Hepatitis C Virus Proteins Induce Cirrhosis Antigen Expression on Human Hepatoma Cells In Vitro: Implications for Viral Mechanisms in Hepatitis C Fibrogenesis

Abstract

Liver Biopsy in Modern Medicine 336 associated antigen, Glial Fibrillary Acidic Protein (GFAP), is induced on pro-fibrotic cells such as HSCs, and is a definitive marker of fibrogenic pathway activation in this latter cell type (29). Studies of fibrosis mechanisms in human liver are limited. One longitudinal study, after liver transplantation, reported that increased density of GFAP in liver biopsy specimens predicted subsequent advanced fibrosis or cirrhosis (9). Although cells harboring GFAP were only presumed to be activated HSCs, the study concluded that 30% of cells in cirrhotic livers may be activated HSCs. However, the possibility of a direct effect of HCV on GFAP expression in hepatocytes was not investigated. The present study therefore examined the effect of HCV on hepatic fibrosis marker expression, using two human hepatoma cell line model systems, capable of supporting either non-productive HCV replication (HCV replicon, (22)), or productive HCV infection (genotype 2a infectious clone JFH1; (44)). The study also examined liver biopsy samples from HCV infected patients for the simultaneous presence of GFAP and HCV replicative intermediate RNA. Finally, microarrays were used to analyze expression of multiple cellular genes linked with liver fibrosis, in human hepatoma cell lines plus or minus HCV. The effect of HCV on differential expression of 153 genes (1, 3, 17, 28, 37) either involved in, or associated with, with the process of liver fibrosis, is reported. 2. Methods 2.1 Human liver biopsy specimens Thirty-two liver biopsy specimens, obtained under informed consent and per IRB-approved protocol, were available for study. All 32 subjects had chronic, active (viremic) HCV genotype 1 infections. During procurement, the specimens were immediately preserved in OCT buffer and snap frozen at the bedside. Parallel sections of the liver biopsies were reviewed by a single pathologist who was blinded to HCV status and all other data. Liver fibrosis severity, staged as 0 (no fibrosis) through 4 (cirrhosis), was assigned according to the system described by Batts and Ludwig (5). For the present study, the liver specimens were de-identified for all information except HCV replication status and fibrosis severity. Fresh thin sections were obtained for the GFAP immunostaining experiments described below. Parallel sections of all 32 liver biopsy specimens were assayed for GFAP expression by immunocytochemistry. 29 of the specimens had been previously analyzed for both HCV genomic (G) and replicative intermediate (RI) RNAs by strand-specific in situ hybridization (ISH). Details of the ISH assay, and assay results for a larger sample of hepatitis C cases, were previously reported (31). Of 29 specimens with both GFAP and HCV replication data, HCV RNA was determined as either positive (G+RI+; 20 specimens), or negative (G-RI-; 9 specimens), and GFAP staining level (% of cells per biopsy staining positive for GFAP, or %GFAP) was then analyzed as a function of HCV infection/replication status, and fibrosis stage. 2.2 Hepat...