Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis

Summary

The epithelial to mesenchymal transition has recently been implicated as a source of fibrogenic myofibroblasts in organ fibrosis, particularly in the kidney. There is as yet minimal evidence for the epithelial to mesenchymal transition in the liver. We hypothesized that this process in biliary epithelial cells plays an important role in biliary fibrosis and might be found in patients with especially rapid forms, such as is seen in biliary atresia. We therefore obtained liver tissue from patients with biliary atresia as well as a variety of other pediatric and adult liver diseases. Tissues were immunostained with antibodies against the biliary epithelial cell marker CK19 as well as with antibodies against proteins characteristically expressed by cells undergoing the epithelial to mesenchymal transition, including fibroblast-specific protein 1, the collagen chaperone heat shock protein 47, the intermediate filament protein vimentin, and the transcription factor Snail. The degree of colocalization was quantified using a multispectral imaging system. We observed significant colocalization between CK19 and other markers of the epithelial to mesenchymal transition in biliary atresia as well as other liver diseases associated with significant bile ductular proliferation, including primary biliary cirrhosis. There was minimal colocalization seen in healthy adult and pediatric livers, or in livers not also demonstrating bile ductular proliferation. Multispectral imaging confirmed significant colocalization of the different markers in biliary atresia. In conclusion, we present significant histologic evidence suggesting that the epithelial to mesenchymal transition occurs in human liver fibrosis, particularly in diseases such as biliary atresia and primary biliary cirrhosis with prominent bile ductular proliferation.

Introduction

The epithelial to mesenchymal transition (EMT) is a process in which mature epithelial cells lose the appearance, cell-cell contacts, and unique protein expression patterns of epithelia and acquire the phenotypic characteristics of mesenchymal cells. The role of EMT in embryonic development and tumor metastasis is well established. Only recently, however, has it been shown to play a role in tissue fibrosis, serving as a source of fibrogenic myofibroblasts responsible for abnormal matrix deposition. Most evidence for EMT in fibrosis comes from the kidney. Strutz et al [1] initially reported in 1995 that renal tubular epithelial cells in a mouse model of interstitial fibrosis expressed a calcium-binding protein, fibroblast-specific protein 1 (FSP1, also known as S100 A4), that otherwise appeared to be unique to fibroblasts. There have since been multiple reports that a subset of renal tubular epithelial cells in rodent models of fibrosis and in fibrotic human kidneys express both epithelial and mesenchymal markers, including FSP1, vimentin, the collagen chaperone heat shock protein 47 (HSP47), and the myofibroblast marker α-smooth muscle actin (α-SMA), and appear to be undergoing EMT (for review, see references [2], [3], [4]). Iwano et al [5] used an elegant genetic approach to demonstrate convincingly that EMT occurs in murine kidney fibrosis, lending validity to the results obtained using EMT marker proteins.

Evidence for EMT in organs other than the kidney is still emerging. In the lung, tissue from patients with idiopathic pulmonary fibrosis demonstrated coexpression of both epithelial and mesenchymal markers, suggesting that alveolar epithelial cells are a source of myofibroblasts in this disease [6]. EMT in the liver is less well established. Several groups have now shown that neonatal and adult hepatocytes can undergo EMT in vitro, although there is no published evidence of hepatocyte EMT in vivo [7], [8], [9]. EMT of cells in the biliary tree was clearly demonstrated for the first time by Xia et al [10] who showed that biliary epithelial cells (BECs) in the mouse liver 12 weeks after bile duct ligation express HSP47 and synthesize type I collagen; in vitro, a line of human intrahepatic BECs could be induced to undergo EMT after treatment with transforming growth factor β (TGF-β). In the human liver, co-staining for epithelial and mesenchymal marker proteins suggested the occurrence of EMT in a single case of recurrent primary biliary cirrhosis (PBC) after liver transplantation [11]. Another group demonstrated that the bile ductules of a single human patient with biliary atresia fibrosis expressed α-SMA and therefore concluded that these cells were undergoing EMT [12].

The increasing evidence for EMT in tissue fibrosis suggested that it might contribute to human liver disease. We hypothesized that EMT might play an important role in biliary atresia. Defined pathologically as an inflammatory sclerosing obliteration of the extrahepatic bile ducts with bile ductular proliferation and periportal fibrosis, biliary atresia is the most common indication for liver transplant in children [13]. Although adequate bile drainage can be achieved in many patients with performance of a Kasai portoenterostomy, fibrosis nonetheless generally persists and often will progress even after resolution of cholestasis. The fibrosis seen in biliary atresia is remarkable for rapidity of progression (many infants have cirrhosis at presentation—before 2 months of age). Significant fibrosis complicated by clinical signs of portal hypertension occurs in most cases, and more than 70% require liver transplantation [14]. Because there is pronounced bile ductular proliferation in biliary atresia, particularly early, we hypothesized that BECs in biliary atresia undergo EMT, potentially explaining the rapidity of the associated fibrosis. We therefore undertook a study of human liver tissue from patients with biliary atresia and a variety of other pediatric and adult liver fibroses. We provide evidence for significant EMT in biliary atresia and other diseases with bile duct proliferation.