The Role of Bcl-2 in Ischemia-Reperfusion Injury

描述(由申请人提供)： 有或没有再灌注的缺血发生在各种疾病中，包括心肌梗死、中风、失血性休克和器官移植。我们和其他人已经证明，缺血和再灌注可以导致中性粒细胞(PMN)诱导的再灌注损伤，并且识别白细胞黏附分子的单抗可以改善部分损伤。这些临床前实验导致了一些临床试验，使用抗粘连疗法试图减少心肌梗死、中风和创伤性损伤后的损伤。不幸的是，尽管有非常强大的临床前数据，这些试验并没有显示出保护作用。为了理解这些临床研究，我们在导致临床试验的心肌和脑缺血-再灌注的实验环境中检查了缺血时间，发现缺血时间一般小于1.5小时，大多数时间在30到60分钟之间。临床试验的失败可能可以用临床环境中发生的延长的缺血时间来解释。因此，在初步实验中，我们将小鼠后肢缺血再灌注的缺血时间延长到2小时，并发现确实存在长达60分钟的CD18黏附分子依赖的损伤。然而，当缺血时间增加到90分钟时，CD18依赖的损伤部分完全消除。尽管如此，使用广谱半胱氨酸蛋白酶抑制剂z-VAD或通过阻断补体激活来保护免受损伤，即使是在延长的缺血时间内也是如此。因此，我们建议研究除抗粘连治疗外的其他机制来治疗延长的缺血后再灌注。在这一应用中，我们提出了以下具体目标：1)确定：当缺血时间足够导致CD18非依赖性损伤时，白细胞是否参与组织损伤，以及b)通过阻断caspase激活可以挽救的缺血时间长度。2)探讨不同细胞类型(髓系细胞、淋巴系细胞、肌肉细胞、内皮细胞)中Bcl2的过度表达在大鼠脑缺血再灌注损伤中的作用。3)探讨死亡受体Fas、TNF、R1在大鼠缺血再灌注后DNA链断裂、半胱氨酸天冬氨酸氨基转移酶激活及肌肉损伤中的作用。4)探讨血管内皮细胞凋亡在大鼠缺血再灌注损伤中的作用。

DESCRIPTION (provided by applicant): Ischemia with or without reperfusion occurs in a variety of diseases including myocardial infarction, stroke, hemorrhagic shock and organ transplantation. We and others have shown that ischemia followed by reperfusion can result in a neutrophil (PMN)-induced reperfusion injury and that a portion of the injury can be ameliorated with monoclonal antibodies that recognize leukocyte adhesion molecules. These pre-clinical experiments led to a number of clinical trials using anti-adhesion therapy in an attempt to reduce injury following myocardial infarction, stroke and traumatic injury. Unfortunately, these trials did not demonstrate a protective effect in spite of the very strong pre-clinical data. In an attempt to understand these clinical studies, we examined ischemia times in the experimental setting of myocardial and cerebral ischemia-reperfusion that led to the clinical trials and found ischemia times to be generally less than 1.5 hours with the majority of times being between 30 and 60 min. It is possible that failure of the clinical trials can be explained by the extended ischemia times that occur in the clinical setting. Thus, in preliminary experiments, we increased the ischemia time up to 2 hours in hind limb ischemia and reperfusion in the mouse and found that there was indeed a CD18 adhesion molecule-dependent injury up to 60 minutes. However, when the ischemia time was increased to 90 minutes, the CD18-dependent portion of injury was completely eliminated. Still, treatment with the broad spectrum cysteine protease inhibitor z-VAD or by blocking complement activation protected from injury even for these extended ischemia times. Thus, we propose to examine mechanisms other than anti-adhesion therapy for the treatment of extended ischemia followed by reperfusion. In this application we propose the following specific aims: 1) To determine: a) whether leukocytes contribute to tissue injury when the ischemia time is sufficient to result in CD18-independent injury, and b) the length of ischemia that can be rescued by blocking caspase activation. 2) To determine the effect of Bcl-2 over-expression in various cell types (myeloid, lymphoid, muscle, endothelial) on the injury following extended ischemia and reperfusion. 3) To determine the role of the death receptors Fas and TNF, R1 in DNA strand-breaks, caspase activation and muscle injury following extended ischemia and reperfusion. 4) To determine the role of endothelial cell apoptosis following extended ischemia and subsequent reperfusion.