DOI
https://doi.org/10.25772/7XC9-9660
Defense Date
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Pharmacology & Toxicology
First Advisor
Martin Mangino
Second Advisor
William Dewey
Third Advisor
Dana Selley
Fourth Advisor
Joseph Ritter
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.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
7-30-2025
Comments
Donation after circulatory death (DCD) has expanded the liver donor pool but is associated with ischemic cholangiopathy (IC), a severe complication leading to graft failure and re-transplantation. The mechanisms underlying IC remain poorly defined. This study investigated whether epithelial-to-mesenchymal transition (EMT) in human cholangiocytes exposed to DCD-simulated ischemia is driven by autocrine Transforming Growth Factor-beta 1 (TGFβ1) signaling. Primary human cholangiocyte cultures were exposed to combined warm and cold ischemia to simulate DCD injury or treated with exogenous TGFβ1. EMT was assessed through gene expression (qPCR), protein quantification (ICC, ELISA, flow cytometry), cell viability (Trypan Blue), and migration assays. Four selective ALK5/TGFβRI inhibitors (galunisertib, LY364947, RepSox, and SM16) were tested for their ability to block EMT-associated phenotypes.
Injury increased TGFβ1, vimentin, and SMAD4 expression, while reducing E-cadherin and cell viability. Exogenous TGFβ1 replicated these changes, confirming its sufficiency in driving EMT. Day 4 post-injury was identified as the optimal convergence point for comparative analysis. Galunisertib significantly mitigated EMT features in both models, including reductions in TGFβ1 secretion, SMAD4/vimentin expression, and cell migration, though E-cadherin restoration remained incomplete. Statistical significance was confirmed using ANOVA and post-hoc tests (α ≤ 0.05).
These findings indicate that DCD-related EMT in cholangiocytes is regulated by autocrine TGFβ1 signaling and may be pharmacologically attenuated via ALK5 inhibition. This study supports the hypothesis that EMT contributes to IC pathogenesis and highlights TGFβ receptor blockade as a potential therapeutic strategy to improve outcomes following DCD liver transplantation.