MECHANISMS OF ENDOTHELIAL CELL DEATH IN ISCHEMIC LIVER

缺血性肝脏内皮细胞死亡的机制

• 批准号: 6177629
• 负责人: PIERRE A CLAVIEN
• 金额: $ 5.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2000-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177629
• 关键词: SDS polyacrylamide gel electrophoresis; apoptosis; biological signal transduction; calpain; cell type; ceramides; disease /disorder model; electron microscopy; enzyme activity; enzyme inhibitors; genetic manipulation; genetically modified animals; histopathology; immunocytochemistry; intermolecular interaction; laboratory mouse; laboratory rat; liver ischemia /hypoxia; liver transplantation; northern blottings; pathologic process; polymerase chain reaction; reperfusion; tumor necrosis factor alpha; vascular endothelium

Ischemic liver injury is a major clinical problem in shock, liver surgery and transplantation. Our initial results allow us to implicate apoptosis of sinusoidal endothelial cells (SEC) - an active process of cell suicide - as a central feature of reperfusion injury in rat models of hepatic ischemia. We have also discovered that calpains, a family of intracytoplasmic calcium dependent cysteine proteases, are activated during periods of ischemia and reperfusion (I/Rp) and in part mediate SEC apoptosis. We HYPOTHESIZE that ischemia sensitizes SEC to the effects of pro- apoptotic mediators released at the time of reperfusion, resulting in the apoptosis of SEC, even when the direct ischemic damage is reversible. The overall objective of this project is therefore to elucidate in vivo mechanisms of SEC apoptosis in rodent models of hepatic I/Rp, and design protective strategies to improve viability of the ischemic liver. The first objective is to characterize the temporal dynamics of SEC apoptosis in hepatic injury by monitoring characteristic microscopic, histomorphologic and biochemical markers of apoptotic cell death at successive time points during I/Rp. Second, we will determine the cellular mechanisms of calpain- mediated SEC apoptosis by identifying tissue localization of calpain activity and the relationship between calpain activation and other possible calpain related cellular effectors of apoptosis (caspase and ceramide) in a hepatic I/Rp whole-organ model. Third, we will design protective strategies by inhibiting mediators of SEC apoptosis or by manipulations designed to favor anti-apoptotic activities. We have shown that calpain inhibition is effective in protecting SEC in normothermic I/Rp. Unfortunately, calpain inhibitors are not as effective in cold I/Rp injury, relevant for transplantation, and are not water soluble reagents, limiting their clinical applicability. Because many interacting pathways lead to apoptosis, we anticipate that interventions at multiple levels will be necessary. In addition to using new water soluble calpain inhibitors, we will block FAS and TNFalpha, well characterized extracellular pro-apoptotic mediators that may be active in the I/Rp-injured liver. A potential regulatory role for the Bcl-2 gene, whose product is inhibitory at various levels of the apoptotic process, will be investigated using transgenic mice overexpressing Bcl-2. Our whole-organ model is uniquely suited to these studies, since - if known precedents hold - signaling for apoptosis most likely involves complex interactions among several cell types. Identification of mechanisms of SEC death in hepatic I/Rp injury and the ability to protect these cells may have significant clinical impact by allowing longer and safer preservation prior to transplantation and by lessening ischemic liver damage during non- transplant surgery and shock.

缺血性肝损伤是休克、肝脏手术和移植中的一个主要临床问题。我们的初步结果使我们能够将肝窦内皮细胞（SEC）的凋亡（细胞自杀的活跃过程）作为肝缺血大鼠模型再灌注损伤的核心特征。我们还发现钙蛋白酶（胞质内钙依赖性半胱氨酸蛋白酶家族）在缺血和再灌注 (I/Rp) 期间被激活，并部分介导 SEC 细胞凋亡。我们假设缺血使 SEC 对再灌注时释放的促凋亡介质的作用敏感，导致 SEC 凋亡，即使直接缺血损伤是可逆的。因此，该项目的总体目标是阐明肝 I/Rp 啮齿动物模型中 SEC 细胞凋亡的体内机制，并设计保护策略以提高缺血肝脏的活力。第一个目标是通过监测 I/Rp 期间连续时间点凋亡细胞死亡的特征性微观、组织形态学和生化标记物来表征肝损伤中 SEC 凋亡的时间动态。其次，我们将通过确定肝 I/Rp 整个器官中钙蛋白酶活性的组织定位以及钙蛋白酶激活与其他可能的钙蛋白酶相关细胞凋亡效应物（半胱天冬酶和神经酰胺）之间的关系来确定钙蛋白酶介导的 SEC 细胞凋亡的细胞机制。模型。第三，我们将通过抑制 SEC 细胞凋亡介质或通过旨在支持抗细胞凋亡活性的操作来设计保护策略。我们已经证明，钙蛋白酶抑制可有效保护常温 I/Rp 中的 SEC。不幸的是，钙蛋白酶抑制剂在与移植相关的冷I/Rp损伤中并不那么有效，并且不是水溶性试剂，限制了它们的临床适用性。由于许多相互作用的途径会导致细胞凋亡，因此我们预计需要进行多个层面的干预。除了使用新型水溶性钙蛋白酶抑制剂外，我们还将阻断 FAS 和 TNFα，这两种细胞外促凋亡介质可能在 I/Rp 损伤的肝脏中具有活性。 Bcl-2 基因的潜在调节作用（其产物在细胞凋亡过程的各个水平上具有抑制作用）将使用过度表达 Bcl-2 的转基因小鼠进行研究。我们的全器官模型特别适合这些研究，因为如果已知先例成立的话，细胞凋亡信号很可能涉及几种细胞类型之间的复杂相互作用。鉴定肝 I/Rp 损伤中 SEC 死亡的机制以及保护这些细胞的能力可能会产生重大的临床影响，因为可以在移植前进行更长时间和更安全的保存，并减少非移植手术和休克期间的缺血性肝损伤。