VPA treatment of activated HSCs induced strong inhibition of Acta2 messenger RNA (mRNA) levels (Fig. 3B [+VPA at day 7]) and a significant change in α-SMA protein expression (Fig. 3C). Morphologically, HSCs treated with VPA at day 7 showed a more quiescent phenotype accompanied by a decrease of α-SMA fibers when compared with control HSC cultures (Fig. 3D). We washed away the VPA after 7 days of treatment and analyzed whether the HSCs could still transdifferentiate PD0325901 into myofibroblasts. Three days after the removal of VPA, HSCs expressed higher amounts of Acta2 and regained their characteristic myofibroblastic morphology (Fig. 3B,D [−VPA at day 7]). Next, the in vivo effect of VPA on genes

that were VPA-sensitive in our in vitro experiments was investigated. For this, we analyzed the livers used for the experiments in Fig. 1 in which we show that Acta2 expression is altered by VPA cotreatment (Fig. 1C). RNA analysis by way of qPCR revealed that VPA cotreatment also inhibits the CCl4-induced up-regulation of Lox and Spp1 (Fig. 4A CT99021 [4-week treatment]). To exclude that the observed effect was due to other cell types than HSCs in the fibrotic liver, we isolated HSCs from normal and fibrotic mice with or without VPA in their drinking water. As

described by De Minicis et al.,3 we were able to isolate in vivo–activated HSCs from 2-week CCl4-treated mice. Under these conditions, we observed a CCl4-induced up-regulation of Acta2, Lox, and Spp1 in total liver RNA that is reduced by VPA cotreatment (Fig. 4B). Analyzing freshly isolated HSCs from 2-week CCl4-treated mice showed higher levels of Acta2, Lox, and Spp1 when compared with HSCs isolated from control animals. VPA cotreatment inhibited the CCl4-induced up-regulation of Acta2, Lox, and Spp1 (Fig. 4C). The results described so far were obtained in a prophylactic setup. In order to test the possible therapeutic effect of VPA, we treated mice for 2

weeks with CCl4, followed by 2 weeks of CCl4+VPA cotreatment and compared these with selleck screening library 4-week CCl4-treated mice. Sirius Red stainings showed a significant reduction in collagen deposition when CCl4 treatment was continued in the presence of VPA (Fig. 5A). qPCR analysis for Acta2 in total liver RNA of these mice confirm these results by a reduced expression of Acta2 in the CCl4+VPA mice compared with the CCl4 mice (Fig. 5B). These results suggest that VPA treatment prevents further progression of CCl4-induced fibrosis in mice. To gain insight in the mechanisms involved in the effect of VPA on HSC transdifferentiation, we determined the expression of class I HDACs during normal HSC differentiation in vitro. Whereas HDAC1 and HDAC2 are easily detected in quiescent (D1) HSCs, their protein expression decreases during stellate cell activation. In contrast, HDAC3 seems to be expressed at constant levels, whereas HDAC8 is induced upon HSC transdifferentiation (Fig. 6A).