Orthotopic liver transplantation is a common therapy for many acquired and inherited disorders. Liver damage caused by ischemia and reoxygenation (termed ischemia/reperfusion or I/R) has been proposed to substantially contribute to the initial poor function (IPF) of transplanted livers, acute rejection, and graft failure.. Central mediators of I/R injury in the liver are reactive oxygen species (ROS) generated during reperfusion with oxygenated blood. Of the multiple forms of ROS potentially generated during reperfusion, O2-, H2O2, and OH will be addressed in the proposed studies. These ROS can cause direct damage to cellular proteins, lipids, and DNA, or they can act as intracellular second messengers to activate and/or inhibit signal transduction pathways that determine cell fates by altering the expression patterns of stress response genes. Central to the goals of this grant are to determine: 1) Which ROS are pathophysiologically important in I/R damage, 2) How do ROS lead to activation of AP-1 and NFkB signal transduction pathways following liver I/R, 3) In which subcellular compartments (i.e., nucleus, mitochondria, endoplasmic reticulum, or cytoplasm) do these ROS act to alter the activity of AP-1 and NFkB signal transduction pathways, and 4) Is activation of AP-1 and NFkB beneficial or detrimental to the liver following I/R injury. Several model systems will be used to address these questions including: 1) Recombinant adenoviral vectors to modulate the cellular redox state and inhibit/activate specific signal transduction pathways, 2) transgenic knockout mice deficient in certain signal transduction components, 3) partial lobar, liver I/R mouse model, and 4) a syngeneic rat liver transplant model. Preliminary data has demonstrated that ectopic expression of MnSOD using reombinant adenovirus protects the liver from warm I/R injury and concordantly reduces AP-1 and NFkB activation. The mechanisms of NFkB activation appear to involve redox mediated tyrosine phosphorylation of 1kBalpha. The hypothesis that subcellular compartmentalized ROS are important in mechanisms of liver I/;R injury is supported by the fact that ectopic expression of Cu/ZnSOD does not protect the liver from I/R damage. Based on our findings that OH radicals and AP-1 activation are increased in Cu/ZnSOD but not MnSOD or LacZ expression livers, and inhibition of Rac1 pathways by expression of the dominant inhibitor N17Rac1 also activates AP-1, one novel focus of our mechanistic studies on AP-1 activating following I/R injury will involve Rac1/PI3-kinase /PKK/GSK modulation of c-Jun phosphorylation. Recombinant adenoviral mutants for the proteins in this signal transduction pathway will be used to dissect its importance. Using recombinant expressing dominant inhibitors and transgenic knockout mice to block either AP-1 and NFkB activation, we will attempt to determine the importance of each of these pathways in mediating both acute hepatic toxicity and subacute inflammatory responses following I/R injury. In the last phase of this proposal, studies will be aimed at determining the relevance of findings in our mouse partial lobar warm I/R model to the rat liver transplant model of cold ischemia. In summary, this proposal will provide experimental and mechanistic paradigms for linking ROS formation in the liver following I/R injury to both acute damage and subsequent inflammation. Moreover, these studies may provide clinically relevant gene therapy approaches for minimizing organ damage following transplantation, which may ultimately increase the graft survival in orthotopic liver transplantation.
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