TGFβ-Mediated Epithelial-to-Mesenchymal Transition in Human Cholangiocyte Cell Cultures Following Ischemia Reperfusion Injury

Abstract

Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) is a complication more commonly observed in DCD liver recipients and is associated with inferior outcomes. The molecular mechanisms of IC are largely unknown but a recent study points to possible ischemia-induced genetic reprogramming of the biliary epithelium into mesenchymal-like cells. The main objective of this study was to determine if this epithelial to mesenchymal transition (EMT) in cholangiocytes after exposure to DCD conditions are regulated via Transforming Growth Factor beta (TGFβ) autocrine signaling. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to model DCD liver donation. Another model involved stimulating cells with TGFβ in the absence of ischemia and served as a positive control. Some cells were pretreated with small molecule TGFβ receptor antagonists prior to injury in both models. Before beginning studies, the effects of injury, substrate, and time on total cell number and viability in vitro were characterized. Injured groups exhibited significant increases in total cell number over time, particularly between Day 1 and Day 4, while uninjured populations remained relatively stable. By Day 4, the disparity in cell number and viability between injured and uninjured groups diminished, identifying Day 4 as a critical convergence point for comparative analyses. Substrate had minimal impact on the injury response at Day 4, supporting its use as an optimal time point. For cross-condition comparisons, EMT was characterized by assays of cell migration, cell morphology, and differential gene and protein expressions. Cholangiocytes exposed to DCD conditions and/or TGFβ displayed migratory behaviors and gene expression patterns consistent with EMT. Cell viability and E-cadherin expression fell while migration and expression of vimentin and TGFβ increased. Each of these effects in the DCD model were significantly mitigated by selective small molecule serine-threonine kinase inhibitors of TGFβ receptor signaling. Similarly, selective TGFβ receptor inhibitors were successful at mitigating TGFβ synthesis and release, vimentin, and SMAD4 elevated expressions, but did not restore E-cadherin expression in cholangiocytes undergoing EMT by exogenous TGFβ. Our findings show that EMT occurring in human cholangiocytes after exposure to DCD conditions is modulated by upstream signaling from autocrine derived TGFβ, and small molecule serine-threonine kinase inhibitors may be potential therapeutic targets for cholangiopathy after DCD liver transplantation