Rosuvastatin and Migration of LDL-Loaded VSMC Figure 7. Rosuvastatin induced actin-cytoskeleton reorganization, cell adhesion and migration in agLDL-VSMC is dependent on the RhoA/ROCK signaling pathway. agLDL-VSMC incubated with rosuvastatin in absence/presence of inhibitors for ROCK (Y27632) or MLCK (ML9) were analyzed for (A) filamentous actin organization during cell attachment. Note that rosuvastatin-promoted filamentous actin organization in LDL- VSMC was prevented by Y27632 (B) cell attachment (expressed as percentage of the control group) (*) vs agLDL and (#) vs agLDL+ rosuvastatin group, p,0.01 (Mann Whitney test). (C) wound repairing (*) vs agLDL p,0.01 (Mann Whitney test) and (#) vs agLDL+ rosuvastatin group, p,0.01 (Mann Whitney test). In B and C, bar diagrams represent the mean6SEM of 3 independent experiments. doi:10.1371/journal.pone.0038935.g007 Figure 8. Scheme for signaling pathways regulating MRLC- phosphorylation in human VSMC. Potential mechanisms involving agLDL and rosuvastatin effects are indicated. MRLC: Myosin regulatory light chain; MLCK: Myosin light chain kinase. MLC-P: Myosin light chain phosphatase; ROCK: Rho-associated protein kinase; PAK: p21-activated protein kinase. RhoA: Small G proteins; agLDL: aggregated low density lipoproteins. doi:10.1371/journal.pone.0038935.g008 and treated as usual before harvesting, either with Tris 40 mM (cytosol extract) or with lysis buffer provided by the RhoA activation assay kit. Protein concentration was determined immediately (Precision Red Advanced Protein Assay Reagent, ADV02, Cytoskeleton) and equal amount of extracts were incubated with GST-Rhotekin sepharose beads in order to pull down active RhoA. Pulled down beads were washed twice and resuspended in Laemmli sample buffer. The complete pulled down sample, containing only active RhoA, was analyzed by western blot, as described above. Statistical analysis Results are presented as mean6SEM and the number of experiments is shown in every case (see figure legends). Statistical differences between groups were analyzed by parametric tests as T-Student, one-way analysis of variance (ANOVA) or two-way ANOVA, followed by the Fisher’s PLSD test for group differences or by non-parametric tests (Kruskal Wallis or Mann-Whitney), as indicated. P value of 0.05 or less was considered significant. Results Rosuvastatin prevents LDL-induced impairment of VSMC- migration Our previous studies have shown that agLDL hamper mobility of human coronary VSMC [18–19]. Here, treatment of VSMC with 10 mM rosuvastatin prevented LDL-induced impairment in migration kinetics and repaired the cell-depleted areas (Fig. 1A). Differences in cell migration due to treatment with rosuvastatin were already observed 2 hour after wounding (Fig. 1B, Table S1). PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38935