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

Abstract

Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial-to-mesenchymal transition (EMT) to become matrix-producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp-Cre × Rosa26-YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor-β1 alone or in combination with tumor necrosis factor-α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E-cadherin, and expression of α-smooth muscle actin (α-SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp-Cre × Rosa26-YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl(4) ) (3 weeks), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α-SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions.

Conclusion: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis.

Figure 1

Cre recombination efficiency in Alfp-Cre x Rosa26-YFP mouse livers is high. Representative split color and merged panels from immunostaining of liver sections following 3 weeks of DDC treatment. (A-D) YFP (Cy5, green) nearly completely co-localizes with K19 staining (Cy3, red). (YFP+K19)/K19 ratio = 1041 cells/1044 cells (99.7%). (E-H) YFP (Cy5, green) and A6 (Cy3, red). (YFP+A6)/A6 ratio = 903 cells/916 cells (98.6%). (I-L) YFP (Cy5, green) and HNF4α (Cy3, red). (YFP+HNF4α)/HNF4α ratio = 899 cells/905 cells (99.3%). Similar co-localization between YFP and either K19 or HNF4α was observed for BDL and CCl₄ models (not shown). Blue = DAPI; * = representative bile ducts; v = portal vein. Scale bar = 10 μm.

Figure 2

Cultured primary cholangiocytes can undergo EMT. Primary cholangiocytes were isolated from Alfp-Cre x Rosa26-YFP mice and cultured on type I collagen. (A) Phase contrast microscopy demonstrating fibroblast-like morphologic changes in cells treated with TGF-β1 alone and in combination with TNF-α for 72 hours compared with vehicle plus TGF-β receptor kinase-1 inhibitor (Inh) and TGF-β1 plus inhibitor. Scale bar = 50 μm. (B) K19 immunostaining (Cy3, red) of primary cholangiocytes completely co-localizes with YFP staining (Cy5, green). Blue = DAPI; scale bar = 20 μm. (C) Treatment with TGF-β1 alone and in combination with TNF-α resulted in intracellular relocalization of E-cadherin (arrowheads) and upregulation of α-SMA. Following combined treatment, rare cells with α-SMA stress fibers can be identified (arrow). These cells also express YFP, confirming their epithelial origin (inset). Scale bar = 20 μm. (D) Relative quantification of α-SMA mRNA expression by qRT-PCR. Graph represents average of 3 independent experiments each carried out in triplicate using cells isolated from the same animal. *P < 0.05 compared with vehicle controls.

Figure 3

Mouse livers demonstrate significant Sirius red staining in fibrosis models. (A) Relative quantification of Sirius red staining, expressed as fold-increase over control. *P < 0.05 compared with controls. (B) Sirius red staining of representative control section with Fast Green counterstain. (C-E) Representative sections from stained livers (C) after 3 weeks of DDC treatment, (D) 2 weeks post-BDL, and (E) 8 weeks post-BDL. Scale bar = 200 μm.

Figure 4

No evidence of epithelial-mesenchymal marker co-expression in mouse livers 2 weeks post-BDL. Split color and merged panels from representative mouse liver sections from 3 animals, stained with antibodies against (A-D) S100A4; (E-H) vimentin; (I-L) α-SMA; and (M-P) pro-collagen 1α2 (Pro-Col). Green = YFP (Cy5), red = mesenchymal marker (Cy3), blue = DAPI; * = representative bile ducts; v = portal vein. Scale bar = 20 μm.

Figure 5

No evidence of epithelial-mesenchymal marker co-expression in mouse livers 8 weeks post-BDL. Split color and merged panels from representative mouse liver sections from 2 animals, stained with antibodies against (A-D) S100A4; (E-H) vimentin; (I-L) α-SMA; and (M-P) pro-collagen 1α2 (Pro-Col). Green = YFP (Cy5), red = mesenchymal marker (Cy3), blue = DAPI. Scattered YFP-negative, S100A4-positive cells were seen in bile ducts (arrows), likely representing infiltrating lymphocytes. * = representative bile ducts; v = portal vein. Scale bar = 20 μm.

Figure 6

No evidence of epithelial-mesenchymal marker co-expression in mouse livers after 3 weeks of DDC treatment. Split color and merged panels from representative mouse liver sections from 2 animals, stained with antibodies against (A-D) S100A4; (E-H) vimentin; (I-L) α-SMA; and (M-P) pro-collagen 1α2 (Pro-Col). Green = YFP (Cy5), red = mesenchymal marker (Cy3), blue = DAPI. * = representative bile ducts; v = portal vein. Scale bar = 20 μm.

Figure 7

No evidence of YFP co-expression with the hepatic stellate marker desmin in murine models of biliary injury. Representative merged images of liver sections from mice (A) 8 weeks post-BDL, (B) after 3 weeks of CCl₄ treatment, and (C) after 3 weeks of DDC treatment. Note the distinct staining of long, thin cells in the sinusoids. Green = YFP (Cy5), red = desmin (Cy3), blue = DAPI. * = representative bile ducts; v = portal vein. Scale bar = 20 μm.