Functional RhoA plays a key role in regulating molecular responses linked to actin dynamics [60]. RhoA-GTP by binding to the Rho-binding domain (RBD) of ROCK disrupts the interaction between the catalytic N-terminal and the inhibitory C-terminal regions of the enzyme, stimulating in turn the phosphotransferase activity of ROCK [61]. Thus, RhoA regulates MRLC-phosphor- ylation through its downstream effector ROCK and through MLC-phosphatase. In addition, ROCK may also phosphorylate Ser19 in MRLC, the same residue that is phosphorylated by MLC kinase (MLCK) [43]. In LDL-loaded-VSMC, ROCK inhibition completely reverted rosuvastatin induced MRLC-phosphoryla- tion, whereas MLC-kinase (MLCK) inhibition did not induced a significant effect. Taken together, these results indicate that rosuvastatin induces MRLC-phosphorylation, enhances cytoskel- eton organization and promotes cell adhesion and migration of LDL-loaded-VSMC through a mechanism involving activation of the RhoA/ROCK downstream pathway. MLCK activity is inhibited by phosphorylated p21-activated kinase (P-PAK) [42]. Rosuvastatin did not revert the increase in phosphorylated PAK induced by agLDL, thus emphasizing the relevance of the ROCK signalling pathway in MRLC-phosphor- ylation. Thus, we propose that agLDL induced MRLC-dephos- phorylation in VSMC is linked to high levels of P-PAK and to a low MLCK-activity, whereas rosuvastatin counteracts the agLDL effect on P-MRLC by a mechanism that mainly depends on the increase of cytosolic RhoA-GTP and its target ROCK, which in turn blocks MLC-phophatase activity and activates MRLC- phosphorylation (Fig. 8). In control cells, without LDL, there is a redundancy in the mechanisms involved in MRLC-phosphor- ylation that overcome the effect of rosuvastatin during cell migration. Indeed, rosuvastatin, in the absence of LDL, does not induce migration in VSMC, in agreement with the inhibitory effect in motility attributed by others to rosuvastatin in absence of LDL [62–63]. Moreover, rosuvastatin might affect VSMC migration through additional mechanisms directly involving activation/inhibition of other small G-proteins, which will require future studies. In summary, current imaging methods in clinical studies [27] have shown that rosuvastatin reduces atherosclerotic plaque burden and the vulnerability of the plaques. Here, we demonstrate that these effects of rosuvastatin could be mediated by counter- acting the impairing effects of LDL in MRLC phosphorylation and cell migration/repair function of lipid-loaded-VSMC. 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Cells were labelled for P- MRLC (Alexa Fluor 488-conjugated, signal in green) and F-actin (Alexa-Fluor 633 conjugated phalloidin, signal in red). (TIF) Figure S2 Increase of RhoA levels upon rosuvastatin treatment in the cytosol of human VSMC. RhoA and b- actin in cytosol, cytoskeleton-membrane and membrane extracts of VSMC with/without LDL and treatment with rosuvastatin or vehicle for 24 hours. The histograms show results expressed as % of the controls after normalization for b-actin. Results are given as mean6SEM of 3 independent experiments. (TIF) Figure S3 Rosuvastatin decreases p50RhoGAP level in LDL-treated VSMC. Representative 2-DE gel of the urea/ detergent-soluble fraction of human VSMC. Enlarged images correspond to the gel area where p50RhoGAP (pI 5.9, MW 50 kDa) was detected. Rosuvastatin markedly decreases the labelling signal for p50RhoGAP (agLDL+rosuvastatin) in VSMC treated with agLDL. 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