The Role of Bcl-2 in Ischemia-Reperfusion Injury

描述（由申请人提供）： 有或无再灌注的缺血发生在多种疾病中，包括心肌梗死、中风、失血性休克和器官移植。我们和其他人已经表明，缺血后再灌注可导致中性粒细胞（PMN）诱导的再灌注损伤，并且部分损伤可通过识别白细胞粘附分子的单克隆抗体得到改善。这些临床前实验导致了许多使用抗粘连疗法的临床试验，试图减少心肌梗死、中风和创伤性损伤后的损伤。不幸的是，尽管有非常强的临床前数据，这些试验并没有证明保护作用。为了理解这些临床研究，我们在心肌和脑缺血的实验环境中检查了缺血时间，再灌注导致临床试验，并发现缺血时间通常小于1.5小时，大多数时间在30和60分钟之间。临床设置。因此，在初步实验中，我们在小鼠后肢缺血和再灌注中将缺血时间增加至2小时，并发现确实存在长达60分钟的CD 18粘附分子依赖性损伤。然而，当缺血时间增加到90分钟时，损伤的CD 18依赖性部分被完全消除。尽管如此，用广谱半胱氨酸蛋白酶抑制剂z-VAD或通过阻断补体活化的治疗即使在这些延长的缺血时间内也保护免受损伤。因此，我们建议检查机制以外的抗粘连治疗延长缺血再灌注。在本申请中，我们提出了以下具体目的：1）确定：a）当缺血时间足以导致CD 18非依赖性损伤时，白细胞是否有助于组织损伤，和B）可以通过阻断半胱天冬酶激活来挽救的缺血时间长度。2)确定Bcl-2在各种细胞类型（骨髓、淋巴、肌肉、内皮）中的过表达对延长缺血和再灌注后损伤的影响。3)目的探讨死亡受体Fas和TNF、R1在缺血再灌注损伤中的作用。4)探讨内皮细胞凋亡在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.