Discussion The RhoA-Rho kinase signaling pathway plays a key role in the SPC-induced differentiation of hASCs to SMCs. Inhibition of the RhoA-Rho kinase pathway by over-expression of DN-RhoA or pre-treat- ment with Y27632 completely abrogated SPC-in- duced α-SMA expression (Jeon et al., 2008). It has been documented that the RhoA/Rho kinase-depend- ent pathway regulates transcription of SMC-specif- ic genes (Mack et al., 2001; Wamhoff et al., 2004). Sphingosine-1-phosphate increased RhoA activity and expression level of α-SMA in smooth muscle cells, and that inhibition of Rho kinase by Y27632 attenuated the sphingosine-1-phosphate-stimulated α-SMA expression (Lockman et al., 2004). In the present study, we demonstrated that simvastatin treatment blocked SPC-induced translocation of RhoA to membrane fractions and abrogated SPC-in- duced α-SMA expression in hASCs. Inactive GDP-bound RhoA becomes activated by mem - brane translocation and subsequent exchange of GDP and GTP in the presence of guanine nucleo- tide exchange factor (Bokoch et al., 1994). Statins lead to the deprivation of mevalonate and down- stream metabolites such as mevalonate, GGPP and FPP by blocking HMG-CoA reductase (Nakagami et al., 2003). GGPP and FPP are transferred to pro- teins such as small GTPases by GGPP and FPP, respectively (Wong et al., 2002; Graaf et al., 2004). Statins has been reported to inhibit membrane lo- calization and GTP binding activity of RhoA (Kusama et al., 2001; Yoshida et al., 2001). Taken together, these results support the notion that abla- tion of RhoA-Rho kinase signaling cascade is re- sponsible for simvastatin-induced inhibition of SPC-induced differentiation of hASCs to SMCs. We have previously showed that SPC-induced activation of the autocrine TGF-β1 signaling loop is responsible for the sustained phosphorylation of Smad2 on day 4 (Jeon et al., 2006). In the present study, we demonstrated for the first time that in- hibition of Rho kinase by treatment with Y27632 or simvastatin blocked SPC-induced secretion of TGF-β1 and sustained Smad2 phosphorylation. Although the molecular mechanisms by which acti- vation of Rho kinase regulates expression of TGF-β1 are largely elusive, a possible role of Rho kinase in the regulation of TGF-β1 is supported by a report that mechanical stretch of human airway SMCs augmented TGF-β1 expression through a RhoA-Rho kinase-dependent mechanism (Mohamed and Boriek, 2010). Inhibition of Rho kinase by treat- ment with Y27632 has been reported to abrogate renal fibrosis-associated increase of α-SMA ex- pression and TGF-β1-induced myofibroblastic dif- Effect of simvastatin on differentiation of MSCs 163 ferentiation of gingival fibroblasts (Nagatoya et al., 2002; Smith et al., 2006). Consistently, inhibition of RhoA by C3 exoenzyme or DN-RhoA and pharma- cological inhibition of Rho kinase by pretreatment with Y27632 blocked TGF-β1-induced expression of α-SMA in Monc-1 neural crest stem cells (Chen et al., 2006). Taken together with these results, the present study suggests that Rho kinase is involved in the SPC-induced autocrine secretion of TGF-β1 and subsequent activation of TGF-β receptor and Smad2 phosphorylation. Differentiation and phenotypic modulation of vas- cular smooth muscle cells (VSMCs) are implicated in the development of vascular diseases including atherosclerosis (Rzucidlo et al., 2007). Accumulating evidence suggests that MSCs from various tissues reside in a perivascular location and can be identi- fied as pericytes that play a key role in develop- ment of microvessels (Covas et al., 2008; Feng et al., 2010). It has been reported that hASCs also originate from perivascular cells (Cai et al., 2011). Moreover, several studies suggest that pericytes surrounding capillaries and microvessels but also adventitial cells located around larger arteries and veins natively express MSC markers and multi- potent differentiation potentials similar to MSCs (Chen et al., 2009; Corselli et al., 2010). Therefore, these results suggest that MSCs may play a role in the vascular development and vascular diseases. SPC has been reported to exist in high-density lip- oprotein (HDL) particles (Tolle et al., 2008). Increasing body of evidence demonstrate that HDL exerts an- ti-atherogenic and anti-inflammatory effects (Nofer et al., 2004; Scanu and Edelstein, 2008). HDL in- duced expression of TGF-β2, an anti-inflammatory cytokine, in human umbilical vein endothelial cells and SPC mimicked the effect of HDL (Norata et al., 2005). These results suggest that SPC included in HDL may promote differentiation of tissue-resident MSCs or pericytes to SMCs, and that statins can interfere the differentiation of MSCs to SMCs. Similar to the results of the present study, it has been reported that TGF-β1-induced α-SMA ex- pression in human tenon fibroblasts was attenu- ated by treatment with lovastatin, a member of the statin family (Meyer-Ter-Vehn et al., 2008). However, lovastatin treatment has been shown to stimulate α-SMA expression in VSMCs and prevent pheno- typic dedifferentiation of VSMCs (Wada et al., 2008; Wagner et al., 2010). These results raise a possibility that statins differentially affect ex- pression of SMC markers in different cell types. To clarify functional role of statins on MSC differ- entiation, it is necessary to determine further whether statins can affect differentiation of MSCs to SMCs using in vivo vascular disease animal