As shown in Fig. 6A, the specific ROCK inhibitor Y27632 (10 mM) abolished the increase in P-MRLC induced by rosuvas- tatin in agLDL-VSMC (phosphorylation ratio agLDL: 0.3460.00; agLDL+rosuvastatin: 0.9260.01, agLDL+rosuvastatin+Y27632: 0.3360.04; P,0.01). Addition of the MLC-kinase inhibitor ML-9 induced a non-significant reduction of P-MRLC (agLDL+rosu- vastatin+ML9: 0.7060.16; P = NS vs agLDL+rosuvastatin). The levels of phosphorylated-PAK1 (P-PAK1) were 2fold increased (p,0.05) in agLDL- VSMC with respect to control cells, an effect not reversed by rosuvastatin (10 mM) (Fig. 6B). In a subset of experiments, VSMC were incubated with agLDL and treated with/without rosuvastatin for 16 hours, and then were seeded and allowed to attach on glass plates for 3 hours. Confocal analysis of actin fibres labelled with Alexa Fluor-633 phalloidin (Fig. 7A) demonstrated that rosuvastatin strongly promoted filamentous actin organization in LDL-treated VSMC, whereas co-incubation with 10 mM Y27632 prevented the effect of rosuvastatin. Three hours after seeding, 7363% of the plated agLDL-VSMC were attached in the presence of rosuvastatin whereas only 3064% in the absence of rosuvastatin (P,0.01) (Fig. 7B). Co-incubation of rosuvastatin with 10 mM Y27632 decreased cell attachment to the level of the agLDL-VSMC (22610%; P,0.01 vs LDL+rosuvastatin group; P = NS vs LDL- group). The ROCK inhibitor Y27632 consistently reduced the revers- ing effect of rosuvastatin on LDL-induced impairment in cell migration (Fig. 7C). Thus, when agLDL treated cells were co- incubated with Y27362 and rosuvastatin the percentage of wound coverage did not differ significantly from the basal levels observed in the absence of rosuvastatin. In addition, Y27362 retarded the induction of migration (2 hours) during an in vitro wound healing assay in control VSMC, but no significant difference was seen at longer times. Y27362 had also no effect when VSMC were treated only with rosuvastatin. Discussion Atherosclerotic plaques with large lipid cores and paucity of VSMC have an increased risk of rupture. We have recently demonstrated that LDL impair VSMC migration kinetics and induce changes in cytoskeleton proteins [18–19], mechanisms by which LDL could contribute to the relative loss of human VSMC in vulnerable plaques. The role of infiltrated LDL in vascular remodelling and repair is a key issue to understand progression of atherosclerosis. Modified LDL are found within the diseased vessel in close contact with smooth muscle cells. These infiltrated LDL aggregate and accumulate by binding to extracellular matrix proteoglycans [9,11,13] becoming targets for oxidation, glycation, and other enzymatic modifications. Therefore, aggregation is the initial modification suffered by infiltrated LDL affecting VSMC function and phenotype. Indeed, in addition to cytoeskeleton protein modifications, LDL downregulate expression of LDLR whereas upregulate expression of LRP1, a receptor for agLDL in VSMC [14–15]. Thus, LRP1 by internalizing significant amounts of cholesteryl esters from agLDL contributes to the transformation of VSMC into ‘‘foam cell-like’’ cells [15–17]. Overexpression of LRP1 has been found in atherosclerotic plaques in animal models and in human plaques of increasing severity [44–46]. HMG-CoA reductase inhibitors have shown to reduce clinical cardiovascular disease presentation [23–24]. Among them, rosuvastatin, the latest introduced, has shown efficacy in primary presentation [26] and regression or halt of disease burden [27,47]. Several studies have shown that statins favourably alter the fibromuscular composition of the plaques both in hyperlipidaemic Rosuvastatin and Migration of LDL-Loaded VSMC ApoE2/2 mice with increased collagen content and increased SMC-a-actin [34–35] and human carotid plaques [33]. In this study, we aimed at investigating the mechanisms by which rosuvastatin exert plaque remodelling effects and report that rosuvastatin elicits a counteracting regulation of the impairing effects of LDL on VSMC function and migration kinetics. Here, we report for the first time that rosuvastatin prevents in a dose-dependent manner the agLDL-induced dephosphorylation of MRLC and the agLDL-detrimental effects on actin fiber formation. Additionally, rosuvastatin affects intracellular localiza- tion of phosphorylated-MRLC that is found concentrated at the front edge of the migrating cells in the treated VSMC. Phosphorylated-MRLC activates myosin II through its ATPase motor domain, a critical regulator of cytokinesis and as consequence of cell migration [8,29]. Rho GTPases are key regulators of cytoskeleton dynamics in a wide variety of morphogenetic events, including cell migration [48–49]. Earlier studies in migrating cells had linked Rac1 to the protrusion of the leading edge and RhoA to the contractility control at the back of the cell [50]. However, as shown here recently RhoA has been reported to accumulate at the front of migrating cells. To this respect, a Rab13 dependent trafficking of RhoA from the junctions to the leading edges has been shown in migrating endothelial cells [51]. Besides, active RhoA has been detected in lamellipodia and filopodia of transendothelial migrat- ing T cells where it was involved via mDIA or ROCK signalling in regulation of actin-myosin-mediated protrusion and retraction events at the leading edge [52]. Additionally, a spatio-temporal dynamic process coordinated with other Rho GTPases as Rac and Cdc42 has been also reported [49]. HMG-CoA reductase inhibitors block the synthesis of farnesyl pyrophosphate (FPPP) and geranylgeranyl pyrophosphate (GGPP) isoprenoids and thereby disrupt posttranslational isoprenylation and intracellular translocation from cytoplasm to membrane of isoprenylated proteins involved in cell signalling, such as the members of the Rho family [53]. We have recently demonstrated that simvastatin abrogates agLDL-induced translocation of RhoA to cell membrane in VSMC [54]. Here, we further report that rosuvastatin leads to a highly significant increase in functional GTP-RhoA in the cytosol of VSMC, along with a several-fold decrease in the amount of RhoA partitioning in the membrane compartment. These new findings in VSMC are consistent with those of previous studies in monocytes, HEL cells and endothelial cells showing that statins promote Rho-GTP binding and activation [55–56]. Besides, we report a significant increase of RhoA protein at the front edge of migrating LDL-loaded-VSMC in response to rosuvastatin. All together, our results suggest that anchoring of active RhoA in the cell membrane is not required for the increase in cell motility induced by rosuvastatin in LDL-loaded cells. The functional relevance of this finding is underscored by studies showing that unprenylated G-proteins are also biologically active [55,57–58]. Further studies are needed to determine the mechanism underlying rosuvastatin-mediated cytosolic RhoA activation. One potential explanation is that HMG-CoA reductase inhibitors disrupt the interaction of small G-proteins with its negative regulator RhoGDI (Rho guanine nucleotide dissociation inhibitor) [56], as they do with GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis. These interactions depend on protein isoprenylation [59]. Interestingly, in our study, supplementation of VSMC cultures with GGPP prevented rosuvastatin-induced RhoA-GTP increase in the cellular cytosol. Beside, we observed higher p51GAP levels in LDL-loaded-VSMC in the absence than in the presence of rosuvastatin. PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38935