Oxidative-stress-induced biosignatures: a predictor of allograft dysfunction?

Abstract

BackgroundChronic graft dysfunction is the commonest cause of graft loss and is affected by perioperative factors. In liver transplantation, perioperative oxidative stress precipitates a senescence-associated secretory phenotype in biliary epithelial cells and this could be one of the factors responsible for fibrosis and allograft dysfunction. We aimed to examine effects of oxidative stress on biliary epithelial cells and assess the transcriptome of time-zero liver transplant biopsies for biomarkers of allograft dysfunction. MethodsTo model oxidative stress, the biliary epithelial cell H69 cell line was exposed to hydrogen peroxide in vitro. Immunofluorescence was performed post injury for protein expression associated with senescence and epithelial-mesenchymal transition. Similarly gene expression was analysed with both Taqman real-time PCR and a SYBR Green custom gene array (Thermo Fisher, Waltham, MA, USA). These experiments were repeated in triplicate. Similarly, the transcriptome of six time-zero liver transplant biopsy samples were analysed to examine in-vivo effects. A one-way ANOVA was performed for statistical analysis. FindingsOxidative stress induced gene expression upregulation of senescence marker p21 (p<0·001) and transforming growth factor β2 (TGFβ2) (p<0·001) during the first 48 h after injury to biliary epithelial cells. After this time these cells had increased expression of mesenchymal proteins (α smooth muscle actin and vimentin, p<0·001) with a decrease in epithelial markers (ZO-1 and E-cadherin, p<0·001). In-vitro pharmacological blockade of the ALK5 component of the TGFβ receptor abrogated this response. Data from the time-zero patient biopsy samples highlighted further potential candidate biomarkers, but none reached statistical significance. InterpretationOur results show that oxidative stress induces a senescence phenotype that triggers an epithelial dedifferentiation in biliary epithelial cells through TGFβ2 production. Marginal organs are particularly susceptible to the damage from oxidative stress. In conjunction with normothermic reperfusion, these targets might provide a unique opportunity in the pretransplant optimisation of donor organs, potentially minimising the impact of ischaemia reperfusion injury and allograft dysfunction. FundingNewcastle University Shire Bursary, Wellcome Trust Clinical Research Training Fellowship.