The Role of Bcl-2 in Ischemia-Reperfusion Injury

Bcl-2 在缺血再灌注损伤中的作用

• 批准号: 6611548
• 负责人: ROBERT K WINN
• 金额: $ 30.02万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611548
• 关键词: BCL2 gene /protein; DNA damage; apoptosis; biological signal transduction; cell line; clinical research; cysteine endopeptidases; genetically modified animals; histochemistry /cytochemistry; human tissue; injury; ischemia; laboratory mouse; leukocytes; neutrophil; polymerase chain reaction; reperfusion; smooth muscle; striated muscles; tissue /cell culture; transfection

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.

描述（由申请人提供）： 伴有或不伴有再灌注的缺血发生在多种疾病中，包括心肌梗塞、中风、失血性休克和器官移植。我们和其他人已经证明，缺血再灌注可导致中性粒细胞 (PMN) 诱导的再灌注损伤，并且部分损伤可以通过识别白细胞粘附分子的单克隆抗体得到改善。这些临床前实验引发了许多使用抗粘连疗法的临床试验，试图减少心肌梗塞、中风和外伤后的损伤。不幸的是，尽管有非常有力的临床前数据，但这些试验并未证明具有保护作用。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，发现缺血时间通常小于 1.5 小时，大多数时间在 30 至 30 小时之间。 60 分钟 临床试验的失败可能是由于临床环境中发生的缺血时间延长所致。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至2小时，发现确实存在长达60分钟的CD18粘附分子依赖性损伤。然而，当缺血时间增加到90分钟时，CD18依赖性损伤部分被完全消除。尽管如此，使用广谱半胱氨酸蛋白酶抑制剂 z-VAD 或阻断补体激活进行治疗，即使在缺血时间延长的情况下也能免受损伤。因此，我们建议检查抗粘连疗法以外的机制来治疗长期缺血再灌注。在本申请中，我们提出以下具体目标：1) 确定：a) 当缺血时间足以导致 CD18 独立损伤时，白细胞是否会导致组织损伤，b) 可以通过阻断来挽救的缺血时间长度半胱天冬酶激活。 2) 确定Bcl-2在各种细胞类型（骨髓细胞、淋巴细胞、肌肉细胞、内皮细胞）中过度表达对长期缺血和再灌注后损伤的影响。 3) 确定死亡受体 Fas 和 TNF、R1 在长时间缺血和再灌注后 DNA 链断裂、半胱天冬酶激活和肌肉损伤中的作用。 4)确定长期缺血和随后的再灌注后内皮细胞凋亡的作用。