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（由适用提供）：急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）是全身性炎症状况的破坏性后果，在美国，每年遭受75,000人死亡，每年遭受近200,000人。 ALI的标志是炎症引起的内皮细胞（EC）屏障的破坏，该内皮细胞（EC）屏障将肺脉管系统排列，导致液体，蛋白质和细胞渗入肺部的渗漏导致呼吸衰竭。 EC屏障功能受屏障干扰性收缩力和屏障保护力之间的平衡调节，两种竞争力与多种结合蛋白相关的EC肌动蛋白细胞骨架。我们的工作定义了肌动蛋白结合蛋白Cortactin在确定细胞骨架结构确定EC渗透性方面的重要作用。该提议的中心假设是，ALI中的肺血管泄漏受肺EC水平的皮质素功能调节。在这种背景下，使用跨科学学科的综合方法提出了研究，以检查cortactin对体外和体内Ali发病机理的贡献。特定的目标＃1将表征皮质素对体外肺EC细胞骨架结构和渗透性的功能作用。皮质素缺乏症（通过siRNA）和过表达对以下参数的影响将在培养的人类肺EC中确定：1）薄片脂植物结构和动力学，2）皮质肌动蛋白结构，3）连接复杂结构，4）渗透率。将采用多种方式，包括共聚焦显微镜，活细胞成像，kymography，新型的原子力显微镜显微镜测量接头复杂粘合强度和细胞弹性特性，生化测定以及EC渗透率的体外模型。具体的目标＃2将确定皮质素单核苷酸多态性（SNP）与炎症性肺损伤及其对肺EC的功能作用的关联。我们小组的初步研究已经确定了与患者ALI相关的Cortactin基因中的启动子和编码SNP。将对CTTN启动子和编码区域中的SNP进行复制队列中Ali-sasociation的验证，其对Cortactin表达和体外肺内皮功能的影响将被表征为SA＃1中的概述。特定的目标＃3将翻译表征皮质素和与疾病相关的皮质素SNP对小鼠炎性肺损伤的影响。将采用两种小鼠ALI模型：1）气管内LPS； 2）通风引起的肺损伤。在这些模型中降低皮质素表达的效果将使用cortactin +/-小鼠和体内siRNA确定，而在将使用具有ACE抗体靶向脂质体递送的野生型和SNP Cortactin的过表达在小鼠肺中的影响。将生成一种EC特异性的有条件敲除小鼠。这些研究将机械地表征皮尔塔素在ALI发病机理中的重要作用，并为预防或减少血管泄漏的潜在治疗方法提供新的见解。