PROLIFERATION AND PHENOTYPIC MODULATION OF PORTAL FIBROBLASTS IN THE EARLY STAGES OF CHOLESTATIC FIBROSIS IN THE RAT

The animal model of hepatic fibrosis induced by bile duct ligation rep resents an experimental model of human chronic biliary fibrosis. Much attention has been given to the hepatic stellate cell (HSC), or perisi nusoidal cell, as the source of the extracellular matrix proteins. How ever, in the bile duct ligation model, mesenchymal cells other than HS C may be involved in the early stages of fibrosis development. The cur rent study examined, in Sprague-Dawley rats, proliferation in differen t liver cell subpopulations as well as expression of alpha-smooth musc le (SM) actin and desmin in portal fibroblasts and HSC at 6 hours and 1,2,3, and 7 days after bile duct ligation. Kinetics of liver cell pro liferation and of phenotypic modulation of portal fibroblasts and HSC (expression of alpha-SM actin and desmin) was evaluated by immunocytoc hemistry, immunofluorescence, and immunoelectron microscopy using immu nogold technique. In sham-operated animals, the evaluation of prolifer ation in various liver cell subpopulations revealed nonsignificant cha nges compared with nonoperated rats. alpha-SM actin was detected in ve ssel walls but was absent in cells of portal tract and parenchyma. Des min was expressed in vessel walls and in some fibroblastic cells of po rial stroma (8.2 cells/unit area) as well as in HSC in acinar Zones 1 and 3 (15.6 cells/unit area and 7.1 cells/unit area, respectively). In bile duct-ligated rats, 24 and 48 hours after ligation, marked prolif erations of bile duct epithelial cells (labeling indices 36.8% and 29. 5%, respectively) and of periductular fibroblasts (labeling indices 16 .7% and 31.0%, respectively) were observed; thereafter, proliferation decreased for both populations (labeling indices at 7 days 12.0% and 1 1.6%, respectively). HSC proliferation increased gradually until the t hird day (labeling index 18.8%) and then leveled off. Immunocytochemis try and immunoelectron microscopy revealed a significant number of cel ls expressing alpha-SM actin 72 hours after bile duct ligation in the stroma adjacent to proliferating ductules. The number of alpha-SM acti n-positive cells increased until the seventh day (251.6 cells/unit are a). At all times examined, the distribution of alpha-SM actin was rest ricted to the connective tissue stroma adjacent to proliferating ductu les; alpha-SM actin was not expressed in HSC of the lobule. An expansi on of desmin expression was noted in fibroblastic cells in stroma surr ounding proliferating ductules until 72 hours after bile duct ligation (74.7 cells/unit area) followed by a plateau. At this time, desmin ex pression increased also in HSC; as in controls, the number of positive cells was greater in Zone 1 (31.8 cells/unit area) than in Zone 3 (18 .5 cells/unit area). Double immunofluorescence staining detected by co nfocal microscopy showed that the majority of portal fibroblastic cell s expressing alpha-SM actin was desmin negative 48 hours after bile du ct ligation. From 72 hours, portal fibroblastic cells coexpressing alp ha-SM actin and desmin appeared, and their proportion increased until 7 days. The present findings indicate that in the early phase of bile duct ligation, there is a marked and transient proliferation of bile d uct epithelial cells associated with proliferation of portal periductu lar fibroblasts, which rapidly express alpha-SM actin. This fibroblast ic population may play a dominant role in the early porial fibrosis af ter bile duct ligation.