The knockout of telomere-independent RAP1 attenuates hepatic ischemia reperfusion injury by suppressing neutrophil activation

Abstract

BACKGROUND AND AIM: Telomere-independent Rap1, a component of the telosome protein complex, is a critical regulator of NF-κB and mediates inflammatory responses. However, the role of Rap1 on liver injury has not been clearly explored. In this study, we aim to investigate the roles of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying molecular mechanism. METHODS: The intragraft Rap1 expression profile, inflammatory cytokines/chemokines, and neutrophil infiltration were examined in human liver graft biopsies. The role of Rap1 in liver injury was also explored in Rap1 knockout mice with hepatic IRI. Histological changes, liver function, expressions of neutrophil chemoattractants and adhesion factors, together with neutrophil infiltration were compared between Rap1 knockout mice and wild type controls. The interaction between hepatic endothelial cells and neutrophil migration/activation was further explored under the regulation of RAP1. In addition, primary neutrophils isolated from Rap1 knockout or wild type mice were activated by fMLP. The migration and activation of neutrophil were compared between those primary neutrophils from Rap1 knockout mice and wild type controls. RESULTS: Over-expression of intragraft Rap1 was associated with lots of neutrophil infiltration (36.2/HPF in liver graft vs 3.5/HPF in normal liver, P=0.00) and high expressions of inflammatory cytokines/chemokines in human liver graft after transplantation. High expression of Rap1 was mainly expressed in neutrophils in liver graft. In mouse model, sinusoidal damage, liver function (ALT: 395.59 vs 280.67 U/L, p=0.024; AST: 522.886 vs 368.628 U/L, p=0.025), neutrophil infiltration (19.7 vs 9.2/HPF, p=0.001), expressions of neutrophil chemoattractants (CXCL2: 30.9 vs 12.7, p=0.037) and adhesion factors (ICAM1: 4.2 vs 2.4, p=0.049; VCAM1:4.5 vs 2.1 folds of normal liver; p=0.026), and activation of NF-κB were attenuated in Rap1 knockout mice. Furthermore, the knockout of Rap1 decreased the expressions of neutrophil chemoattractants and adhesion factors in endothelial cells and further suppressed neutrophil migration and activation. CONCLUSION: The knockout of Rap1 attenuates hepatic IRI by suppressing the neutrophil migration and activation. Rap1 might be the potential treatment target for prevention of liver graft injury.