Donor graft adenoviral iNOS gene transfer improve transplant survival and ischemia / reperfusion injury

Abstract

Background: Stress response genes, such as inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)-1, are known to be upregulated by ischemia/reperfusion (I/R) injury. We have previously shown that the L-arginine/NOS pathway has a protective effect on hepatic cold I/R injury. In this study, to examine whether hepatic iNOS gene transfer could improve transplant survival following severe I/R injury, donor grafts underwent prolonged cold-storage for 24 hrs prior to transplant. Methods: Adenovirus encoding iNOS (AdiNOS) or control gene vector (AdLacZ) was delivered to the liver by donor intravenous pretreatment 4 days before harvesting. Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 24 hrs preservation in cold UW. Serum LFTs, histopathology, hepatic ICAM-1 mRNA expression, and neutrophil infiltration were assessed after reperfusion. L-NIL and tin-protoporphyrin IX (SnPP) were used as selective inhibitors for iNOS and HO-1, respectively. Survival data (24 hrs preservation) was analyzed by Kaplan-Meier and log-rank x2 tests. Results: AdiNOS, but not AdLacZ, treatment at a relatively low titer (2 × 109 pfu) produced abundant iNOS protein expression in the donor liver before harvesting and in the recipient isograft as early as 1 hr post-reperfusion, indicating iNOS expression was due to the transduced AdiNOS and not induction of endogenous iNOS. Using the prolonged cold storage (24 hrs) model of severe I/R injury, animals treated with AdiNOS showed significantly improved survival from 30% to 70% (table, # indicates p <0.05 vs. saline or AdLacZ groups). AdiNOS delivery also markedly decreased serum AST/ALT levels and tissue necrosis. Hepatic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (mean±SE, n=3-6 animals per group, p < 0.05). HO-1 protein expression was induced in the AdiNOS-treated, but not AdLacZ-treated, donor liver grafts. Administration of L-NIL (30 mg/kg iv) or SnPP (10 μM/kg iv) partially abrogated the protection afforded by AdiNOS (data not shown). Conclusion: These results demonstrate that AdiNOS pretreatment decreases hepatic transplant preservation injury. With severe I/R injury due to prolonged cold storage, AdiNOS delivery also improves transplant survival, indicating the clinical relevance of this treatment modality. Hepatic HO-1 induction by iNOS transduction may account in part for the protective effect of donor pre-treatment with AdiNOS. Tabled 1 Two-week animal survival after liver transplantation. Treatment n Survival (days) 14-days survival Saline 10 1, 1, 1, 2, 3, 3, 4, >14 ×3 3/10 (30.0%) AdiNOS 10 2, 4, 5, >14 × 7 7/10 (70.0%)# AdLacZ 8 1, 1, 2, 2, 3, 5, >14 × 2 2/8 (25.0%) Open table in a new tab Tabled 1 Serum AST (18 hr CIT) (IU/L) Necrotic area (%) ICAM-1 mRNA (fold increase) Neutrophil (/mm2) HO-1 band density (/normal liver) Group 6h 24h 48h 48h 3h 48h Donor liver 3h Ad-LacZ 2314 ±324 3434 ±814 1064 ±405 10.3 ±6.8 13.3 ±4.7 61.9 ±17.7 1.1 ±0.5 1.8 ±0.6 Ad-iNOS 1367 ±163* 1402 ±276* 296 ±50 0.6 ±0.9* 7.3 ±3.6* 13.9 ±8.8* 5.7 ±1.0* 3.1 ±0.3 Open table in a new tab