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: 7056689
• 负责人: ROBERT K WINN
• 金额: $ 29.31万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056689
• 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）： 有或没有再灌注的缺血发生在多种疾病中，包括心肌梗塞，中风，出血性休克和器官移植。我们和其他人已经表明，缺血随后再灌注会导致中性粒细胞（PMN）诱导的再灌注损伤，并且可以用识别白细胞粘附分子的单克隆抗体来改善一部分损伤。这些临床前实验导致了许多临床试验，使用抗粘附疗法，试图减少心肌梗塞，中风和创伤性损伤后的损伤。不幸的是，尽管存在非常强大的临床前数据，但这些试验并未表现出保护作用。为了理解这些临床研究，我们在心肌和脑缺血 - 恢复灌注的实验环境中检查了缺血时间，这导致了临床试验，并发现缺血时间通常小于1.5小时，大部分时间在30至60分钟之间。 临床试验的失败可能通过临床环境中发生的延长缺血时间来解释。因此，在初步实验中，我们在小鼠缺血和再灌注中增加了2小时的缺血时间，并发现确实存在CD18粘附分子依赖性损伤长达60分钟。但是，当缺血时间增加到90分钟时，完全消除了依赖CD18的损伤部分。尽管如此，即使在这些扩展的缺血时间，也可以通过广谱半胱氨酸蛋白酶抑制剂Z-VAD进行治疗，或通过阻止补体激活。因此，我们建议检查除抗粘附疗法以外的其他机制，以治疗扩展缺血，然后再灌注。在此应用中，我们提出以下特定目的：1）确定：a）当缺血时间足以导致CD18独立损伤时，白细胞是否有助于组织损伤，b）可以通过阻止caspase激活来营救的缺血长度。 2）确定Bcl-2过表达在各种细胞类型（髓样，淋巴样，肌肉，内皮）中的影响对延长缺血和再灌注后损伤的影响。 3）确定死亡受体Fas和TNF的作用，R1在DNA链破裂中，胱天蛋白酶激活和肌肉损伤扩展和再灌注后。 4）确定延长缺血和随后再灌注后内皮细胞凋亡的作用。