Regulation of Pulmonary Vascular Permeability by Integrin AlphaVBeta5

描述（由申请人提供）：急性肺损伤是一种常见的疾病，没有已知的有效的药物治疗方法。急性肺损伤发生的主要因素之一是肺血管通透性的增加。整合素5亚基基因零突变纯合的小鼠在呼吸机诱导的肺损伤模型中免受肺水肿的影响，针对整合素5的阻断单克隆抗体在同一模型中保护野生型小鼠，也保护大鼠免受单侧肺缺血和再灌注诱导的肺水肿。阻断或基因消融av - 5整合素可以保护培养的内皮细胞免受单层通透性增加的影响，也可以防止rhoa诱导的肌动蛋白应激纤维的形成和内皮粘附连接成分的磷酸化。在目前的建议中，我们将更详细地探索将av - 5整合素与血管通透性调节增加联系起来的分子途径。我们将使用显性阴性和组成性活性构建体、siRNA敲低和敲除小鼠的内皮细胞来测试这种整合素通过与小gtpase、Ga12和Ga13以及已知的G蛋白激活剂整合素相关蛋白（IAP）相互作用调节血管通透性的假设。然后，我们将通过研究表达这些蛋白质的全局或条件敲除的小鼠呼吸机诱导的肺损伤，来检查这些蛋白质在体内的作用。由于我们的初步数据表明，av¿5是包含Ga13的多蛋白复合物的中心成分，以及rhoA依赖信号通路的许多成分，我们将研究该复合物的哪些成分依赖于av¿5进行组装，以及重组具有该整合素野生型，截断型和嵌合形式的¿5敲除小鼠的内皮细胞需要哪些区域。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，我们将进一步研究在另外三种体内模型（气管内博莱霉素、气管内内毒素以及低剂量内毒素和中等容量通气的组合）中发现的av¿5和任何其他关键成分的作用。通过提出的研究，我们希望确定av¿5或该途径的其他成分是否成为干预急性肺损伤的有吸引力的靶点。

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.