Mechanisms of Reperfusion-induced Endothelial Injury

再灌注引起的内皮损伤的机制

• 批准号: 7626709
• 负责人: CUIHUA ZHANG
• 金额: $ 35.76万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7626709
• 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

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）：该赠款提案的总体目的是了解心肌缺血/再灌注（I/R）伤害后内皮功能障碍的基础。我们建议在心肌I/R损伤期间内皮功能障碍是由肿瘤坏死因子-Alpha（TNF-Alpha）过度表达引起的。第一个目标将随意确定心肌I/R后TNF-Alpha在内皮损伤中的作用。 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.我们将确定在再灌注时施用TNF-Alpha抗体是否会防止内皮功能障碍，如保留内皮功能和维持内皮屏障功能通过预防炎症细胞的流入所示。该方案旨在模仿临床上相关的范式，在该范式中，可以在冠状动脉旁路后进行重新定性程序或恢复流量时进行中和针对TNF-Alpha的抗体。我们还将在I/R期间建立表达TNF-Alpha的细胞类型。第二个目标将确定基础条件和I/R损伤期间活性氧（ROS）的产生和来源。在心肌I/R后，该目标将在诱导内皮损伤的诱导中建立TNF-Alpha和ROS之间的联系。我们将确定在再灌注时施用TNF-Alpha抗体是否会阻止ROS产生。我们将确定在TNF ++/++小鼠中ROS的产生是否升高，还是在TNF小鼠中降低。我们进一步提出，TNF-Alpha将通过激活黄嘌呤氧化酶和NADPH氧化酶刺激这些化学物种的产生。我们还将建立TNF-α与使用电子顺磁共振光谱范围的超氧化物生成酶的激活之间的联系，以定量测量O2。暴露于不同剂量的TNF-α时，由冠状动脉动脉产生。第三个目标将确定TNF-Alpha表达是否影响内皮细胞中精氨酸酶的表达。该目标将集中在TNF ++/++小鼠将显示精氨酸酶表达升高的主张上。而TNF - / - 小鼠与WT对照动物相比将表现出精氨酸酶表达水平降低。我们利用涉及体外显微镜，荧光和O2的EPR分析的方法组合。生产，真实NO的电化学检测，RNA转录物的实时PCR以及蛋白质印迹以评估模型中关键蛋白的表达。我们认为，这项研究将为治疗I/R损伤和相关疾病（Bypass后并发症）提供新的方法。我们的目标是，这些研究将对I/R伤害后内皮功能障碍的基础有清晰的了解，我们希望这种更好的理解将促进治疗的新途径，以消除这种并发症。