使用的方法包括基因敲除小鼠、siRNA诱导的蛋白质表达抑制、跨细胞和细胞旁途径的内皮渗透性评估、活内皮细胞中小窝介导的运输的成像分析以及相关信号传导途径的生化评估。完整小鼠肺的研究将通过利用小鼠肺内皮细胞的研究来补充，以提供对PMN激活的内皮通透性增加的信号基础的更详细和机制的理解。这些研究将提供一个新的视角到PMN激活诱导的肺跨内皮通透性增加的机制，希望确定新的分子靶点和更好地设计针对治疗富含蛋白质的肺水肿和急性肺损伤的策略。项目叙述：血管损伤和富含蛋白质的肺水肿形成是与炎性疾病如缺血/再灌注损伤和脓毒症相关的危及生命的并发症。我们推测，通过小窝机制增加的跨内皮白蛋白转运有助于炎症过程中嗜酸性粒细胞诱导的内皮细胞通透性增高。因此，该项目解决了小窝介导的白蛋白摄取和转胞吞作用的作用相比，细胞旁泄漏的血浆蛋白在嗜酸性粒细胞介导的内皮通透性增加和肺损伤。我们将评估中性粒细胞激活Src激酶在调节小窝蛋白-1，发动蛋白-2和eNOS功能中的作用，并确定白蛋白转运和NO产生增加在调节跨细胞和连接通透性途径中的后果。