Liver Preservation for Transplantation

描述(由申请人提供)：延迟的实质(肝细胞)杀伤发生在肝移植后约4小时，可导致移植功能障碍和失败。延迟性实质细胞损伤发生的机制尚不清楚。我们的初步实验表明，线粒体通透性转变(MPT)的开始在延迟性实质细胞杀伤中起着关键作用。因此，我们的具体目标是：1)阐明线粒体内膜通透性在移植物迟发性肝细胞损伤中的作用和机制；2)确定MPT与移植物微循环障碍、白细胞黏附和炎症的关系；3)研究c-jun氨基末端激酶亚型(JNK1和JNK2)在移植物MPT发病和肝移植失败中的作用；4)验证HIF-1/Pro羟化酶氧敏信号级联激活阻断MPT，将移植肝的保存/再灌注损伤降至最低的假说。我们的研究利用新兴的活体多光子显微镜技术，在活体大鼠和小鼠的肝移植中显示单个肝细胞及其细胞器中的参数指示荧光团。具体而言，我们将确定移植肝的线粒体功能障碍是否与MPT的发病有关，以及功能障碍是否会导致坏死和细胞凋亡；ROS的形成增强如何导致MPT的发病；以及线粒体钙调节失调是否与MPT的发病有关。此外，我们将评估相互竞争的假说，即再灌注导致微循环障碍和炎症，从而促进氧化应激和MPT的发生，或者MPT依赖的细胞死亡激活炎症和微循环障碍。在之前的支持期间，我们证明了JNK的激活发生在肝移植后，药物抑制JNK可以减少移植物损伤，提高移植物存活率。两种JNK亚型JNK1和JNK2在肝脏中表达。我们将比较JNK1和JNK2缺陷型移植肝的损伤和存活情况，并确定JNK亚型缺陷对移植肝MPT诱导、微循环障碍和炎症反应的影响。在初步实验的基础上，我们期望证明JNK2促进MPT的发生和肝细胞对移植肝的损伤。在最后一系列实验中，我们将评估3，4-二羟基苯甲酸乙酯(EDHB)对移植肝损伤的影响，包括抑制MPT、减少氧化应激、减少非实质细胞损伤和增加血红素加氧酶-1(HO-1)的表达。这些实验将有助于更全面地了解供肝保存/再灌注损伤的发病机制，从而改善边缘供肝的功能和存活。在几乎所有的人类肝移植中，转氨酶的升高远远超过正常上限，这表明每一次肝移植都需要保存/再灌流。因此，来自该项目的新信息有可能改善所有人类肝脏移植的结果。延迟的肝实质(肝细胞)杀伤几乎发生在每一个人的肝移植中，当严重时是最初功能不佳和移植物失败的主要原因，需要重新移植。这个项目将解决延迟肝细胞杀伤的潜在机制，并测试线粒体通透性转变的开始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所谓的边缘供者肝脏，而且实际上是每一种肝移植的临床结果。

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.