Insulin promoted the mineralization of the matrix in calcifying vascular smooth muscle cells (CVSMCs).

<p>CVSMCs were cultured in DMEM that contained 10% fetal bovine serum (FBS) in 24-well plates, in the presence of vehicle or 10 nM insulin for 20 days. Mineralization of the matrix was determined by Alizarin red S staining. (A) Representative microscopic view at a magnification of ×100. Left panel showed CVSMCs incubated with vehicle, and the right panel showed CVSMCs incubated with 10 nM insulin for 12 days. (B) Quantification of Alizarin red S stain through extraction with cetyl-pyridinium chloride. The amount of released dye was quantified by spectrophotometry at 540 nm. The bars represent mean ± standard deviation (SD) (<i>n</i> = 4; **<i>p</i><0.01 vs. control). (C) Cells were treated with vehicle (serum-free medium) or insulin at 1, 5, 10, or 100 nM for 48 h in serum-free DMEM. ALP activity were determined and normalized to cell total protein. The dose–response of insulin on ALP activity in cultured CVSMCs. (D) Cells were treated with vehicle (serum-free medium) or insulin at 1, 5, 10, or 100 nM for 48 h in serum-free DMEM. Osteocalcin secretion were determined and normalized to cell total protein. The dose–response of insulin on osteocalcin secretion in cultured CVSMCs. The bars represent mean ± standard deviation (SD) (**<i>p</i><0.01 vs. control, *<i>p</i><0.05 vs. control, <i>n</i> = 4).