Mechanisms of Reperfusion-induced Endothelial Injury

描述(由申请人提供)：这项资助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损伤时的内皮功能障碍是由肿瘤坏死因子-α(TNF-α)过度表达引起的。第一个目的是随随便便地确定肿瘤坏死因子-α在心肌I/R后内皮损伤中的作用。我们认为，在肿瘤坏死因子-α基因敲除(TNF-/-)小鼠中，心肌再灌注对内皮细胞的损伤是轻微的，在肿瘤坏死因子-α过度表达(TNF++/++)小鼠中是严重的，在杂合子(肿瘤坏死因子-/++)小鼠中是中等的。我们将确定在再灌流时给予抗肿瘤坏死因子-α抗体是否能防止内皮功能障碍，如通过保护内皮功能和通过阻止炎症细胞的涌入来维持内皮屏障功能所表明的那样。该方案旨在模拟临床相关的范例，在该范例中，抗肿瘤坏死因子-α的中和抗体可以在冠脉搭桥术后的再通过程或恢复血流时递送。我们还将建立I/R期间表达肿瘤坏死因子-α的细胞类型(S)。第二个目的是确定基础条件下和I/R损伤期间活性氧(ROS)的产生和来源。这一目的将建立在心肌I/R后诱导内皮损伤中肿瘤坏死因子-α和ROS之间的联系。我们将确定在再灌注时给予抗肿瘤坏死因子-α抗体是否可以阻止ROS的产生。我们将确定是在肿瘤坏死因子++/++小鼠体内ROS的产生增加，还是在肿瘤坏死因子小鼠中减少。我们进一步提出，肿瘤坏死因子-α将通过激活黄嘌呤氧化酶和NADPH氧化酶来刺激这些化学物种的产生。我们还将使用电子顺磁共振光谱来定量测量O2，从而建立肿瘤坏死因子-α与超氧化物产生酶的激活之间的联系。暴露于不同剂量的肿瘤坏死因子-α时，冠状动脉小动脉的生成量。第三个目的将确定肿瘤坏死因子-α的表达是否影响内皮细胞中精氨酸酶的表达。这一目标将集中在这一命题上，即肿瘤坏死因子++/++小鼠将表现出精氨酸酶的表达升高；而与WT对照组动物相比，肿瘤坏死因子-/-小鼠将表现出精氨酸酶表达水平的降低。我们利用了一系列的方法，包括体外显微镜、荧光和氧的EPR分析。制备、电化学检测真实的NO、实时聚合酶链式反应和蛋白质印迹以评估关键蛋白在模型中的表达。我们相信，这项研究将为治疗I/R损伤和相关疾病(搭桥后并发症)提供一种新的方法。我们的目标是，这些研究将提供对I/R损伤后内皮功能障碍的基础的清楚了解，我们希望这种更好的理解将促进对抗这一并发症的新的治疗途径。

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.