Cortactin in Regulation of Pulmonary Vascular Permeability

 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，在美国每年有近20万人受到影响，其中75，000人死亡。ALI的标志是炎症诱导的内衬肺脉管系统的内皮细胞（EC）屏障的破坏，导致由于流体、蛋白质和细胞渗漏到肺的气隙中而导致呼吸衰竭。EC屏障功能受屏障破坏细胞收缩力和屏障保护束缚力之间的平衡调节，这两种竞争力通过多种结合蛋白与EC肌动蛋白细胞骨架连接。我们的工作已经确定了肌动蛋白结合蛋白，corneum，在调节细胞骨架结构，决定EC通透性的重要作用。这一建议的中心假设是，肺血管渗漏在ALI是由coronin功能在肺EC的水平。在此背景下，提出了使用跨学科的综合方法来研究corneumin对体外和体内ALI发病机制的贡献的研究。具体目标#1将表征coronin对体外肺EC细胞骨架结构和渗透性的功能作用。将在培养的人肺EC中确定皮质素缺乏（通过siRNA）和过表达对以下参数的影响：1）板状伪足结构和动力学，2）皮质肌动蛋白结构，3）连接复合物结构，4）渗透性。将采用多种方式，包括共聚焦显微镜，活细胞成像，kymography，新的原子力显微镜连接复合物的粘附强度和细胞弹性性能的测量，生化分析，并在EC渗透性的体外模型。具体目标#2将确定coronin单核苷酸多态性（SNP）与炎性肺损伤的相关性及其对肺EC的功能影响。我们小组的初步研究已经确定了与ALI患者相关的corneum基因的启动子和编码SNP。将对CTTN启动子和编码区中的SNP进行重复队列中ALI相关性的验证，并将如SA#1中概述的那样表征它们对coronin表达和肺内皮功能的体外作用。具体目标#3将跨国地表征corneumn和疾病相关的corneumn 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）呼吸机诱导的肺损伤。这些模型中降低的corneumen表达的作用将使用corneumen +/-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 corneum蛋白在小鼠肺中的过表达的作用。将产生EC特异性条件性coronin敲除小鼠。这些研究将从机制上描述coronin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。

 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.