160 Exp. Mol. Med. Vol. 44(2), 159-166, 2012 (Kinner et al., 2002; Wang et al., 2004), mechan- ical stress (Kobayashi et al., 2004), and direct con- tact with vascular endothelial cells in vitro (Ball et al., 2004). Moreover, injected bone marrow-derived MSCs have been reported to have differentiated in- to SMCs and to have contributed to the remodeling of vasculature in vivo (Davani et al., 2003; Gojo et al., 2003; Yoon et al., 2005). In a previous study, we showed that sphingosylphosphorylcholine (SPC) increased the expression levels of α-SMA and oth- er smooth muscle-specific proteins in human adi- pose tissue-derived mesenchymal stem cells (hASCs) via an autocrine TGF-β/Smad2-dependent mechanism (Jeon et al., 2006). In addition, we have previously reported that SPC stimulated the small GTPase RhoA and that the RhoA-Rho kin- ase pathway played a key role in SPC-induced dif- ferentiation of hASCs to SMCs. RhoA-Rho kinase pathway plays a key role in SMC differentiation by regulating the integrity of the actin cytoskeleton and MRTF-dependent gene transcription (Cen et al., 2004; Miano et al., 2007). Therefore, SPC-in- duced SMC differentiation of MSCs would be an ideal model for the study of vascular diseases-associated SMC differentiation. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG- CoA) reductase inhibitors (statins) reportedly exert beneficial effects in patients with cardiovascular diseases via pleiotropic functions, including reduc- tion of plaque inflammation and platelet ag- Figure 1. Effect of simvastatin on SPC-induced expression of smooth muscle markers in hASCs. (A) hASCs were treated with serum-free me- dium containing 2 μM SPC or vehicles (0.1% DMSO, w/o) in the pres- ence of indicated concentrations of simvastatin for 4 days. Expression levels of α-SMA, calponin, and GAPDH were determined by Western blotting. (B) Inhibitory effects of simvastatin on SPC-induced α-SMA ex- pression in hASCs were further determined by immunostaining with an- ti-α-SMA antibody. Scale bar = 50 μm. Representative data from three independent experiments are shown. gregation, enhanced plaque stability and endothe- lial function, and inhibition of SMC proliferation and increased apoptosis (Calabro and Yeh, 2005; Liao, 2005). Accumulating evidence suggests that sta- tins attenuate neointimal formation and vascular remodeling by blocking the activation of the Rho family of small G proteins (Rolfe et al., 2005). Statins inhibit the activity of HMG-CoA reductase which catalyses the conversion of HMG-CoA into mevalonate during cholesterol biosynthesis. Me- valonate can be converted into farnesylpyrophos- phate (FPP) and geranylgeranylpyrophosphate (GGPP), 2 isoprenoid residues that can be anch- ored onto several intracellular proteins through far- nesylation or geranylgeranylation (Wong et al., 2002; Graaf et al., 2004). Simvastatin has been re- ported to inhibit the relocalization of RhoA to cell membranes and the resulting activation of RhoA by blocking geranylgeranylation (Laufs et al., 1999). However, whether statins can affect the SPC-in- duced differentiation of MSCs to SMCs has not been studied. In the present study, we show for the first time that simvastatin inhibits the differentiation of hASCs into SMCs by blocking RhoA-Rho kinase- dependent activation of autocrine TGF-β/Smad2 signaling pathway. Results Simvastatin inhibits SPC-induced differentiation of hASCs to SMCs To explore whether statin can affect SPC-induced differentiation of hASCs to SMCs, we examined the effect of simvastatin on the SPC-induced ex- pression of smooth muscle-specific markers, in- cluding α-SMA and calponin. As shown in Figure 1, SPC treatment increased the expression of α-SMA and calponin in hASCs, and simvastatin dose-de- pendently attenuated SPC-induced expression of α-SMA and calponin with a complete inhibition at a 1 μM concentration, suggesting simvastatin has an inhibitory effect on the SPC-induced differentiation of hASCs to SMCs. To confirm these results, we determined the ef- fects of simvastatin on α-SMA expression and ac- tin filament formation using immunocytochemistry. As shown in Figure 1B, treatment of hASCs with 2 μM SPC for 4 days increased α-SMA expression levels, and pretreatment of the cells with simvasta- tin completely abrogated SPC-induced expression of α-SMA in hASCs.