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

描述(由申请人提供)：通过ICAM-1黏附于内皮细胞的多形核白细胞(PMN)的激活导致肺血管内皮细胞通透性增加，导致富含蛋白质的肺水肿形成和急性肺损伤。然而，PMN诱导的内皮通透性增加的机制还不完全清楚。我们的支持数据表明，小凹介导的白蛋白跨细胞转运增加可能有助于内皮通透性的增加，从而可能是导致肺微血管渗漏的一个因素。在这一更新应用中，我们将讨论PMN在诱导白蛋白的小窝依赖的细胞转运激活和促进水肿形成中的作用。这些研究将验证以下假设：(I)PMN的fMLP激活刺激内皮细胞小窝介导的白蛋白转运，并介导内皮细胞通透性增加和肺损伤；(Ii)PMN激活内皮细胞中的Src激酶，通过PI3-激酶和Akt信号调节eNOS介导的NO产生，以及PMN-内皮相互作用产生的NO调节跨细胞和连接通透性途径，从而增加肺微血管通透性。我们将描述负责内皮通透性激活的信号通路及其在肺水肿形成机制中的后果。将使用的方法包括基因敲除小鼠，siRNA诱导的蛋白表达抑制，跨细胞和细胞旁通路的内皮通透性评估，小窝介导的活内皮细胞运输的成像分析，以及相关信号通路的生化评估。对完整小鼠肺的研究将得到利用小鼠肺内皮细胞的研究的补充，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更机械的理解。这些研究将为探讨PMN活化导致肺内皮细胞通透性增加的机制提供新的视角，以期发现新的分子靶点，并更好地设计针对富含蛋白质的肺水肿和急性肺损伤的治疗策略。项目简介：血管损伤和富含蛋白质的肺水肿形成是与炎症性疾病相关的危及生命的并发症，如缺血/再灌注损伤和脓毒症。我们假设炎症过程中中性粒细胞诱导的内皮细胞高通透性是通过空泡机制增加的跨内皮白蛋白转运。因此，该项目探讨了与中性粒细胞介导的内皮通透性增加和肺损伤中血浆蛋白的细胞旁渗漏相比，小窝介导的白蛋白摄取和跨细胞作用的作用。我们将评估内皮细胞中性粒细胞激活Src激酶在调节小窝蛋白-1、动力蛋白-2和eNOS功能中的作用，并确定白蛋白转运增加和NO产生在调节跨细胞和连接通透性途径中的后果。

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.