*Purpose: Liver ischemia-reperfusion injury (IRI) is a major risk factor of early graft dysfunction and predisposes to rejection in orthotopic liver transplantation (OLT). Although Notch1 regulates macrophage activation in warm hepatic IRI, little is known as to how hepatocyte-specific Notch1 might affect liver IRI. We generated hepatocyte-specific Notch1-deficent (hNotch1-KO) mice (C57/BL6) to study putative role of hepatocyte Notch1 in IR-stressed livers. In parallel, we screened human liver transplant (LT) biopsies.

*Methods: Primary mouse hepatocytes isolated from WT (C57/BL6) and hNotch1-KO mice were exposed to hypoxia (1%, 3h) and reoxygeneration. At 3h and 6h of reoxygeneration, hepatocyte cultures were analyzed by Western blots. In the clinical arm, human hepatic biopsies obtained at 2h post-LT (n=55) were screened for Notch1, ATG5, ATG7, LC3B, Bcl-xL and Bcl2 (Western blots) and pro-inflammatory phenotype (RT-PCR). The correlation between hepatic Notch1 expression and clinical parameters (serum transaminases, LT survival) were assessed.

*Results: Unlike WT counterparts, Notch1-deficient hepatocytes were sensitive to oxidative stress, evidenced by enhanced HMGB1/Histone H3 release into culture media at 3h and 6h after reoxygeneration. Disruption of Notch1 signaling augmented the expression of BclxL, Bcl2 (anti-apoptotic markers), ATG5, ATG7, VPS34 (classical phosphatidylinositol 3-kinase) and LC3B (autophagy markers), but enhanced cleaved caspase 3 and DR5 (death receptor 5) expression. Silencing DR5 (siRNA) in Notch1-deficient hepatocytes inhibited cleaved caspase 3 and increased LC3B expression, leading to cytoprotection against the oxidative stress. In the clinical arm, the hepatic expression of Notch1 correlated negatively with serum transaminase release at postoperative day 1. In addition, Notch1 positively correlated with ATG5 (p<0.0001), ATG7 (p<0.0001), LC3B (p=0.0168), Bcl2 (p=0.0162) and BclxL (p<0.0001) expression. When contrasted into low (n=28) vs. high (n=27) Notch1 expression groups, Notch1 low human LTs showed enhanced innate/adaptive immune activation (RT-PCR) and inferior overall graft survival (p=0.0445, 3 year: 66.3% vs 87.9%).

*Conclusions: Disruption of hepatocyte-specific Notch1 under IR-stress exerts cytodestructive function via DR5 signaling. By documenting the protective role of Notch1 in stressed hepatocytes, our findings add new insights to the complex function of Notch1 in LT recipients.

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