Regulation of Pulmonary Vascular Permeability by Integrin AlphaVBeta5

描述（由申请方提供）：急性肺损伤是一种常见疾病，尚无已知有效的药物治疗。急性肺损伤发生的主要因素之一是肺血管通透性增加。在主要研究者的实验室中产生的整联蛋白5亚基基因中的无效突变的纯合小鼠在呼吸机诱导的肺损伤模型中被保护免于肺水肿，并且针对α ν β 5整联蛋白的阻断性单克隆抗体在该相同模型中保护野生型小鼠，并且还保护大鼠免于由单侧肺缺血和再灌注诱导的肺水肿。α v β 5整合素的阻断或基因消融可保护培养的内皮细胞免于单层通透性增加，以及rhoA诱导的肌动蛋白应力纤维的形成和内皮粘附连接组分的磷酸化。在目前的提议中，我们将更详细地探索连接α v β 5整联蛋白与调节血管通透性增加的分子途径。我们将使用显性负性和组成型活性结构，siRNA敲除和敲除小鼠的内皮细胞来测试这种整合素通过与小GTP酶，Ga 12和Ga 13和已知的G蛋白激活剂整合素相关蛋白（IAP）相互作用来调节血管通透性的假设。然后，我们将通过研究呼吸机诱导的肺损伤小鼠表达的全球或条件敲除这些蛋白质的每一个检查在体内的作用。由于我们的初步数据表明，av <$5是一个多蛋白复合物的核心组成部分，该复合物含有Ga 13，以及rhoA依赖性信号通路的许多组成部分，我们将研究该复合物的哪些组成部分依赖于av <$5进行组装，以及av <$5亚基的哪些区域是由重组内皮细胞所必需的。5敲除小鼠与野生型，截短和嵌合形式的这种整合素。最后，为了确定我们正在研究的途径是否与非心源性肺水肿广泛相关，我们将进一步检查av？5和任何其他在三种额外的体内模型中发现的关键成分的作用--气管内博莱霉素，气管内毒素和低剂量内毒素和中等容量通气的组合。通过这些研究，我们希望确定av 5或该通路的其他成分是否是急性肺损伤干预的有吸引力的靶点。

DESCRIPTION (provided by applicant): Acute lung injury is a common disorder with no known, effective, pharmacologic treatment. One of the central contributing factors to the development of acute lung injury is an increase in the permeability of the pulmonary vasculature. Mice homozygous for a null mutation in the integrin ¿5 subunit gene, generated in the principal investigator's laboratory, are protected from pulmonary edema in a model of ventilator induced lung injury, and a blocking monoclonal antibody against the av¿5 integrin protects wild type mice in this same model and also protects rats from pulmonary edema induced by unilateral lung ischemia and reperfusion. Blockade or genetic ablation of the av¿5 integrin protects cultured endothelial cells from increases in monolayer permeability and from both the formation of rhoA-induced actin stress fibers and the phosphorylation of components of the endothelial adherens junction. In the current proposal, we will explore the molecular pathways linking the av¿5 integrin to regulated increases in vascular permeability in more detail. We will use dominant negative and constitutively active constructs, siRNA knockdown and endothelial cells from knockout mice to test the hypotheses that this integrin regulates vascular permeability through interaction with the small GTPases, Ga12 and Ga13 and the known G protein activator integrin associated protein (IAP). We will then examine the in vivo roles for each of these proteins by studying ventilator-induced lung injury in mice expressing global or conditional knockouts of each of these proteins. Because our preliminary data suggest that av¿5 is a central component of a multi-protein complex that contains Ga13, and many of the components of the rhoA dependent signaling pathway, we will examine which components of this complex depend on av¿5 for assembly and which regions of the ¿5 subunit are required by reconstituting endothelial cells from ¿5 knockout mice with wild type, truncated and chimeric forms of this integrin. Finally, to determine whether the pathways we are studying are broadly relevant to non-cardiogenic pulmonary edema, we will further examine the role of av¿5 and any other component found to be critical in three additional in vivo models - intratracheal bleomycin, intratracheal endotoxin and the combination of low dose endotoxin and moderate volume ventilation. Through the proposed studies we hope to determine whether av¿5 or other components of this pathway are attractive targets for intervention in acute lung injury.