ABSTRACT Orthotopic liver transplantation (OLT) is the gold standard of care in patients with end-stage liver disease and those with tumors of hepatic origin. However, the scarcity of donor organs prompted the use of extended criteria “marginal” livers, which are particularly susceptible to ischemia-reperfusion injury (IRI), which predispose to acute/chronic rejection, and may require re-transplantation. We have introduced the concept of organ “rejuvenation”, i.e., conversion of the donor liver from the state of IRI-hypersensitivity to the homeostatic state of IRI-resistance. We have also proposed that SIRT1 deacetylase serves as a rheostat linking IR-stress with liver rejuvenation in both, mouse and human OLT. We have recently identified new regulators of hepatic resistance against warm vs. cold ischemia stress responses, i.e., Human Antigen R (HuR) and Hypoxia-Inducible Factor (HIF-1α). We have also discovered Ikaros (IKZF1), acts as a macrophage activation marker and exacerbates liver IRI. Importantly, we also found that preserved hepatocellular function/improved clinical outcomes in human OLT patients were associated with increased HuR/HIF-1α but depressed Ikaros levels in the liver biopsy samples. We hypothesize that crosstalk between hepatocyte HuR / HIF-1α and macrophage Ikaros provides a new means to regulate the adaptation of donor livers to IR-stress and reperfusion-mediated hepatic damage. Specific Aim 1: Determine mechanisms of hepatocyte HuR / HIF-1α crosstalk with SIRT1 in IRI-OLT. Hypothesis: Hepatocyte SIRT1 activation, controlled by distinct hypoxia/reoxygenation (H/R) requirements for HuR (warm H/R) vs. HIF-1α (cold H/R), provide new means to regulate adaptation of donor livers to IR-stress. 1.1: SIRT1 function is dependent on HuR signaling for anti-inflammatory responses in oxidative stress. 1.2: HuR/HIF-1α signaling in a mouse model of hepatic ischemia is temperature stress-dependent. 1.3: HIF-1α controls cold-induced IRI-OLT. 1.4: HuR controls warm-induced IRI-OLT. Specific Aim 2: Delineate mechanisms of macrophage Ikaros crosstalk with SIRT1 in IRI-OLT. Hypothesis: Macrophage Ikaros signaling exacerbates IRI-OLT by repressing SIRT1 transcription and M2 macrophage polarization. 2.1: Ikaros-SIRT1 myeloid axis influences hepatic HuR/HIF1α hypoxia sensing circuit in IRI-OLT. 2.2: Macrophage Ikaros signaling depends on SIRT1 transcription for M2 polarization. Specific Aim 3: Define mechanism of human liver rejuvenation under hypothermic machine preservation. Hypothesis: Manipulation of HIF-1α / SIRT1 axis during ex-vivo HMP improves hepatocellular function to rejuvenate human livers declined for transplantation due to preexisting poor quality. 3.1: Pharmacological stabilizer of HIF-1α protein synergizes with SIRT1 to improve human liver function. 3.2: Preconditioning with PHD-inhibitor, which activates/stabilizes HIF-1α, synergizes with enhanced SIRT1 signaling to ameliorate inflammation, promote cytoprotection, and rejuvenate hu...