A Histone Deacetylase Inhibitor, Trichostatin A, Suppresses Myofibroblastic Differentiation of Rat Hepatic Stellate Cells in Primary Culture

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Abstract
Hepatic stellate cells are the major cellular sources of extracellular matrix in chronic liver diseases leading to fibrosis. We explored the antifibrogenic effect of two histone deacetylase inhibitors, sodium butyrate and trichostatin A (TSA), on this cell type in vitro. Primary hepatic stellate cells as well as culture activated cells were exposed to butyrate (0.01–1 mmol/L) or TSA (1–100 nmol7sol;L); their effect on collagen types I and III and smooth muscle α–actin was examined by quantitative immunoprecipitation and by Northern analysis. Their antiproliferative effect was examined by3H–thymidine incorporation and cell counting. Hyperacetylation of histones was demonstrated by acid urea/Triton–X–100 (AUT) polyacrylamide gel electrophoresis. Possible cytotoxic effects were judged on stellate cells by evaluating de novo total protein synthesis, and on hepatocytes by measuring lactate dehydrogenase (LDH) leakage, albumin secretion, and epoxide hydrolase and ethoxycoumarin O–deethylase activity. TSA at 100 nmol/L and butyrate at 1 mmol/L retarded the morphological changes characteristic for activation of primary stellate cells. TSA at 100 nmol/ inhibited synthesis of collagen types I and III and smooth muscle α–actin by 62%, 70%, and 88%. Butyrate at 1 mmol/L showed a modest inhibitory effect on collagen type III and smooth muscle α–actin, but had no effect on collagen type I. Northern analysis suggested that these inhibitory effects on collagen type III and smooth muscle α–actin were transcriptional, while the effect on collagen type I was largely posttranscriptional. At 100 nmol/L, TSA strongly suppressed proliferation of primary hepatic stellate cells. Inhibition of activation of stellate cells was preceded by hyperacetylation of histone H4. When tested on cells at day 14 in culture, butyrate had no inhibitory effects on the synthesis of collagens or smooth muscle α–actin. One hundred or 10 nmol/L TSA modestly inhibited the synthesis of collagens type I (-24%,-22%) and III (-34%,-22%), and smooth muscle α–actin (-27%,-12%). We conclude that TSA inhibits transdifferentiation of stellate cells into myofibroblasts by interfering with the level of acetylation of histone H4.