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Mechanisms of Reperfusion-induced Endothelial Injury

再灌注引起的内皮损伤的机制

基本信息

批准号：
7796786
负责人：
Michael A HILL
金额：
$ 35.73万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-06-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7796786
关键词：
Acetylcholine Acute myocardial infarction Adenosine Diphosphate Affect Allopurinol Animals Antibodies Applications Grants Arginine Attenuated Biological Preservation Blood Vessels Bypass Caliber Cardiac Cardiomyopathies Cells Chemicals Complication Control Animal Coronary Coronary Artery Bypass Data Detection Development Disease Dose Electrocardiogram Electron Spin Resonance Spectroscopy Endothelial Cells Endothelium Enzymes Experimental Models Exposure to Fluorescence Microscopy Fluorescent Dyes Free Radicals Functional disorder Generations Goals Heart Heterozygote Hydrogen Peroxide In Vitro Inflammatory Injury Investigation Ischemia Knock-out Knockout Mice Knowledge Lead Link Maintenance Measurement Measures Mediating Modeling Mus Myocardial Myocardial Ischemia Myocardial Reperfusion Injury Myocardium NADPH Oxidase Nitric Oxide Nitric Oxide Synthase Oxidases Peroxonitrite Play Principal Investigator Procedures Production Proteins Protocols documentation Publications RNA Reactive Oxygen Species Reperfusion Injury Reperfusion Therapy Reporting Research Personnel Role Smooth Muscle Myocytes Solutions Source Superoxides System TNF gene Testing Thrombolytic Therapy Time Transcript Transgenic Organisms Tumor Necrosis Factor-alpha Vascular Smooth Muscle Western Blotting Wild Type Mouse Xanthine Oxidase arginase arteriole base cell type ceramide-activated protein kinase clinically relevant combat cytokine design dihydroethidium enzyme activity fluorescence imaging heuristics human TNF protein inhibitor/antagonist insight mast cell mimetics neutralizing antibody novel novel strategies prevent programs receptor response restoration tempol tetrahydrobiopterin xanthine oxidase inhibitor

项目摘要

DESCRIPTION (provided by applicant): The overall aim of this grant proposal is to understand the basis of endothelial dysfunction following myocardial ischemia/reperfusion (I/R) injury. We propose that endothelial dysfunction during myocardial I/R injury is caused by over-expression of tumor necrosis factor-alpha (TNF-alpha). The first aim will casually determine the role of TNF-alpha in endothelial injury following myocardial I/R. We propose that myocardial reperfusion injury to the endothelium will be modest in TNF-alpha knockout (TNF-/-) mice, severe in TNF-alpha over-expression (TNF++/++) mice, and moderate in heterozyqote (TNF-/++, cross of TNF-/- and TNF++/++) mice. We will determine if administration of an antibody to TNF-alpha at the time of reperfusion will prevent endothelial dysfunction as indicated by preservation of endothelial function and maintenance of endothelial barrier function by preventing influx of inflammatory cells. This protocol is designed to mimic a clinically relevant paradigm in which a neutralizing antibody against TNF-alpha could be delivered at the time of a recanalization procedure or restoration of flow following coronary bypass. We will also establish the cell type(s) expressing TNF-alpha during I/R. The second aim will determine the production and sources of reactive oxygen species (ROS) during basal conditions and during I/R injury. This aim will establish the link between TNF-alpha and ROS in the induction of endothelial injury following myocardial I/R. We will determine if administration of an antibody to TNF-alpha at the time of reperfusion will prevent ROS generation. We will establish if the production of ROS is elevated in TNF++/++ mice, or is reduced in TNF mice. We further propose that TNF-alpha will stimulate production of these chemical species by activation of xanthine oxidase and NADPH oxidase. We will also establish the link between TNF-alpha and the activation of superoxide generating enzymes using electron paramagnetic resonance spectroscopy to quantitatively measure O2. production from coronary arterioles during exposure to varying doses of TNF-alpha. The third aim will determine if TNF-alpha expression affects the expression of arginase in endothelial cells. This aim will focus on the proposition that TNF++/++ mice will show elevated arginase expression; whereas, TNF-/- mice will demonstrate reduced levels of arginase expression compared to WT control animals. We utilize a combination of approaches involving in vitro microscopy, fluorescence and EPR analysis of O2. production, electrochemical detection of authentic NO, real time PCR of RNA transcripts, and Western Blotting to evaluate expression of key proteins in the models. We believe that this study will provide a new approach for the treatment of I/R injury and related disorders (post-bypass complications). It is our goal that these studies will provide a clear understanding of the basis for endothelial dysfunction following I/R injury and we hope that this better understanding will facilitate new avenues of therapy to combat this complication.

描述（由申请人提供）：本基金提案的总体目标是了解心肌缺血/再灌注（I/R）损伤后内皮功能障碍的基础。我们认为，心肌I/R损伤时内皮功能障碍是由肿瘤坏死因子-α（TNF-α）的过度表达引起的。第一个目标是偶然确定TNF-α在心肌I/R后内皮损伤中的作用。我们认为TNF-α基因敲除（TNF-/-）小鼠的心肌再灌注内皮损伤是中度的，TNF-α过度表达（TNF++/++）小鼠的心肌再灌注内皮损伤是重度的，杂合（TNF-/++，TNF-/-和TNF++/++的交叉）小鼠的心肌再灌注内皮损伤是中度的。我们将确定在再灌注时给予TNF-α抗体是否会预防内皮功能障碍，如通过防止炎性细胞流入来保护内皮功能和维持内皮屏障功能所示。该方案旨在模拟临床相关范例，其中可以在冠状动脉搭桥术后再通手术或恢复血流时递送针对TNF-α的中和抗体。我们还将确定I/R期间表达TNF-α的细胞类型。第二个目标将确定在基础条件和I/R损伤期间活性氧（ROS）的产生和来源。这一目标将建立TNF-α和ROS在心肌I/R后诱导内皮损伤中的联系。我们将确定在再灌注时给予TNF-α抗体是否会阻止ROS的产生。我们将确定ROS的产生在TNF++/++小鼠中是否升高，或在TNF小鼠中是否降低。我们进一步提出，TNF-α将通过激活黄嘌呤氧化酶和NADPH氧化酶刺激这些化学物质的产生。我们还将建立TNF-α和超氧化物生成酶的激活之间的联系，使用电子顺磁共振光谱定量测量O2。在暴露于不同剂量的TNF-α期间从冠状小动脉产生。第三个目标是确定TNF-α的表达是否影响内皮细胞中TNF-α酶的表达。该目的将集中于TNF++/++小鼠将显示出升高的TNF-α表达的命题;而TNF-/-小鼠将显示出与WT对照动物相比降低的TNF-α表达水平。我们利用的方法，包括在体外显微镜，荧光和EPR分析O2的组合。产生、真实NO的电化学检测、RNA转录物的真实的时间PCR和Western印迹以评估模型中关键蛋白质的表达。我们相信这项研究将为I/R损伤和相关疾病（搭桥术后并发症）的治疗提供一种新的方法。我们的目标是，这些研究将提供一个清晰的理解的基础上内皮功能障碍后I/R损伤，我们希望这种更好的理解将促进新的治疗途径，以打击这种并发症。