Oxidants & Caspases: Initiation of Reperfusion Injury

描述(由申请人提供)：caspase或其调节因子的治疗操作在控制缺血/再灌流中的细胞损伤方面提供了重要的前景。然而，由于阻断特定的caspase可以通过替代或补偿途径导致细胞凋亡，因此了解单个caspase和那些直接控制caspase活性或提供导致所谓的caspase非依赖的下游细胞死亡机制的分子的作用是至关重要的。这一设想的核心假设是，在心肌细胞缺血/再灌注(I/R)后，活性氧(ROS)的产生和caspase-2的激活导致线粒体的扰动，导致细胞色素c(Cytc)的释放，电子传递链中断和细胞凋亡。利用细胞和动物模型、RNA干扰去除特定蛋白和特定基因敲除小鼠，我们建议研究特定的caspase及其调节因子，包括Smac/DIABLO和凋亡抑制蛋白(IAPs)在两个主要的特定目的中的作用：1)确定caspase-2、线粒体ROS和Bax在启动缺血/再灌注诱导的心肌细胞损伤中的交互作用。我们假设IR导致线粒体ROS的产生和Bax和caspase-2的激活。在这些研究中，我们还将检查特定的ROS，如超氧化物和过氧化氢，以及抗氧化处理对Bax和caspase-2激活的影响。进一步，我们假设caspase-2活性直接作用于线粒体和/或通过Bid裂解导致细胞色素c的释放和进一步的ROS产生；2)确定caspase-2在促凋亡的线粒体蛋白Smac/Diablo的释放中的作用，它对IAP阻断caspase活性的作用以及它们在放大I/R诱导的细胞凋亡中的相关作用。我们推测，在I/R刺激的心肌细胞中，caspase可以导致细胞色素c和IAP抑制的Smac/DIABLO等线粒体蛋白的差异释放，并影响caspase介导的I/R诱导的细胞死亡的放大。更好地了解caspase的特定活性以及caspase、线粒体ROS和作用于线粒体或从线粒体释放的内源性调节因子之间的生理相互作用，将是评估这些靶点的治疗潜力的关键。

DESCRIPTION (provided by applicant): Therapeutic manipulation of caspases or their regulators offers significant promise in controlling cellular injury in ischemia/reperfusion. Because blockade of specific caspases can result in apoptotic progression via alternative or compensatory pathways, however, it is critical to understand the roles of individual caspases and those molecules which directly control caspase activities or which provide "short-circuits" leading to so-called "caspase independent" downstream mechanisms of cell death. The central hypothesis of this proposal is that following ischemia/reperfusion (I/R) in cardiomyocytes, generation of reactive oxygen species (ROS) and caspase-2 activation lead to mitochondrial perturbations resulting in release of cytochrome c (cyt c) and disruption of the electron transport chain and apoptotic cell death. Using a combination of cellular and animal models, depletion of specific proteins via RNA interference and specific knockout mice, we propose to study the roles of specific caspases and their regulators, including Smac/DIABLO and inhibitor of apoptosis proteins (IAPs) in two major specific aims: 1) Determine the interactive roles of caspase-2, mitochondrial ROS, and Bax and Bid ininitiating ischemia/reperfusion-induced injury to cardiomyocytes. We hypothesize that IR results in generation of mitochondrial ROS and the activation of Bax and caspase-2. In these studies we will examine also the effects of specific ROS, such as superoxide and hydrogen peroxide, and antioxidant treatment on the activation of Bax and caspase-2. Further, we postulate that active caspase-2 acts directly on the mitochondria and/or via Bid cleavage to cause the release of cyt c and further ROS generation; 2) Determine the role of caspase-2 in the release of pro-apoptotic mitochiondrial protein Smac/DIABLO, its effects on IAP blockade of caspase activity and their relative roles in amplification of I/R-induced apoptosis. We hypothesize that in I/Rstimulated cardiomyocytes, caspases can cause differential release of apoptogenic mitochondrial proteins, such as cyt c and the IAP-inhibiting Smac/DIABLO, and that these affect caspase-mediated amplification of I/R-induced cell death. A better understanding of specific caspase activity and the physiological interplay between caspases, mitochondrial ROS, and the endogenous regulators which act on or are released from the mitochondria will be critical in assessing the therapeutic potential of these targets.