Abstract
Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) after DCD liver transplantation causes inferior outcomes. The molecular mechanisms of IC are currently unknown but may depend on ischemia-induced genetic reprograming of the biliary epithelium to mesenchymal-like cells. The main objective of this study was to determine if cholangiocytes undergo epithelial to mesenchymal transition (EMT) after exposure to DCD conditions and if this causally contributes to the phenotype of IC. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to mimic DCD liver donation. EMT was tested by assays of cell migration, cell morphology, and differential gene expression. Transplantation of syngeneic rat livers recovered under DCD conditions were evaluated for EMT changes by immunohistochemistry. Human cholangiocytes exposed to DCD conditions displayed migratory behavior and gene expression patterns consistent with EMT. E-cadherin and CK-7 expressions fell while N-cadherin, vimentin, TGFβ, and SNAIL rose, starting 24 hours and peaking 1–3 weeks after exposure. Cholangiocyte morphology changed from cuboidal (epithelial) before to spindle shaped (mesenchymal) a week after ischemia. These changes were blocked by pretreating cells with the Transforming Growth Factor beta (TGFβ) receptor antagonist Galunisertib (1 μM). Finally, rats with liver isografts cold stored for 20 hours in UW solution and exposed to warm ischemia (30 minutes) at recovery had elevated plasma bilirubin 1 week after transplantation and the liver tissue showed immunohistochemical evidence of early cholangiocyte EMT. Our findings show EMT occurs after exposure of human cholangiocytes to DCD conditions, which may be initiated by upstream signaling from autocrine derived TGFβ to cause mesenchymal specific morphological and migratory changes.
Highlights
Liver transplantation is the only potentially curative treatment for patients with end stage liver disease
Cells that were exposed to prolonged cold storage for 24 hours with or without prior warm ischemia for one hour displayed a mesenchymal morphology compared to their baseline cuboidal epithelial cell appearance
The molecular mechanisms of these phenotypic changes are unknown, it is hypothesized that the biliary epithelia first undergo an Epithelial to Mesenchymal Transition (EMT) that establishes the genetic reprogramming of the cells to transform them into cells with a mesenchymal phenotype
Summary
Liver transplantation is the only potentially curative treatment for patients with end stage liver disease. The increased warm ischemia time (WIT) with DCD transplants leads to significantly lower graft and recipient survival rates compared to DBD. This is due to higher rates of early allograft dysfunction, acute organ failure, and severe biliary complications (2–5). Ischemic cholangiopathy (IC), defined as non-anastomotic strictures of the intra- or extrahepatic biliary tree in the presence of a patent hepatic artery [2,3,4], is the most common serious biliary complication It has been reported in 4–12% and up to 34% within 6 and 12 months of DCD transplantation, respectively [5]. IC is a feared complication of liver transplantation because it can cause biliary sepsis, requiring multiple hospitalizations for invasive diagnostic and therapeutic interventions and eventually require re-transplantation, all of which reduce patient and graft survival [6,7,8,9]
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