Fibrosis is a common endpoint of clinical trials in chronic hepatitis C, and liver biopsy remains the gold standard for fibrosis evaluation. However, variability in the distribution of fibrosis within the liver is a potential limitation. Our aim was to assess the heterogeneity of liver fibrosis and its influence on the accuracy of assessment of fibrosis with liver biopsy. Surgical samples of livers from patients with chronic hepatitis C were studied. Measurement of fibrosis was performed on the whole section by using both image analysis and METAVIR score (reference value). From the digitized image of the whole section, virtual biopsy specimens of increasing length were produced. Fibrosis was assessed independently on each individual virtual biopsy specimen. Results were compared with the reference value according to the length of the biopsy specimen. By using image analysis, the coefficient of variation of fibrosis measurement with 15-mm long biopsy specimens was 55%; and for biopsy specimens of 25-mm length it was 45%. By using the METAVIR scoring system, 65% of biopsies 15 mm in length were categorized correctly according to the reference value. This increased to 75% for a 25-mm liver biopsy specimen without any substantial benefit for longer biopsy specimens. Sampling variability of fibrosis is a significant limitation in the assessment of fibrosis with liver biopsy. In conclusion, this study suggests that a length of at least 25 mm is necessary to evaluate fibrosis accurately with a semiquantitative score. Sampling variability becomes a major limitation when using more accurate methods such as automated image analysis.

Nonalcoholic steatohepatitis (NASH) may progress to liver fibrosis and cirrhosis. Mechanisms directly involved in the development of fibrosis have been poorly investigated. Because connective tissue growth factor (CTGF) is an intermediate key molecule involved in the pathogenesis of fibrosing chronic liver diseases and is potentially induced by hyperglycemia, the aims of this study were to (1) study the expression of CTGF in vivo both in human liver biopsy specimens of patients with NASH and in an experimental model of obesity and type II diabetes (Zucker rats); and (2) analyze the effects of hyperglycemia and insulin in vitro on hepatic stellate cells. In vivo, CTGF overexpression was observed in the liver tissue of all of the 16 patients with NASH. CTGF immunostaining was mild in 7 cases (44%) and moderate or strong in 9 cases (56%). Staining was mainly detected in the liver extracellular matrix in parallel with the amount of liver fibrosis. Liver from fa/fa rats also showed CTGF overexpression by comparison with Fa/fa rats both at the messenger RNA (mRNA) level (3-fold increase) and protein level. In vitro, both CTGF mRNA and protein were significantly increased when hepatic stellate cells were incubated with either glucose or insulin. A slight increase in type I procollagen mRNA level was also observed in hepatic stellate cells incubated with glucose. In conclusion, this study suggests that hyperglycemia and insulin are key-factors in the progression of fibrosis in patients with NASH through the upregulation of CTGF. (HEPATOLOGY 2001;34:738-744.)

Connective tissue growth factor (CTGF) stimulates in vitro fibroblast proliferation and extracellular matrix synthesis. The aim of this study was to assess the role of CTGF in liver fibrogenesis. CTGF expression was investigated both at the protein and mRNA level in biopsies of chronic liver diseases, in experimental models of liver fibrosis, and in hepatic stellate cells in culture. CTGF immunostaining was observed in most human liver biopsies with significant fibrosis. An increase of CTGF immunostaining was associated with a higher score of fibrosis both in the group of chronic hepatitis C ( 2 ‫؍‬ 9.3; P F .01) and in the non-hepatitis C group ( 2 ‫؍‬ 7.2; P F .02). In situ hybridization showed CTGF mRNA expression in spindle cells in both the fibrous septa and sinusoidal lining. In experimental models of liver fibrosis, CTGF accumulated in parallel with the development of septal fibrosis and cirrhosis. Quantification of CTGF mRNA by a real-time reversetranscription polymerase chain reaction (RT-PCR) assay showed a significant increase of CTGF mRNA in both CCl 4 -induced and bile duct-ligated rat models of liver fibrosis. Expression of CTGF protein and mRNA was definitively assigned to hepatic stellate cells, because CTGF was detected by Western blot both in lysate and supernatant of a hepatic stellate cell line derived from rats. These cells also displayed CTGF protein and mRNA as shown by immunohistochemistry and in situ hybridization. In conclusion, this study shows that CTGF is strongly expressed during liver fibrogenesis, and hepatic stellate cells seem to be the major cellular sources of CTGF in the liver. (HEPATOL-OGY 1999;30:968-976.)