Cortactin in Regulation of Pulmonary Vascular Permeability

Cortactin 调节肺血管通透性

基本信息

批准号：
9130397
负责人：
STEVEN M DUDEK
金额：
$ 42.16万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9130397
关键词：
Actin-Binding Protein Actins Acute Acute Lung Injury Adhesives Adult Respiratory Distress Syndrome Antibodies Atomic Force Microscopy Binding Proteins Biochemical Biological Assay Blood Vessels Candidate Disease Gene Cell Culture Techniques Cell Shape Cells Cessation of life Code Complex Confocal Microscopy Cytoskeleton Discipline Disease EMS1 gene Endothelial Cells Endothelium Equilibrium Extravasation Fiber Figs - dietary Health Human In Vitro Inflammation Inflammatory Injury Investigation Knockout Mice Kymography Laboratories Life Link Liposomes Liquid substance Lung Lung diseases MYLK gene Measurement Mechanical Ventilators Mediating Modality Modeling Molecular Molecular Target Mus Pathogenesis Patients Peripheral Permeability Pre-Clinical Model Process Property Protein Array Proteins Regulation Respiratory Failure Role Sepsis Single Nucleotide Polymorphism Small Interfering RNA Structure Syndrome Techniques Therapeutic Transgenes Translating Validation Ventilator-induced lung injury Work base biophysical properties cellular imaging cohort human EMS1 protein in vitro Model in vivo insight lung injury mouse model novel overexpression pre-clinical prevent promoter pulmonary vascular permeability therapeutic target tool translational study

项目摘要

DESCRIPTION (provided by applicant): The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions 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 respiratory failure from the leakage of fluid, protein, and cells into th airspaces of the lung. EC barrier function is regulated by a balance between barrier-disrupting cellular contractile forces and barrier-protective tethering forces, with both competing forces linked to the EC actin cytoskeleton by a variety binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in regulating the cytoskeletal structures hat determine EC permeability. The central hypothesis of this proposal is that pulmonary vascular leak in ALI is regulated by cortactin function at the level of lung EC. With this background, studies are proposed using integrated approaches across scientific disciplines to examine the contribution of cortactin to ALI pathogenesis in vitro and in vivo. Specific Aim #1 will characterize the functional effects of cortactin on pulmonary EC cytoskeletal structure and permeability in vitro. The effects of cortactin deficiency (via siRNA) and overexpression on the following parameters will be determined in cultured human pulmonary EC: 1) lamellipodia structure and dynamics, 2) cortical actin structure, 3) junctional complex structure, 4) permeability. Multiple modalities will be employed, including confocal microscopy, live cell imaging, kymography, novel atomic force microscopy measurements of junctional complex adhesive strength and cell elastic properties, biochemical assays, and in vitro models of EC permeability. Specific Aim #2 will determine the association of cortactin single nucleotide polymorphisms (SNPs) with inflammatory lung injury and their functional effects on pulmonary EC. Preliminary studies by our group have identified promoter and coding SNPs in the cortactin gene that are associated with ALI in patients. Validation of ALI-association in a replicate cohort will be performed for SNPs in the CTTN promoter and coding region, and their effects on cortactin expression and lung endothelial function in vitro will be characterized as outlined in SA#1. Specific Aim #3 will transnationally characterize the effects of cortactin and disease-associated cortactin SNPs on inflammatory lung injury in mice. Two models of ALI in mice will be employed: 1) intratracheal LPS; 2) ventilator-induced lung injury. The effects of decreased cortactin expression in these models will be determined using cortactin +/- mice and in vivo siRNA, while the effects of overexpression of wild type and SNP cortactin in mouse lungs will be explored using ACE- antibody targeted liposomal delivery. An EC-specific conditional cortactin knockout mouse will be generated. These studies will mechanistically characterize the important role of cortactin in the pathogenesis of ALI and provide new insights into potential therapeutic approaches to prevent or reduce vascular leak.

描述（由申请人提供）：急性肺损伤/急性呼吸窘迫综合征 (ALI/ARDS) 是全身炎症性疾病的毁灭性后果，每年影响美国近 200,000 人，导致 75,000 人死亡。 ALI 的特点是炎症。诱导肺血管内皮细胞 (EC) 屏障破坏，导致渗漏导致呼吸衰竭EC屏障功能通过破坏屏障的细胞收缩力和屏障保护束缚力之间的平衡来调节，这两种竞争力通过多种结合与EC肌动蛋白细胞骨架相关。我们的工作明确了肌动蛋白结合蛋白皮质蛋白在调节细胞骨架结构中的重要作用，该结构决定了 EC 的通透性。 ALI 在肺 EC 水平上受到 Cortactin 功能的调节，因此建议使用跨科学学科的综合方法来研究 Cortactin 在体外和体内对 ALI 发病机制的贡献，具体目标 #1 将表征功能效应。 Cortactin 对肺 EC 细胞骨架结构和渗透性的体外影响将在培养的人肺 EC 中确定 Cortactin 缺乏（通过 siRNA）和过度表达对以下参数的影响： 1) 片状伪足结构和动力学，2) 皮质肌动蛋白结构，3) 连接复合体结构，4) 将采用多种模式，包括共聚焦显微镜、活细胞成像、kymography、新型原子力显微镜测量连接复合体粘附强度和细胞弹性特性、生化测定和 EC 渗透性的体外模型将确定 cortactin 单核苷酸的关联。我们小组的初步研究已经确定了 cortactin 基因中与患者 ALI 相关的启动子和编码 SNP，并将在重复队列中验证 ALI 相关性。对 CTTN 启动子和编码区中的 SNP 进行的研究，以及它们对体外皮质蛋白表达和肺内皮功能的影响将如 SA#1 中所述进行表征。将采用两种小鼠模型来表征 Cortactin 和疾病相关的 Cortactin SNP 对小鼠炎症性肺损伤的影响：1）气管内 LPS；2）呼吸机诱导的肺损伤。这些模型将使用 cortactin +/- 小鼠和体内 siRNA 来确定，而将使用 ACE 抗体靶向脂质体递送来探索野生型和 SNP cortactin 在小鼠肺部过度表达的影响，将产生 EC 特异性条件性 cortactin 敲除小鼠，这些研究将在机制上表征 cortactin 在 ALI 发病机制中的重要作用。并为预防或减少血管渗漏的潜在治疗方法提供新的见解。