*Purpose: The clinical relevance and tissue-specific role of Notch1 activation, a highly conserved transmembrane receptor involved in cell fate decision, proliferation and regeneration, in orthotopic liver transplantation (OLT) remains to be elucidated. We utilized a clinically-relevant mouse OLT model, and screened human liver transplant biopsies (Bx) to dissect the role of hepatic Notch1 signaling in IR-stressed OLT.

*Methods: In the clinical arm, hepatic Bx were obtained from liver transplant patients (n=60) at 2h post-reperfusion (prior to abdominal closure). The expression of Notch1 intracellular domain (NICD) target gene was evaluated by Western blots. In the experimental arm, WT or myeloid-specific Notch1 deficient (mN1-KO) livers (C57/BL6) subjected to extended (18h) ex-vivo cold storage were transplanted into syngeneic WT recipients; OLT and sera samples were collected at 6h post-reperfusion. Some mouse recipients were treated with a putative Notch1 activator, Serelaxin (SER, 5μg/kg i.v. at reperfusion).

*Results: Post-reperfusion NICD levels in clinical OLT Bx correlated negatively with mRNA levels coding for TLR4 (r=-0.334, p=0.013), CXCL10 (r=-0.286, p=0.034) and Cathepsin G (neutrophil marker; r=-0.343, p=0.010); as well as with sAST (r=-0.381, p=0.003) and sALT (r=-0.367, p=0.004) levels at POD1. Liver Bx with high-NCID levels (n=30) were characterized by decreased expression of TLR4/CXCL10/Cathepsin G (p<0.05), along with lower sAST (252±37 vs 858±373 IU/L, p=0.029) and sALT (256±34 vs 434±65 IU/L, p=0.037) at POD1; and showed improved rejection-free OLT survival (3-years: 87% vs 68%, p=0.076, median follow-up of 1287 days) as compared to low-NICD group (n=30). Treatment of mouse OLT with SER: 1/ increased NICD expression; 2/ decreased sAST levels (1315±520 vs 3204±1539 IU/L, n=10/10, p<0.001), Suzuki's histological grading (p<0.05), frequency of TUNEL+ cells (p<0.05); 3/ inhibited neutrophil/macrophage infiltration (p<0.05); 4/ depressed CXCL10/MCP1/CXCL2 mRNA levels (p<0.05); and 5/ improved overall survival (2-weeks: 60% vs 31%, p=0.016), all documenting the therapeutic potential of OLT-specific Notch1 activation. In marked contrast to WT liver grafts, myeloid-deficient Notch1 livers (mN1-KO>WT) suffered exaggerated IR-damage, evidenced by sALT (9127±4276 vs 4633±2678 IU/L, n=7/7, p=0.020), Suzuki’s histological grading (p<0.05), frequency of TUNEL+ cells (p<0.05); increased neutrophil/macrophage infiltration (p<0.05); and enhanced CXCL10/MCP1/CXCL2 (p<0.05) mRNA levels.

*Conclusions: This translational study highlights the cytoprotective role of hepatic Notch1 activation, especially in donor liver Kupffer cells, to regulate innate immune-driven inflammation response and preserve OLT function. These findings provide the rationale for Notch1 inducing novel therapeutic strategies in IR-stressed liver transplant recipients.

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