In vitro and in vivo models to investigate the mechanisms of liver fibrosis and cholestasis

Abstract

Chronic liver disease (CLD) is a leading cause of mortality around the world and can arise from a range of injuries or insults. Untreated CLD results in liver fibrosis and ultimately can lead to the development of liver cancer. Primary liver cancers include hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Every year approximately 700,00 people die of liver cancer, making it the third most common cause of cancer death, worldwide (Ferlay et al., 2010). To date, drugs targeting these aggressive cancers have shown minimal benefit in clinical trials and cause a range of side effects. Therefore, improved insights into the underlying mechanisms and pathogenesis of these diseases will facilitate the development of novel drugs.

Considering the significance of chronic hepatitis C virus (HCV) infection in the development of liver fibrosis, and to identify potential therapeutic targets, we examined the release of major fibrogenic cytokines from HCV infected cells and monitored their effect on activation of pro-fibrotic hepatic stellate cells. We demonstrated the release of fibrogenic cytokines in several HCV cell culture models, including a subgenomic replicon (SGR) and two full length virus clones, JFH1 and Jc1. We found that TGF-131 and CTGF expression was up regulated more in Jc1 infected cells than in cells containing the SGR or in JFH1 infected cells.

Next, we examined the effect of these fibrogenic cytokines on hepatic stellate cells, using the human LX2 cell line and primary rat stellate cells. We demonstrated that the supernatant of Jc1 infected hepatic cells induced up-regulation of fibrotic markers in stellate cells, compared to supernatant from uninfected Huh7 cells. In summary, our results suggest a role for HCV induced cytokines in the development of liver fibrosis in people with chronic hepatitis C.

In addition to HCV induced liver fibrosis, a major focus of this thesis is the inflammatory response to cholestasis. Diverse aetiologies result in liver cholestatic disease, which causes toxicity in the liver and ultimately leads to liver fibrosis and frequently liver cancer. A prominent ductular reaction occurs in response to biliary obstruction in cholestatic diseases. This ductular reaction involves the differentiation of bipotentialliver progenitor cells {LPCs) into hepatocytes and cholangiocytes, as well as proliferation of cholangiocytes and liver progenitor cells {LPCs). To date, the mediators of cholangiocyte proliferation in ductular reaction have not been well established. Therefore, we sought to determine the role of Notch and NF-KB signalling in cholangiocyte proliferation. In one animal model, bile duct ligation {BDL) was undertaken in rats to induce ductular reaction. Relative mRNA {qPCR) and protein {western blot) expression of key signalling and downstream molecules were used to determine activation of the Notch and NF-KB pathways. In complementary experiments, expression and localization of Notch and NF-KB signalling molecules was determined by immunofluorescence {IF). Our data suggests activation of both Notch and NF-KB signalling during the proliferation of biliary epithelial cells in cholestatic liver injury. Next, we used a mouse model of cholangiocyte proliferation involving a diethoxycarbonyl dihydrocollidine (DDC) diet, which causes biliary obstruction and cholestasis. We confirmed activation of NF-KB and Notch signalling in this model by real time PCR (qPCR) and found significant up regulation of Notch and canonical NF-KB pathway markers, but no change in the non-canonical NF-KB pathway.

Because ductular reaction occurs in response to a range of chronic liver injuries, we decided to study the effect of HCV infection on ductular reaction. We hypothesised that dual insults (HCV and DDC) may exacerbate ductular reaction, so investigated cholangiocyte proliferation and ductular reaction in HCV transgenic (SL139) mice exposed to the DDC diet. In preliminary experiments we observed no change in the expression of Notch or NF-KB signalling in SL139 mice compared to wild type, either by histology or western blot analysis.

Finally, we studied the effect of HCV infection on Notch and NF-KB signalling in vitro, as HCV has been proposed to convert infected hepatocytes to biliary cell lineage, resulting in cholangiocarcinoma. We examined activation of the Notch and NF-KB pathways in Jc1 infected Huh? cells, and found significant up regulation of both pathways. Taken together, our data suggest that Notch and NF-KB signalling may play a significant role in cholestatic diseases, including development of cholangiocarcinoma, and could serve as potential therapeutic targets.