Studies on mechanisms in liver fibrosis

Abstract: The aim of the present study was to elucidate mechanisms involved in liver fibrogenesis, the wound healing response of the liver to chronic injury. A role for TNF-[alpha] and all three isoforms of TGF-[beta] in the pathogenesis of hepatitis C related liver disease was suggested by the finding of increased expression of TGF-[beta]1-3, latent TGF-[beta] binding protein (LTBP), and TNF-[alpha] by immunohistochemistry in areas of inflammation and fibrosis in liver tissue from untreated patients with hepatitis C. In patients responding to therapy, TGF-[beta]1 expression decreased significantly, but remained unchanged in non-responders. The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in liver tissue from patients with cystic fibrosis (CF), was assessed by immunohistochemistry and confocal laser scanning microscopy. In non- CF controls, CFTR expression was confined to the luminal surface of bile duct cells. In patients carrying the [delta]F508 mutation, bile duct cells displayed aberrant cytoplasmic CFTR immunolocalization, and in two patients homozygous for R764X and for 1112delT, no CFTR immunoreactivity was detected. In CF patients prominent inflammatory infiltrates and ICAM-1 expression on bile ducts were rare findings, while smooth muscle [alpha]-actin positive myofibroblasts were identified within fibrotic areas around bile ducts. The results are consistent with an impairment of AF508 CFTR processing in intrahepatic biliary epithelium, and furthermore suggest that bile duct cells may interact directly with hepatic stellate cells (HSCs), the principal fibrogenic cell type in liver. We undertook dynamic tests, using a Transwell chernotaxis filter assay, in order to examine the possibility that bile ducts may stimulate chemoattraction of HSCs. The results showed that bile duct segments isolated from cholestatic rats triggered migration of HSCs, and that this effect was mediated mainly by PDGF-BB, indicating that chernotaxis of HSCs towards bile duct structures may contribute to the development of periductular fibrosis in cholestatic disorders. We next analyzed the initial proliferative response of HSCs to rat cholestatic liver injury. Induction of HSC proliferation, as assessed by in vivo incorporation of bromodeoxyuridine and by flow cytometry analysis of freshly isolated HSCs occurred between 24 and 48 hours, and reached a plateau as soon as 48 hours after bile duct ligation. Induction of proliferation in HSCs coincided with the upregulation of PDGFR-[beta] protein on their cell surface, but preceded that of smooth muscle [alpha]-actin. ST1571, a PDGF receptor tyrosine kinase inhibitor, administrated by intraperitoneal injections, reduced the proliferation of HSCs 48 hours after bile duct ligation, by 60%. Thus, cholestatic liver injury elicits a very early proliferative response in HSCs that is mainly mediated by PDGF, and that precedes HSC phenotypic transition into myofibroblasts. Finally phenotypic characterization of myofibroblasts arising in vivo in the initial stage of biliary fibrosis due to bile duct ligation, indicated that portal myofibroblasts accumulating together with fibrosis around bile ducts derive from cells in the portal tract, and not from HSCs. Furthermore, in vitro, fibroblast-like cells outgrew from bile duct segments and underwent transition into myofibroblastic cells with fibrogenic potential, as evidenced by the expression of smooth muscle [alpha]-actin and of [alpha]1 (I) collagen mRNA.