PROJECT SUMMARY/ABSTRACT Hepatic ischemia/reperfusion injury (IRI), an innate immune-driven inflammation response, is a major obstacle limiting the success of orthotopic liver transplantation (OLT) in patients with end-stage liver disease and those with tumors of hepatic origin. Although significant progress has been made in better appreciation of the liver inflammatory cascade by IR-stress, much less is known about its resolution, which may affect not only the severity of tissue injury itself but also, more importantly, the long-term outcomes. Recent studies document striking cytoprotective functions of hrRLX (recombinant human relaxin-2) against IR-stress in mouse OLT models via hepatocyte glucocorticoid receptor (GR) signaling; while polarizing macrophage activation via Notch1 promoted IRI-OLT resistance. These experimental findings, supported by a clinical evidence of enhanced GR/Notch1 phenotype needed for IRI resistance in human OLT, prompted to propose that rhRLX may function as a novel GR agonist and glucocorticoid (GC) mimetic in liver transplantation. Pilot studies also point to anti- fibrotic functions of the cognate RXFP1 receptor, a GR-independent RLX-2 binding partner. As the conventional murine OLT model offers a limited translational utility, this project will be dissecting GR – RXFP1 molecular interplay in mice expressing human RXFP1 gene; as well as testing new concepts of RXFP1-driven hepatic rejuvenation of discarded human livers during hypothermic machine preservation. A newly discovered divergent role of GR–RXFP1 signaling axis in the “acute” and “resolution” phase of IRI-OLT inflammation, has prompted to put forth a novel and heretofore untested overall hypothesis, that: 1/ pharmacological rhRLX-induced GR enhancement will rescue OLT from acute IR-insult; while 2/ harnessing GR-independent rhRLX signaling via its cognate receptor, RXFP1, will promote homeostatic/anti-fibrotic functions in the inflammation resolution phase. Two interlocked aims explore this hypothesis: Aim 1: Delineate molecular mechanisms of rhRLX – GR hepatocellular protection in OLT (acute IRI-inflammation phase). Aim 1.1: Test hypothesis that hepatocellular Keap1-dependent Nrf2 signaling is indispensable for rhRLX – GR axis to prevent DAMPs release and innate inflammation in cold-stored donor livers. Aim 1.2: Test hypothesis that SIRT1 enhances GR-induced hepatocyte regenerative functions/autophagy in IR-stressed OLT. Aim 2. Delineate molecular mechanisms of Notch1 / RXFP1 anti-fibrotic functions in OLT (IRI-inflammation “resolution” phase). Aim 2.1: Test hypothesis that Notch1 (macrophage) – RXFP1 (T cell) cross-regulation is essential in the resolution of IRI – OLT inflammation. Aim 2.2: Test hypothesis that the activation of human RLX receptor exerts anti-fibrotic functions in the resolution of IRI-OLT inflammation in humanized RXFP1 “knockin” mouse system. Aim 2.3: Test hypothesis that activation of RXFP1 receptor during hypothermic machine preservat...