Figure 4.

ERK signaling mediates Runx2 activation and VSMC calcification. (A) Expression of phosphorylation ERK1/2 was determined in VSMCs exposed to 10 ng·mL⁻¹ OMV in osteogenic media by western blot analysis. Representative results from three independent experiments in duplicate are shown (upper). VSMCs were pretreated with ERK1/2 inhibitor (PD98059) 20 μm for 30 min and then exposed to 10 ng·mL⁻¹ OMV in osteogenic media. The treatment was repeated every 3 days, and cells were treated for 2 weeks. Expression of p‐ERK1/2 was determined in VSMCs exposed to 10 ng·mL⁻¹ OMV cotreated with PD98059 by western blot analysis (lower). (B) VSMC calcification was determined by Alizarin Red staining. (C) Effect of MAPK inhibitor on the expression of Runx2, ALP, ColIA1, OC, and SM22α was determined by RT‐PCR. VSMCs were pretreated with PD98059 (20 μm) for 30 min and then exposed to 10 ng·mL⁻¹ OMV for 2 weeks in osteogenic media. The results are presented as the fold increase relative to the control group (mean ± SD, n = 3). Representative results from five independent experiments in duplicate are shown (*P < 0.01, compared with control).