Liver Preservation for Transplantation

描述（由申请人提供）：肝移植物中发生延迟性实质（肝细胞）杀伤，约4小时后开始，可导致移植物功能障碍和衰竭。迟发性实质细胞损伤的机制仍然知之甚少。我们的初步实验表明，线粒体通透性转换（MPT）的开始在延迟实质细胞杀伤中起着关键作用。因此，我们的具体目标是：1）阐明线粒体内膜透化在肝移植延迟性肝细胞损伤中的作用和机制; 2）确定MPT与微循环障碍、白细胞粘附和移植肝炎症的关系; 3）表征c-Jun N-末端激酶亚型如何（JNK 1和JNK 2）有助于MPT的发生和肝移植失败和4）为了检验HIF-1/脯氨酰羟化酶氧-感测信号级联阻断MPT并使对肝移植物的储存/再灌注损伤最小化。我们的研究利用活体多光子显微镜的新兴技术来可视化活体大鼠和小鼠肝移植物中单个肝细胞及其细胞器内的参数指示荧光团。具体来说，我们将确定肝移植物中的线粒体功能障碍是否是由于MPT的发生，以及功能障碍是否会导致坏死和细胞凋亡;增强的活性氧形成如何促进MPT的发生;以及线粒体钙调节异常是否有助于MPT的发生。此外，我们将评估竞争的假设，再灌注引起微循环障碍和炎症，促进氧化应激和MPT的发病或MPT依赖性细胞死亡激活炎症和微循环障碍。在前期的支持中，我们发现JNK激活发生在肝移植后，药物抑制JNK可减少移植物损伤，提高移植物存活率。两种JNK亚型，JNK 1和JNK 2，在肝脏中表达。我们将比较JNK 1和JNK 2缺陷肝移植物的损伤和存活，并确定JNK亚型缺陷对MPT诱导、微循环障碍和移植肝移植物中炎症反应的影响。基于初步的实验，我们期望表明JNK 2促进MPT的发生和肝细胞损伤肝移植物。在最后一系列的实验中，我们将评估乙基-3，4-二羟基苯甲酸酯（EDHB），脯氨酰羟化酶抑制剂，激活氧传感级联反应，对肝移植损伤和保护机制的特点，包括MPT抑制，减少氧化应激，减少非实质细胞损伤和血红素加氧酶-1（HO-1）的表达增加的影响。这些实验将导致更全面地了解与改善边缘供体肝脏的功能和存活相关的储存/再灌注损伤的肝移植损伤的发病机制。几乎在每例人肝移植中，转氨酶的增加都远远超过正常上限，表明每例肝移植物的储存/再灌注。因此，该项目的新信息有可能改善所有人类肝脏移植的结果。延迟性肝实质（肝细胞）杀伤发生在几乎每一个人类肝移植，当严重时，是最初的功能差和移植失败需要再次移植的主要原因。该项目将解决延迟肝细胞杀伤的潜在机制，并测试线粒体渗透性转变的发生具有关键作用的假设。从拟议的实验中获得的信息将有助于改善临床结果，不仅是所谓的边缘供体肝脏，但几乎每一个肝移植。

DESCRIPTION (provided by applicant): Delayed parenchymal (hepatocellular) killing occurs in liver grafts that begins after about 4 h and can lead to graft dysfunction and failure. The mechanisms of delayed parenchymal cell injury develops remain poorly understood. Our preliminary experiments indicate that the onset of the mitochondrial permeability transition (MPT) plays a key role in delayed parenchymal cell killing. Accordingly our specific aims are to 1) elucidate the role and mechanisms of mitochondrial inner membrane permeabilization in delayed hepatocellular injury to liver grafts; 2) determine the relationship of the MPT with microcirculatory disturbances, leukocyte adhesion and inflammation to liver grafts; 3) characterize how c-Jun N-terminal kinase isoforms (JNK1 and JNK2) contribute to MPT onset and liver graft failure and 4) to test the hypothesis that activation of the HIF-1/prolyl hydroxylase oxygen-sensing signal cascade blocks the MPT and minimizes storage/reperfusion injury to liver grafts. Our studies utilize the emerging technology of intravital multiphoton microscopy to visualize parameter- indicating fluorophores inside individual hepatocytes and their organelles in liver grafts of living rats and mice. Specifically, we will determine whether mitochondrial dysfunction in liver grafts is due to MPT onset and if dysfunction leads to necrosis and apoptosis; how enhanced ROS formation contributes to MPT onset; and if mitochondrial calcium dysregulation contributes to MPT onset. Additionally, we will evaluate competing hypotheses that reperfusion causes microcirculatory disturbances and inflammation that promote oxidative stress and MPT onset or that MPT-dependent cell death activates inflammation and microcirculatory disturbances. During the previous period of support, we showed that JNK activation occurs after liver transplantation and that pharmacological inhibition of JNK decreases graft injury and improves graft survival. Two JNK isoforms, JNK1 and JNK2, are expressed in liver. We will compare injury and survival of JNK1 and JNK2 deficient liver grafts and determine the effects of JNK isoform deficiency on MPT induction, microcirculatory disturbances and the inflammatory response in transplanted liver grafts. Based on preliminary experiments, we expect to show that JNK2 promotes MPT onset and hepatocellular injury to liver grafts. In a final series of experiments, we will assess the effects of ethyl-3,4-dihydroxybenzoate (EDHB), a prolyl hydroxylase inhibitor that activates the oxygen sensing cascade, on liver graft injury and characterize mechanisms of protection, including MPT inhibition, decreased oxidative stress, decreased nonparenchymal cell injury and increased expression of heme oxygenase-1 (HO-1). These experiments will lead to a fuller understanding of the pathogenesis of liver graft injury from storage/reperfusion injury relevant to improving the function and survival of marginal donor livers. Transaminases increase well beyond the upper limit of normal in virtually every human liver transplantation, indicating storage/reperfusion to every liver graft. Thus, new information from this project has the potential of improving the outcome of all human liver transplantations. Delayed parenchymal (hepatocellular) killing occurs in virtually every human liver transplantation and when severe is a principal cause of initial poor function and graft failure requiring retransplanation. This project will address mechanisms underlying delayed hepatocellular killing and test the hypothesis that onset of the mitochondrial permeability transition has a key role. The information gained from the proposed experiments will be useful for improving clinical outcomes of not only so-called marginal donor livers but of virtually every liver graft.