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. ? ? ?
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