of mineralization inhibitors [Ketteler et al., 2003; Lomashvili et al., 2005], apoptotic mechanisms [Reynolds et al., 2004], and current therapies such as calcium-based phosphate binders and vitamin D sterols [Goodman et al., 2000b; Wolisi and Moe, 2005]. The interrelationships between these factors, the precise timing of molecular events, and other possible factors are currently unknown. While vitamin D is necessary for patient health and to control secondary HPT in dialysis patients, the harm of too little or too much vitamin D is becoming increasingly appreciated. In pre-clinical CKD-MBD models, pharmacological levels of the vitamin D sterol calcitriol increased VC [Haffner et al., 2005; Wu-Wong et al., 2006; Cardus et al., 2007], but it was not assessed whether this was a result of direct effects on vascular smooth muscle cell (SMC) in the setting of deranged minerals, indirect via increased mineral levels, or both. Early reports of in vitro model systems demonstrated that bovine and human VSMC spontaneously converted into chondro/osseous phenotypes that calciﬁed [Bostrom et al., 1993; Watson et al., 1994; Shanahan and Weissberg, 1999; Tyson et al., 2003]. Calciﬁcation was accelerated by phosphate [Shioi et al., 1995; Jono et al., 2000; Reynolds et al., 2004] and more-so by phosphate þ dexamethasone [Mori et al., 1999] or vitamin D [Jono et al., 1998]. However, subsequent reports on direct effects of vitamin D sterols on SMC calciﬁcation were conﬂicting. We reported that vitamin D sterols (calcitriol and paricalcitol) increased phosphate-induced calciﬁca- tion and chondro/osteoblastic gene expression [Shalhoub et al., 2006], whereas others showed no effect of calcitriol or paricalcitol on phosphate-induced SMC calciﬁcation [Wolisi and Moe, 2005; Wu-Wong et al., 2006], or effects by one but not another vitamin D sterol [Cardus et al., 2007]. Calcimimetics are allosteric small molecule activators of the calcium sensing receptor (CaSR) predominantly expressed on parathyroid glands. Calcimimetics, which decrease PTH and serum Ca and P levels in dialysis patients [Block et al., 2004; Lindberg et al., 2005], did not appear to induce VC in a rodent uremic model [Henley et al., 2005]. Whether this lack of calciﬁcation was the result of direct or indirect effects of calcimimetics on vascular cells could not be assessed in vivo. Some have described calcimimetic responses in SMCs in the absence of the parathyroid CaSR, whereas others have described the CaSR in SMCs and effects on mineralization [Farzaneh-Far et al., 2000; Smajilovic et al., 2006; Molostvov et al., 2007; Alam et al., 2009; Ivanovski et al., 2009]. In vitro, matrix gla protein (MGP) reporter activity increased in cultured rat SMCs in response to calcium and CaSR agonists [Farzaneh-Far et al., 2000] and this response was attributable to a CaSR-like receptor, rather than the CaSR. Others demonstrated calcium- and CaSR agonist- induced responses, CaSR mRNA and/or protein in rat and human SMCs and delay or inhibition of mineralization after calcimimetic R-568 exposure [Smajilovic et al., 2006; Molostvov et al., 2007; Ivanovski et al., 2009; Alam et al., 2009]. However, other reports showed no effect of calcimimetics on phosphate-induced calciﬁca- tion in bovine aorta SMCs [Shalhoub et al., 2006] and undetectable CaSR transcripts [Farzaneh-Far et al., 2000; Shalhoub et al., 2006]. Thus, reports on the presence of the CaSR and response to calcimimetics in SMC are conﬂicting, limiting the assessment of direct effects of calcimimetics on SMCs. Thus, we felt examining the effects of calcimimetics in the presence or absence of the CaSR warranted. To elucidate possible interrelationships between mineral imbal- ance and secondary HPT therapeutics, we determined the gene expression proﬁle in response to calcitriol, paricalcitol, and the calcimimetic R-568 in an in vitro model of human coronary artery SMC (CASMC) calciﬁcation. Vitamin D sterols exacerbated phosphate-induced calciﬁcation in human CASMC, while modulat- ing multiple genes containing vitamin D receptor (VDR) response elements that were representative of multiple gene networks including mineralization, apoptotic, osteo/chondrocytic, and car- diovascular pathways. In contrast, the calcimimetic R-568 did not affect CASMC calciﬁcation or gene expression. Future studies will dissect which vitamin D-regulated genes are involved in protective and pro-calciﬁcation processes. These results may have implications for CKD-MBD patients on dialysis that are at increased VC risk and may be exposed to more than optimal phosphate and vitamin D sterol concentrations. MATERIALS AND METHODS MATERIALS Tissue culture plates were purchased from Falcon-Becton Dickinson (Franklin Lakes, NJ), phenol-red free Dulbecco’s modiﬁed Eagle’s medium (DMEM) from Cambrex Biosciences (Walkersville, MD); penicillin/streptomycin/glutamine, sodium pyruvate, and fungizone from GIBCO-Invitrogen (Grand Island, NY), fetal bovine serum (FBS) from Terra Cell (Etobicoke, Canada), calcitriol from Calbiochem- EMD Biosciences (La Jolla, CA), and paricalcitol from Abbott (North Chicago, IL). MILLIPLEXTM MAP kits to measure transforming growth factor beta (TGFb2) and hepatocyte growth factor (HGF) were obtained from Millipore (Billerica, MA). CELLS Primary human CASMC from two donors (catalog number CC-2583: lot number 3F0246, donor 1; and lot number 00077, donor 2) were obtained from Clonetics (Walkersville, MD). Cell cultures were expanded in ‘‘expansion medium’’ (D-MEM/15% FBS/1 Na pyruvate/penicillin/streptomycin; EM) as previously reported [Shioi et al., 1995]. CASMC from donor 1, but not donor 2, mineralized in EM in the presence of 10 mM beta-glycerolphosphate (BGP) within 2 weeks, and was used for subsequent studies. All experiments were performed on passage 7 cells, and results are from the average of 2–3 independent experiments. Cells for each dose–response and microarray experiment were plated at the same time from the same batch of cells, but in different size wells: for calcium accumulation dose–response (in 24 wells, n ¼ 4); for microarrays (in 6 wells, n ¼ 1). Thus, three independent experiments were performed sequentially, at different times, each with a dose–response and microarray component, so that gene expression could be correlated to calciﬁcation. TREATMENT PROTOCOL FOR CASMC CALCIFICATION WITH VITAMIN D STEROLS AND CALCIMIMETIC R-568 Cells were seeded in 24-well plates (3,500 cells/cm2) in EM. Each experiment was performed in duplicate on separate plates (n ¼ 2 912 VITAMIN D STEROLS AFFECT CORONARY ASMC GENES JOURNAL OF CELLULAR BIOCHEMISTRY