*Purpose: While hepatocytes are affected by ischemia-reperfusion injury (IRI) especially under normothermic stress, liver sinusoidal endothelial cells (LSECs) are particularly vulnerable to cold storage-induced IR-insult. Although hepatic endothelium damage during cold preservation is considered a trigger for full-blown liver IRI, little is known about the molecular mechanisms by which LSECs may affect IRI outcomes in transplant recipients. A recent experimental study showed cold stress activated ferroptosis through accumulation of lipid reactive oxygen species. Here, we aimed to assess the role of hepatic ferroptosis during ex-vivo donor organ cold storage, a major problem in the context of orthotopic liver transplantation (OLT).

*Methods: Ferroptosis inhibitor (Fer-1) was incubated with wild type (WT) mouse liver grafts subjected to extended cold storage (4°C/18 hours in UW). Hepatocytes and LSECs were isolated from WT or NRF2 knockout (KO) livers to investigate the influence of cold-stress on liver cells function. In parallel, human liver graft biopsies (pre-reperfusion; n=50) were collected to evaluate ferroptosis in human OLT.

*Results: The IR-damage in mouse OLT significantly decreased in donor livers stored with Fer-1 compared to untreated controls, evidenced by AST/ALT levels and Suzuki’s scores (p<0.01). Immunofluorescence of cold stored-donor livers revealed that malondialdehyde (MDA) expressed mainly in LSECs. Cell death assay and MDA expression in hepatocytes and LSECs showed LSECs were particularly sensitive to cold stress-induced cell damage. However, adjunctive use of Fer-1 significantly reduced cell death programs in LSECs (4.5±0.8 vs. 23.3±4.5 cells/HPF, p<0.05). Further, NRF2 KO confirmed LSECs to be particularly susceptible to cold stress (cell death: WT vs NRF2KO, p<0.001). In human donor liver biopsies, the expression of NRF2 significantly correlated with GPX4, a key regulator of ferroptosis, which was negatively correlated with mRNA levels coding for TLR2, TLR9, IL-17, CXCL10, and cathepsin G in human OLT samples.

*Conclusions: This is the first evidence that ferroptosis death pathway via NRF2-mediated signaling is instrumental to the susceptibility of LSECs in cold liver preservation-mediated stress. This study provides the basis of novel therapeutic strategies in OLT by using adjunctive ferroptosis inhibitor for the protection of LSECs in cold preserved donor livers.

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