Epithelial-mesenchymal transition of biliary epithelial cells during cholestatic fibrosis

Introduction: Epithelial-mesenchymal transition (EMT) is a process in which epithelial cell layers lose polarity plus cell-cell contacts and undergo cytoskeleton reorganization. This phenomenon has been observed in the pathogenesis of cancer. In addition, recently, EMT was also shown to be involved in renal fibrosis upon ureteral obstruction. In order to study whether biliary epithelial cells (BEC) are able to undergo EMT during fibrogenesis we analyzed liver tissues in a bile-duct ligation (BDL) model of rats and human liver biopsies with cholestatic fibrosis.

Methods: Liver tissue of sham operated and BDL-rats was investigated by immunochemistry. Antibodies against epithelial (E-cadherin, cytokeratin, β-catenin) and mesenchymal (vimentin, α-smooth-muscle-actin (α-SMA), desmin) marker proteins were used for immunhistochemical detection and subsequent analysis using confocal microscopy. The involvement of TGF-β transmitted signaling pathways was shown by detection of phosphorylated (p-)smad2. In addition, 60 liver biopsies from patients with cholestatic fibrosis were used to analyze similar processes in the human liver. To study contribution of BEC in extracellular matrix (ECM) production we performed laser microdissection combined with real-time PCR.

Results: In rats, periportal fibrogenesis and prominent biliary epithelial proliferation was induced by BDL. Immunohistochemistry with antibodies against mesenchymal marker proteins revealed α-SMA, desmin and vimentin staining of vascular smooth muscle cells, sinusoidal and periportal myofibroblasts. However, in addition, immunochemistry using vimentin antibodies showed pronounced staining of BEC. In order to prove mesenchymal and epithelial specific characteristics of BEC, we analysed double-stained sections by confocal laser microscopy. Human and murine vimentin-positive BEC showed additionally a defined immunostaining for epithelial marker proteins such as E-cadherin, cytokeratins and β-catenin. Nuclear localization of p-smad2 indicates that vimentin-positve BEC are TGF-β activated. Further, we observed collagen I mRNA expression in laser microdissected bile ducts.

Conclusion: In summary, bile duct cells gain mesenchymal specific characteristics in the presence of activated TGF-β signaling pathways during fibrogenic processes due to cholestasis. The detection of collagen I mRNA in bile duct cells leads to the suggestion that they participate in extracellular matrix production.

EMT - fibrosis - liver