Cortactin in Regulation of Pulmonary Vascular Permeability

Cortactin 调节肺血管通透性

• 批准号: 7896592
• 负责人: STEVEN M DUDEK
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF ILLINOIS AT 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/7896592
• 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; Cytoskeleton; DNA Sequence Rearrangement; EMS1 gene; Endothelial Cells; Equilibrium; Extravasation; Generations; In Vitro; Inflammation; Inflammatory; Injury; Laboratories; Link; Liquid substance; Lung; Membrane; Membrane Microdomains; Modeling; Molecular Biology; Molecular Biology Techniques; Molecular Target; Mutation; 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）是全身性炎症性疾病（如败血症）的毁灭性后果，在美国每年有近20万人受其折磨，其中75，000人死亡。ALI的标志是炎症诱导的肺血管内皮细胞（EC）屏障的破坏，导致液体、蛋白质和细胞渗漏到肺的气隙中。我们的实验室已经广泛研究了维持和增强EC屏障功能的机制，作为识别可能的治疗靶点的工具。EC屏障调节的当前范例表明屏障破坏细胞收缩力与屏障保护细胞-细胞和细胞-基质束缚力之间存在平衡。在这个模型中的两个竞争力是密切联系的肌动蛋白为基础的内皮细胞骨架的各种肌动蛋白结合蛋白。我们的工作已经确定了肌动蛋白结合蛋白，corneumn，在解决阶段的血管通透性与这一关键功能发生通过EC细胞骨架重排的重要作用。关于损伤后EC屏障功能恢复的机制知之甚少。因此，corneumn是一个有吸引力的新疗法的分子靶点，并保证密集的结构/功能的研究，我们提出在这个应用程序。在此背景下，PI建议研究corneumn调节EC细胞骨架重排导致ALI综合征期间屏障功能改变的假设。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，TGF β 1），以集中于在屏障恢复阶段期间corneumen的作用。转基因动物模型的ALI将扩展这些研究在体内。在SA#2中，我们将研究皮质肌动蛋白和连接蛋白重排的作用，调节肺内皮屏障功能，使用新的原子力显微镜（AFM）和其他技术，功能性地表征皮质蛋白的外周细胞骨架重排参与屏障恢复的作用，重点是皮质肌动蛋白结构，连接复合物的形成，和脂筏信号。在SA#3中，我们将通过分子生物学和蛋白质组学技术的组合来表征我们在coronin基因中鉴定的ALI相关编码单核苷酸多态性（SNP）的功能后果。这一目的将确定这种ALI相关SNP对coronin功能的机制作用，因为它涉及使用上述体外和转基因动物技术的内皮渗透性和屏障恢复。