Cortactin in Regulation of Pulmonary Vascular Permeability

Cortactin 调节肺血管通透性

• 批准号: 7244759
• 负责人: STEVEN M DUDEK
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244759
• 关键词: Actin-Binding Protein; Actins; Acute; Acute Lung Injury; Adhesives; Adult Respiratory Distress Syndrome; Animal Model; Asparagine; Atomic Force Microscopy; Cell Shape; Cells; Cessation of life; Code; Complex; Condition; Cytoskeleton; DNA Sequence Rearrangement; Disruption; EMS1 gene; Endothelial Cells; Equilibrium; Extravasation; Generations; In Vitro; Inflammation; Inflammatory; Injury; Laboratories; Link; Liquid substance; Lung; Membrane; Membrane Microdomains; Microscopy; Modeling; Molecular Biology; Molecular Biology Techniques; Molecular Target; Mutation; Newborn Respiratory Distress Syndrome; Patients; Peripheral; Permeability; Phase; Predisposition; Protein Array; Proteins; Proteomics; Recovery; Regulation; Research Personnel; Resolution; Role; Sepsis; Serine; Signal Transduction; Single Nucleotide Polymorphism; Small Interfering RNA; Structure; Syndrome; Techniques; Thrombin; Transgenic Animals; Variant; Vascular Permeabilities; Work; base; human EMS1 protein; in vitro Model; in vivo; novel; programs; pulmonary vascular permeability; therapeutic target; tool; translational approach

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.

描述（由申请人提供）：急性肺损伤/急性呼吸窘迫综合征 (ALI/ARDS) 是脓毒症等全身炎症性疾病的毁灭性后果，在美国每年影响近 200,000 人，其中 75,000 人死亡。 ALI 的特点是炎症引起的肺血管内皮细胞 (EC) 屏障破坏，导致液体、蛋白质和细胞渗漏到肺腔内。我们的实验室广泛研究了维持和增强 EC 屏障功能的机制，作为识别可能治疗靶点的工具。目前的 EC 屏障调节范式表明，屏障破坏细胞收缩力与屏障保护性细胞-细胞和细胞-基质束缚力之间存在平衡。该模型中的两种竞争力通过多种肌动蛋白结合蛋白与基于肌动蛋白的内皮细胞骨架密切相关。我们的工作明确了肌动蛋白结合蛋白 Cortactin 在血管通透性解决阶段的重要作用，这一关键功能是通过 EC 细胞骨架重排发生的。关于损伤后 EC 屏障功能恢复的机制知之甚少。因此，皮质素是新疗法的一个有吸引力的分子靶标，并保证我们在本申请中提出的深入的结构/功能研究。在此背景下，PI 提议研究这样的假设：cortactin 调节 EC 细胞骨架重排，导致 ALI 综合征期间屏障功能改变。在 SA#1 中，我们将通过分子生物学和蛋白质组学技术，利用屏障破坏的体外模型（例如凝血酶、TGFpl），从机制上表征参与屏障调节的 Cortactin 分子的关键部分，重点关注 Cortactin 在屏障恢复阶段的作用。 ALI 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) 和其他技术来研究 Cortactin 在调节肺内皮屏障功能的皮质肌动蛋白和连接蛋白重排中的作用，以功能性地表征 Cortactin 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、连接复合物形成和脂筏 发信号。在 SA#3 中，我们将通过分子生物学和蛋白质组学技术的结合，描述在 cortactin 基因中鉴定出的 ALI 相关编码单核苷酸多态性 (SNP) 的功能后果。该目标将使用上述体外和转基因动物技术确定这种 ALI 相关 SNP 对 cortactin 功能的机械效应，因为它与内皮通透性和屏障恢复有关。