Lineage tracing demonstrates no evidence of cholangiocyte epithelial‐to‐mesenchymal transition in murine models of hepatic fibrosis

Liver fibrosis results from the deposition of excessive, abnormal extracellular matrix (ECM) by cells that differentiate into fibrogenic myofibroblasts during chronic liver injury. The biliary fibrosis subtype, which arises from persistent cholangiocyte injury, develops primarily in the portal/periportal regions and is characterized by a ductular reaction (a duct-like proliferation of bipotential progenitor cells, also called oval cells). Unlike other forms of fibrosis in which hepatic stellate cells are the dominant source of myofibroblasts, portal fibroblasts are a major myofibroblast precursor population in biliary fibrosis.1,2 A significant ongoing controversy is whether hepatic epithelial cells that undergo an epithelial-to-mesenchymal transition (EMT) represent another candidate myofibroblast precursor pool.3–10 EMT describes the phenomenon whereby epithelial cells adopt the structural and functional characteristics of mesenchymal cells with the acquisition of motility, loss of cell-cell contacts, development of a flat, spindle-like shape, downregulation of epithelial markers such as E-cadherin and keratins, and gain of mesenchymal markers such as vimentin and fibronectin. Substantial experimental evidence supports the occurrence of EMT in embryonic development and tumor metastasis, processes in which the motility phenotype of the transitioned cells is essential.11–13 For tissue fibrosis, however, there are conflicting data on whether or not EMT occurs. Evidence favoring hepatocyte EMT primarily comes from cell culture studies, although an in vivo lineage tracing study also suggested that hepatocytes in mouse models of fibrosis express the putative EMT marker S100A4 (fibroblast-specific protein 1 [FSP1]).14 Evidence favoring biliary EMT, in contrast, comes largely from immunohistochemical studies of fibrotic human and rodent livers that identified cholangiocytes co-expressing epithelial markers (especially the cholangiocyte marker keratin 19 [K19]) and mesenchymal markers (i.e. S100A4, vimentin, and heat shock protein 47 [HSP47]).3–7,14 Notably, few of these studies reported co-expression of cholangiocyte markers with the definitive myofibroblast marker α-smooth muscle actin (α-SMA), and none demonstrated collagen deposition by cholangiocytes or their derivatives. Some studies have proposed that EMT leads to myofibroblast accumulation through a two-stage process. In the first stage, epithelial cells adopt a mesenchymal phenotype, while in the second stage, these mesenchymal cells further transition to myofibroblasts as part of what has been termed an epithelial-to-myofibroblast transition (EMyT).15–17 Although not stated as such in the literature, the debate in liver fibrosis has focused largely on whether epithelial cells undergo EMyT, thereby contributing to the population of fibrogenic myofibroblasts.8–10,15,18 Definitive evaluation of EMT requires the use of lineage tracing technology, whereby epithelial cells are irreversibly tagged with a heritable label that persists regardless of the presence or absence of epithelial markers. (For clarity, the term EMT will be used throughout to refer collectively to both EMT and EMyT). A recent lineage tracing study in which β-galactosidase was expressed under the control of the hepatocyte marker albumin in transgenic mice also expressing a collagen marker provided strong evidence against hepatocyte EMT in the carbon tetrachloride (CCl4) model of fibrosis.8 A similar study carried out with K19-CreERT x Rosa26-YFP (yellow fluorescent protein) mice found no evidence in the CCl4 or bile duct ligation (BDL) models that cholangiocytes ever expressed α-SMA or collagen.9 Although this work demonstrated that labeled K19-positive cells did not become myofibroblasts, the possibility remained that K19-positive cells undergoing EMT were not labeled or that K19-negative cholangiocyte precursors underwent EMT.19–27 Therefore, we undertook lineage tracing studies using Alfp-Cre x Rosa26-YFP mice, enabling us to track the behavior of virtually all bipotential epithelial progenitors and their progeny in liver injury.28,29