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.)
Connective tissue growth factor (CTGF) is a cysteine-rich peptide originally identified as a growth factor secreted by vascular endothelial cells in culture. 1 It belongs to a family of immediate early growth-responsive genes including cyr61 and fisp-12 from mouse, and cef10 and nov from chicken. [2][3][4][5] These genes are thought to regulate cell proliferation, differentiation, and embryogenesis. The physiological function of CTGF both in vitro and in vivo has not yet been elucidated. However, CTGF is considered to be the major mitoattractant produced by human umbilical vascular endothelial cells. 1 Furthermore, results from experimental studies showed that CTGF may be one of the downstream effectors of transforming growth factor  (TGF-). Indeed, CTGF mRNA is induced specifically by TGF-, but not by platelet-derived growth factor (PDGF), epidermal growth factor, or basic fibroblast growth factor. 6,7 Furthermore, a novel TGF-responsive element was described in the CTGF promoter sequence. 8 The role of CTGF in the fibrogenesis process has recently been proposed. First, Northern blot analysis showed that CTGF was expressed at higher levels in atherosclerotic vessels than in normal arteries. 9 Furthermore, the authors showed that the vascular smooth muscle cells that express CTGF were localized predominantly in areas with extracellular matrix accumulation. 9 Igarashi et al. showed that, during wound repair, CTGF participates in the process of tissue regeneration and skin fibrosis as a potential autocrine stimulator released in response to TGF-. 7 Finally, it has recently been suggested that CTGF may be a common growth factor involved in human renal fibrosis. 10 Liver fibrosis represents the main complication of most chronic liver diseases whatever their origin, and cirrhosis constitutes the ultimate stage of this process. The molecular mechanisms of liver fibrosis are not fully understood. However, fibrogenesis, characterized by cell proliferation and accumulation of extracellular matrix components, requires numerous mediators such as TGF- and PDGF. [11][12][13] Aside from these key mediators, other molecules such as CTGF may play a role in the fibrogenesis process. The aim of this study was to investigate the possible involvement of CTGF in liver diseases and its relationship with liver fibrosis. To assess the role of CTGF, we evaluated the expression of CTGF in human biopsy specimens from various chronic liver diseases by immunohistochemistry and in situ hybridization. To gain further insight into the dynamics of CTGF production, we also studied the expression of CTGF at mRNA and protein levels in experimental models of liver fibrosis in rats. Because hepatic stellate cells (HSC) play a crucial role in the development of liver fibrosis through the production of several extracellular matrix components, 14 we studied CTGF expression in cultured HSC.
Abbreviations: CTGF, connective tissue growth factor; HSC, hepatic stellate cell; CCl 4 , carbon tetrachloride; BDL, bile duct ligation; RT, room temperature; C t , threshold cycle.