Rosuvastatin and Migration of LDL-Loaded VSMC Figure 2. Dose-dependent response to rosuvastatin in cell migration of VSMC exposed to LDL. Bar diagram for (A) dose dependent effect of rosuvastatin (2.5–40 mM) in wound regeneration by VSMC treated without/with agLDL 8 hours after wounding. P,0.01 (Kruskal-Wallis test) for rosuvastatin dose-response effect and P,0.001 for simvastatin effect (Man-Whitney test) vs agLDL-group. (B and C) VSMC were treated without or with rosuvastatin (10 mM) since 2 hours before (2 h-pre LDL) or 8 hours after (8 h-post LDL) adding agLDL to the cell culture medium. B: staining with Herxheimer reagent (Sudan III and Sudan IV) (red colour) as a control for intra-cellular accumulation of lipids. Ø refers to agLDL-VSMC non treated with rosuvastatin. C: Results shown as mean6SEM (% of wounded area at time 0) refer % of the wound regenerated area 4 and 8 hours after wounding (n = 4 independent experiments); * p,0.05 compared to the agLDL-group (Man-Whitney test). doi:10.1371/journal.pone.0038935.g002 Hospital de la Santa Creu i Sant Pau, and cultured as described previously [37]. The study was approved by the Institutional Review Committee on Human studies of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) that conforms to the Declaration of Helsinki and tissue samples were collected with written informed consent from the donors. To investigate the effect of rosuvastatin on LDL-loaded VSMC, cell monolayers were preincubated with 10 mM rosuvastatin, concentration that corresponds to the blood level in patients treated with a high therapeutic-doses (40 mg), for 2 hours prior to the addition of 100 mg/mL native-LDL or aggregated-LDL (nLDL, agLDL) and incubated for further 16–24 hours. In a set of experiments, inhibitors of MLCK (ML9, Sigma), and ROCK (Y27632, Sigma) were added for the last 2 hours. For cell migration and immunohistochemical studies during VSMC wound repair, cells were grown on monolayers to confluence on glass bottom dishes (WPI, World Precision Instruments) and a linear wound was induced after the incubation period with/without rosuvastatin (+/2 inhibitors), in the pres- ence/absence of LDL. For proteomic and protein expression studies, treated cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested in PBS containing 5 mM EDTA and stored at 280uC, until used. Human LDL (density 1.019 to 1.063 g/mL) were prepared by ultracentrifugation from pooled sera of normocholesterolemic volunteers. agLDL were generated by vortexing LDL (1 mg/mL), according to the method previously described by Guyton et al [38]. As largely demonstrated, the vortexing-generated aggregated LDL (agLDLs) share structural characteristics, size, and electro- phoretic mobility with the high atherogenic LDL aggregates retained in atherosclerotic arterial intima due to versican fusion [14,38–40]. Intracellular lipid-accumulation in VSMC was PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38935