37, 38 However, the function of individual LOX-like proteins in HSCs is unknown. Studies on rat HSCs have shown that Spp1 is involved in a higher proliferation rate and a higher collagen I expression and migratory capacity of the cells during the activation process in vitro.39 Whereas VPA had a clear effect on Acta2, Lox,

and Spp1, it did not affect expression of the TSA-sensitive genes Arp2, Arp3, Addl70, and Gelsolin11 (data not shown). F-actin staining of HSCs in the presence or absence of VPA also demonstrates that actin remodeling in general is not affected by VPA treatment (Supporting Fig. 1). Removal of VPA led to the onset of classical morphological changes associated with HSC activation, indicating that the inhibitory effects of the drug are reversible (Fig. 3D). The expression of key genes normally up-regulated during in vitro HSC transdifferentiation check details was also inhibited in vivo when CCl4-treated mice were cotreated with VPA.

Stellate cells isolated from mouse livers treated with both CCl4and VPA expressed less Acta2, Lox, and Spp1 when compared with CCl4-treated mice (Fig. 4C). A complete inhibition of HSC activation is not observed, because the expression of several HSC activation markers does not seem to be affected by VPA treatment, indicating that the observed inhibition of liver fibrosis by VPA is most likely due to only a partial inhibition of HSC activation. Whereas it has been reported previously that TSA affects the TGF-β1 signaling in skin fibroblasts,26 we show that VPA treatment does

not affect the early events following TGF-β1 stimulation of mouse HSCs (up-regulation Sodium butyrate of Smad6 and CTLA-4 antibody Smad7), whereas some late responses to TGF-β1 stimulation are affected (Lox and Acta2). The observation that Lox expression, but not Acta2 expression, was influenced by knockdown of all class I HDACs suggests that class I HDACs do play a role during HSC activation, but that class I HDACs are not the only VPA targets in HSCs involved in their activation process. Interestingly, VPA treatment of HSCs also leads to reduced class I HDAC protein levels (Fig. 6), suggesting that in addition to the inhibition of their activity, VPA can also influence their steady state protein levels. Thus far, this effect has only been reported for HDAC2.24 Most likely, the lower HDAC8 levels are a consequence of inhibition of HSC activation, because this HDAC is up-regulated during normal culture conditions (Fig. 6A,B). This overall VPA-induced reduction in HDAC protein levels was not due to transcriptional regulation of these HDACs (data not shown). Studies in human neuroblastoma SH-SY5Y cells have shown that VPA can influence wnt signaling through phosphorylation of GSK3β on Ser-9.40 Although there is some controversy about the exact role of wnt signaling in HSCs, different studies have shown that wnt signaling is important for HSC activation.