Proteomic Profile of Brain Endothelial Cell in Hypoxia

描述(由申请人提供)：神经血管单位的新兴概念强调，血管和实质间隔之间的细胞-细胞相互作用调节对损伤的综合反应。因此，内皮细胞功能障碍可能是中风后脑损伤的上游触发因素。在这项R21项目中，我们建议研究缺氧条件下人脑内皮细胞的蛋白质组学特征，以广泛地表征可溶性和胞浆介质在这一体外卒中模型中的作用。基于我们在氧化应激中发现的新的蛋白酶和底物的令人鼓舞的初步发现，我们假设：与对照组相比，暴露在低氧损伤下的人内皮细胞培养将具有一致和可识别的可溶性蛋白质和底物表达谱，表明在细胞-细胞和细胞-基质信号转导中重要的已知和未知因素的相对上调或下调。我们建议在人脑内皮细胞培养液和裂解液中进行三部分研究，以探讨缺氧-复氧的机制。具体目的1：利用已验证的定量蛋白质组学技术，检测缺氧-复氧前后人内皮细胞培养液的蛋白质表达谱。具体目的2：检测缺氧-复氧前后人内皮细胞培养上清液的下游底物库，以获得缺氧损伤特征的底物库的相对定量。次要目标：扩展目标1和目标2以研究细胞裂解产物。我们希望通过对作为神经血管单位重要组成部分的人脑内皮细胞缺氧-复氧的蛋白质组学定量分析，为发现缺血模型中新的蛋白质提供筛选工具，为发现新的治疗靶点提供场所。

DESCRIPTION (provided by applicant): The emerging concept of the neurovascular unit emphasizes that cell-cell interactions between vascular and parenchymal compartments mediate an integrative response to injury. Thus, endothelial cell dysfunction may be an upstream trigger for brain damage after stroke. For this R21 Project, we propose to investigate the Proteomic Profiles of Human Brain Endothelial Cells in Hypoxia, to broadly characterize the role of soluble and cytosolic mediators in this in vitro stroke model. Building on our encouraging preliminary findings of novel proteinases and substrates found in oxidative stress, we hypothesize that: In comparison to controls, human endothelial cell cultures exposed to hypoxic insult will have consistent and identifiable soluble-protein and substrate expression profiles, indicating relative up- or down-regulation of both known and unknown factors important in cell-cell and cell-matrix signaling. We propose a three-part study in human brain endothelial cell culture media and lysates, to investigate the mechanism of hypoxia-reoxygenation. Specific Aim 1: Using validated techniques of quantitative proteomics technology, examine the protein expression profile of human endothelial cell culture media pre and post hypoxia-reoxygenation. Specific Aim 2: Examine the down-stream substrates repertoire of human endothelial cell culture media pre and post hypoxia-reoxygenation, in order to obtain relative quantification of the substrate repertoire characteristic of hypoxic injury. Secondary Aims: Extend Aim 1 and 2 to study cell lysates. Through quantitative proteomic analysis of hypoxia-reoxygenation in human brain endothelial cells, an important component of the neurovascular unit, we hope to provide a screening tool for the discovery of novel proteins in ischemic models, and provide venues for the discovery of new therapeutic targets.