Indeed, wound repair kinetics (mm2/hour) were significantly higher in cells treated with rosuvastatin than in cells incubated with LDL alone (P,0.01 for both nLDL and agLDL). In contrast, rosuvastatin did not affect wound repair kinetics in the control non-LDL treated group (Fig. 1C; P = NS for control vs control+rosuvastatin group). As shown in Fig. 2A, the effect of rosuvastatin on migration of VSMC exposed to LDL (LDL-VSMC) was dose-dependent (2.5 to 10 mM). Thus, rosuvastatin at 10 mM, concentration that corre- sponds to the blood concentration in patients treated with high therapeutic-doses (40 mg), restored to control levels the VSMC migration that was interfered by LDL. A non-statistically significant increase with respect to 10 mM was found with 40 mM rosuvastatin, concentration above the therapeutic range. Simvastatin elicited a similar effect at a lower concentration according to its lipophilic structure. The effect of rosuvastatin in restoring VSMC migration kinetics was already evident at 4 hours. Additionally, in a set of experiments, VSMC were lipid-loaded (100 mg/mL agLDL for 8 hours [40], Fig. 2B) and then rosuvastatin (10 mM) added for an additional 8 hour-period before wounding. Rosuvastatin restored the migration capacity of VSMC to similar level when treating both lipid-loaded and lipid- unloaded cells (Fig. 2C). The effects of rosuvastatin on the recovery of wound repair function in lipid-loaded-VSMC were not caused by cell prolifer- ation. Indeed, a cell [3H]thymidine incorporation assay demon- strated, as expected, that rosuvastatin (10 mM) significantly reduced (about 80%) proliferation in lipid-loaded VSMC (Table S2). Rosuvastatin reverts the changes induced by LDL on the proteome of MRLC in human coronary VSMC 2-DE and MALDI-TOF techniques were used to compare the proteomic pattern of the non sarcomeric-MRLC isoforms MRLC- 2 (Swiss-Prot number: O14950; observed pI 5.1, observed MW 21.5 kDa) and MRLC-3 (Swiss-Prot number: P19105; observed pI 5.0, observed MW 22.2 kDa) in human VSMC treated with/ without rosuvastatin during exposure to native/aggregated LDL (Fig. 3). MRLC isoforms appeared as a double spot of different pI (MRLC-2: spots 1,2; MRLC-3: spots 3,4; Fig. 3A), which were related to different phosphorylation states. For each MRLC- isoform, the ratio between the intensity level of the spot with lower pI (more phosphorylated forms) and this with higher pI (less phosphorylated forms) was calculated. Treatment of VSMC with rosuvastatin reverted dephosphorylation induced by LDL in MRLC-3 and MRLC-2. The effect was markedly evident for the agLDL-treated group (Fig. 3B). Western blot analysis of agLDL-VSMC treated with different rosuvastatin concentrations revealed a progressive increase in P- MRLC compared to untreated cells (Fig. 3C), being the increase already evident with the lowest concentration of rosuvastatin (2.5 mM). The highest concentration of rosuvastatin (10 mM) increased levels of P-MRLC to 90% of the control group (without agLDL). Rosuvastatin increases P-MRLC levels at the migration front of agLDL-treated VSMC We have also examined the effect of rosuvastatin on the subcellular distribution of P-MRLC by confocal microscopy (Fig. S1). Migrating agLDL-VSMC treated with rosuvastatin shows a P- MRLC labelling signal intensity similar to the control group. Contrarily, the P-MRLC labelling that is very weak at the border Rosuvastatin and Migration of LDL-Loaded VSMC of the wounded area in agLDL treated VSMC, shows a significant increase in presence of rosuvastatin. Rosuvastatin did not show any effect on the P-MRLC in control VSMC migrating into the wounded area. Effect of rosuvastatin on the subcellular distribution of RhoA in human VSMC Because HMG-CoA reductase inhibitors have shown to regulate isoprenylation of small GTP-binding proteins, we analyzed subcellular RhoA levels in cytosol, cytoskeleton-associ- ated and membrane fractions in the lipid loaded VSMC (Fig. S2). Rosuvastatin treatment significantly increased RhoA protein levels in the cytosol of the VSMC (.1.5–2.0fold, p,0.05), while weakly decreased RhoA in the cytoskeleton associated fraction. RhoA levels were markedly lower in the membrane fraction of rosuvastatin treated cells (.8fold decrease, p,0.01). By confocal microscopy analysis, migrating VSMC consistently showed high RhoA levels at the leading edge of the cells, either in the presence or absence of agLDL (Fig. 4A). Rosuvastatin significantly increased RhoA labelling at the migrating front edge (compare Fig. 4B-a and 4B-b with 4B-c and 4A-d). Taking as reference the bulk of RhoA labelling in the control group (Figure 4A), we have defined a 15 mm wide area along the border of the wound (boundary marked by dot lines in Fig. 4B) to quantify the effect of rosuvastatin on the RhoA-level in the migrating VSMC. As shown in Fig. 4C, rosuvastatin induced .2.5fold increase of RhoA either in control and LDL-treated cells (P,0.05 both for control and agLDL-VSMC). Effect of rosuvastatin on RhoA-GTP levels Levels of RhoA-GTP (active RhoA) were analyzed by a pull down assay with GST-rothekin in total cell lysates and cytosol extracts (Fig. 5A). RhoA-GTP was detected in the total cell lysates of both control and LDL-treated VSMC, but never in the cytosolic fraction of these cells. Levels of RhoA-GTP were significantly increased when cells were treated with rosuvastatin. RhoA-GTP levels increased both in total cell lysates and in the cell cytosol- fraction (Fig. 5A). There was no increase in RhoA-GTP in the cytosol of cells coincubated with rosuvastatin and 10 mM geranyl- geraniol pyrophosphate (GGPP) (Fig. 5B). Besides, the level of cytosolic ROCK, the major downstream target of active RhoA, was 2.5fold increased (P,0.01) in agLDL-VSMC treated rosuvastatin (Fig. 5C). By 2-DE we identified the p50RhoGAP (Q07960, pI 5.9, MW 50 kDa), a member of the GTPase activating protein family that catalyzes inactivation of RhoA. This protein was significantly overexpressed by agLDL-loading of VSMC (mean6SE intensity level in control vs agLDL group: 0.2860.01 vs 0.4160.05 AU, P = 0.03, Fig. S3). Treatment with rosuvastatin decreased p50RhoGAP in agLDL-VSMC to levels detected in control cells (agLDL+rosuvastatin: 0.2760.01 AU, P = 0.02 vs agLDL). Rosuvastatin-induced MRLC-phosphorylation, cytoskeleton reorganization, cell adhesion and migration in agLDL-VSMC is dependent on ROCK-signaling We next explored whether the RhoA-downstream pathway was involved in the effect of rosuvastatin on the cytoskeleton dynamics in agLDL-VSMC. MRLC-phosphorylation is mainly influenced by the opposing activities of MLC-kinase (MLCK) and MLC- phosphatase (MLC-P), which in turn are regulated by p21- activated protein kinase (PAK-1) and Rho-dependent kinase (ROCK) [42–43]. PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38935