Cortactin in Regulation of Pulmonary Vascular Permeability

描述(申请人提供)：急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)是全身炎症状态的毁灭性后果，例如败血症，在美国每年有近20万人受到影响，7.5万人死亡。ALI的特点是炎症导致肺血管内皮细胞(EC)屏障的破坏，导致液体、蛋白质和细胞泄漏到肺的空间。我们的实验室广泛研究了维持和增强EC屏障功能的机制，作为确定可能的治疗靶点的工具。目前的EC屏障调节范式表明，在破坏屏障的细胞收缩力量和保护屏障的细胞-细胞和细胞-基质拴系力之间存在平衡。这个模型中的两种相互竞争的力量都通过各种肌动蛋白结合蛋白与以肌动蛋白为基础的内皮细胞骨架紧密相连。我们的工作已经确定了肌动蛋白结合蛋白，皮质动蛋白，在血管通透性的解决阶段发挥了重要作用，这一关键功能通过EC细胞骨架重排发生。损伤后内皮细胞屏障功能恢复的机制知之甚少。因此，Cortactin是新疗法的一个有吸引力的分子靶点，值得我们在这一应用中进行密集的结构/功能研究。在此背景下，PI建议研究Cortactin调节EC细胞骨架重排导致ALI综合征屏障功能改变的假说。在SA#1中，我们将通过使用分子生物学和蛋白质组学技术，利用体外屏障破坏模型(例如，凝血酶、TGFp1)来研究皮质蛋白在屏障恢复阶段的作用，从而从机械上表征皮质蛋白分子参与屏障调节的关键部分。ALI的转基因动物模型将在体内扩展这些研究。在SA#2中，我们将使用新型原子力显微镜(AFM)和其他技术，研究皮质肌动蛋白在调节肺内皮细胞屏障功能的皮质肌动蛋白和连接蛋白重排中的作用，并从功能上表征皮质肌动蛋白在参与屏障恢复的外周细胞骨架重排中的作用，重点关注皮质肌动蛋白结构、连接复合体形成和脂筏信号转导。在SA#3中，我们将通过结合分子生物学和蛋白质组学技术来表征我们在皮质蛋白基因中发现的ALI相关编码单核苷酸多态(SNP)的功能后果。这一目的将确定这种ALI相关的SNP对皮质蛋白功能的机制影响，因为它与上述体外和转基因动物技术的内皮通透性和屏障恢复有关。

DESCRIPTION (provided by applicant): The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions such as sepsis that afflicts almost 200,000 people a year in the US with 75,000 deaths. The hallmark of ALI is inflammation-induced disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature, resulting in leakage of fluid, protein, and cells into the airspaces of the lung. Our laboratory has extensively studied the mechanisms involved in maintaining and enhancing EC barrier function as a tool for identifying possible therapeutic targets. The current paradigm of EC barrier regulation suggests a balance exists between barrier-disrupting cellular contractile forces and barrier-protective cell-cell and cell-matrix tethering forces. Both competing forces in this model are intimately linked to the actin-based endothelial cytoskeleton by a variety of actin-binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in the resolution phase of vascular permeability with this critical function occurring via EC cytoskeletal rearrangement. Very little is known about the mechanisms governing recovery of EC barrier function following injury. Thus, cortactin is an attractive molecular target for novel therapies and warrants the intense structure/function studies we propose in this application. With this background, the PI proposes to investigate the hypothesis that cortactin regulates EC cytoskeletal rearrangements that result in altered barrier function during ALI syndromes. In SA#1 we will mechanistically characterize the key portions of the cortactin molecule involved in barrier regulation through the use of molecular biology and proteomic techniques utilizing in vitro models of barrier disruption (e.g., thrombin, TGFpl) to focus on cortactin's role during the barrier recovery phase. Transgenic animal models of ALI will extend these studies in vivo. In SA#2 we will examine the role of cortactin in cortical actin and junctional protein rearrangements that regulate pulmonary endothelial barrier function using novel atomic force microscopy (AFM) and other techniques to functionally characterize cortactin's role in peripheral cytoskeletal rearrangements involved in barrier recovery, focusing on cortical actin structures, junctional complex formation, and lipid raft signaling. In SA#3 we will characterize the functional consequences of an ALI- associated coding single nucleotide polymorphism (SNP) we have identified in the cortactin gene through a combination of molecular biology and proteomic techniques. This aim will determine the mechanistic effects of this ALI-associated SNP on cortactin function as it pertains to endothelial permeability and barrier recovery using the in vitro and transgenic animal techniques described above.