Notch signaling mediates epithelial-to-mesenchymal transition in the development of marginal liver graft fibrosis

Abstract

BACKGROUND AND AIMS: Although graft size and chronic liver diseases have been proposed as risk factors for post-transplantation liver fibrosis, the underlying mechanisms remained unclear. Our preliminary data indicated that liver repair in small-for-size fatty liver grafts was featured by oval cell activation and their subsequent biliary differentiation. Meanwhile, it was noticed that proliferative biliary cells ensued in this context exhibited features of epithelial-to-mesenchymal transition (EMT) as liver regeneration proceeded and this was associated with increased collagen deposition in the graft. Here, we aim to explore the possible signaling pathway regulating EMT in small-for-size fatty graft fibrosis. MATERIALS AND METHODS: A rat orthotopic liver transplantation model using either small-for-size fatty (40% fatty change) grafts or normal grafts (as control) in cirrhotic recipients was applied. Liver samples were collected at day 7, 14 after transplantation. Intragraft gene expression was assessed by qRT-PCR or immunohistochemisty. Functional studies were conducted using oval cell line (PIL2) and further validation performed in human post-transplantation liver biopsy specimens. RESULTS: Increased mRNA levels of TGFb1 and notch receptor 2 (p<0.05) was detected in small-for-size fatty grafts of the rat model. Immunostaining further revealed that proliferative biliary cells showing features of EMT strongly expressed Notch2 receptor and Notch effector Hes1 at either Day 7 or Day14 after transplantation. In the meantime, enhanced expression of Notch receptor ligand Jag1 was observed in the surrounding stromal cells. Moreover, over-expression of Notch2 receptor was exclusively seen in biliary cells co-expressing epithelial (CK19) and mesenchymal markers (Vimentin) in human small fatty grafts. In vitro study with PIL2 cell line demonstrated that oval cells could be induced to differentiate into cells expressing biliary markers (CK19 and CK7). Addition of TGFb1 to these differentiated oval cells resulted in an EMT phenotype accompanied with up-regulation of Notch2 receptor. However, this effect was significantly attenuated by notch signaling inhibitor γ-secretase. CONCLUSION: Notch signaling mediates epithelial-to-mesenchymal transition of proliferative biliary cells in small-for-size liver fatty grafts. Key components in this pathway may serve as novel targets for the treatment of liver fibrosis after transplantation.