Insulin Induces Calcification of VSMC via RANKL Figure 3. Insulin promoted the mineralization of the matrix in calcifying vascular smooth muscle cells (CVSMCs). 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 6100. 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 6 standard deviation (SD) (n = 4; **p,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 6 standard deviation (SD) (**p,0.01 vs. control, *p,0.05 vs. control, n = 4). doi:10.1371/journal.pone.0029037.g003 Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-meth- yl-3-O-octadecylcarbonate (HIMO) was purchased from Calbio- chem (San Diego, CA, USA). Recombinant rat RANKL was purchased from ABCAM (Cambridge, MA, USA). Cell culture Six-week-old male Sprague Dawley rats were sacrificed, the aorta removed, and calcifying vascular smooth muscle cells (CVSMCs) were isolated as described previously [20,38]. Briefly, the aortas were removed under sterile conditions. The aortas were rinsed several times in Hanks balanced salt solution, then the adventitia was removed and the aortas were minced and digested in 5 ml of digestion solution (0.125 mg/ml elastase, 0.25 mg/ml soybean trypsin inhibitor, 10 mg/ml collagenase I, 2.0 mg/ml crystallized bovine albumin, and 15 mM HEPES) at 37uC for 45 min. The cellular digests were filtered through sterile100-mM nylon mesh, centrifuged at 1000 rpm for 10 min, and washed twice in Dulbecco’s modified Eagle’s medium (DMEM) that contained 10% fetal bovine serum (FBS; Gibco-BRL Corp., NY, USA) before culture in the same medium. CVSMCs were isolated from cultures in which multicellular nodules appeared spontane- ously. Cells were cloned by limiting dilution and single cell harvesting from these nodule-forming cultures. Cloned lines were identified as CVSMCs by their positive staining with monoclonal PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29037