Insulin Induces Calcification of VSMC via RANKL Figure 1. Effect of in vitro incubation of calcifying vascular smooth muscle cells (CVSMCs) with insulin on RANKL expression. Cells were exposed to vehicle or 1–100 nM insulin for 48 h or to 10 nM insulin for 6–48 h. (A) The dose–response of insulin on RANKL mRNA levels measured by real-time polymerase chain reaction (PCR) in cultured CVSMCs. Data are shown as percentage over basal values. (B) The dose–response of insulin on RANKL protein secreted by CVSMCs in the culture medium. (C) Time course of effects of 10 nM insulin on RANKL mRNA levels in cultured CVSMCs. (D) Time course of effects of 10 nM insulin on RANKL protein secretion in the culture medium by CVSMCs. The dots represent the percent of control RANKL levels at various time points. (n = 3, *p,0.05 vs. control, **p,0.01 vs. control.). doi:10.1371/journal.pone.0029037.g001 the RANKL/OPG/RANK system is involved in the pathogenesis of arterial calcification. The first evidence for an involvement of OPG in arterial calcification was derived from OPG knockout mice that displayed severe osteoporosis and arterial medial calcification of the aorta and renal arteries. Furthermore, administration of OPG can inhibit vascular calcification in experimental animals [26,27]. Olesen et al. showed that insulin could accelerate VSMC calcification and was accompanied by an increase in OPG expression [17]. However, their results did not explain if the altered OPG levels during the calcification could directly regulate the calcification process or if it was just a consequence of mineralization. Several studies have suggested that RANKL is involved in VSMC calcification. In addition, RANKL and RANKL transcripts could be detected in calcified arterial lesions from experimental animals [7], and RANKL expression was colocalized and upregulated in areas that displayed arterial calcification [28,29]. In contrast, RANKL increased VSMC calcification by binding to RANK and increased BMP4 produc- tion through activation of the alternative NF-kB pathway [8]. Our recent results demonstrated that RANKL plays a pivotal role in PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29037