STATISTICS Values are presented as mean SE. Signiﬁcance ( P < 0.05) was tested using an ANOVA with a Fischer’s post hoc test (calciﬁcation) or an ANOVA with a Bonferonni correction for multiple testing (as indicated) (gene expression). For the microarray analysis, statistics were performed as previously described [Shalhoub et al., 2006], with the addition of R-568 þ DM group. ‘‘Perfect match’’ oligonucleotide reporters used to determine intensity measurements were log-transformed, scaled using a nonlinear normalization, summarized into a single intensity using the arithmetic mean and referred to as ‘‘gene intensities.’’ For error-weighted ANOVA, the reciprocal of the squared error was used as a weight. Intensities reported as zero or otherwise ﬂagged by the image analysis software were treated as missing. Hierarchical clustering was performed using the correlation matrix as a measure of similarity. Linkage maps were built using the single-linkage method. ANOVA was performed on a subset of genes to test the hypothesis that gene intensity was not altered due to treatment group. In tests involving multiple runs, experimental run number was treated as a random covariate and an error weighted ANOVA was performed using SAS/JMP (Cary, NC). Samples were divided into the following groups: EM þ vehicle, BM þ vehicle, DM þ vehicle, calcitriol (10 8 or 10 11 M) þ DM, paricalcitol (10 8 or 10 11 M) þ DM, or calcimimetic R-568 (10 7 or 10 11 M) þ DM. Signiﬁcantly changed genes at day 7, as well as candidate genes not changed at day 7, were examined for signiﬁcant changes at earlier times (4 h, and 1 and 3 days). It is possible that additional genes were signiﬁcantly changed at the earlier times. VITAMIN D RESPONSE ELEMENT (VDRE) PROMOTER ANALYSIS Upstream promoter sequences of the regulated genes identiﬁed in this study were extracted from UCSC genome server (http:// genome.ucsc.edu/) [Karolchik et al., 2004], which contains sequences 2,500 bp upstream of the transcription starting site. The position weight matrix (PWM) of VDRE was obtained from JASPAR [Sandelin et al., 2004], an open-access database of annotated eukaryotic transcription factor binding proﬁles. The VDRE binding sites in the promoter regions were predicted with the PATSER computer program in bioperl [Stormo et al., 1982; Letondal, 2001; Stajich et al., 2002], a programming interface for bioinfor- matics application programmers, and signiﬁcant putative VDRE sites were selected with the PATSER score cut-off >75. This score is assumed to be exponentially related to the probability of binding. PATSER also calculates a P-value cut-off for each weight matrix using the information content [Staden, 1989]. RESULTS CALCIFICATION OF HUMAN DONOR CASMCS We sought to determine the gene expression changes associated with vitamin D sterol enhancement of VC induced by uremic-like conditions found in secondary HPT using an in vitro model of human CASMC calciﬁcation. To properly model the calciﬁcation of human vascular SMC induced by the mineral imbalance found in Fig. 1. Elevated extracellular calcium and/or phosphate enhanced CASMC mineralization. CASMC were culture expanded for 5 days before exposure to different phosphorus (beta-glycerol-phosphate) and total calcium concentra- tions. Synergistic increase in calciﬁcation with combination of calcium and phosphorus. aP < 0.0001 versus 1.8/1.9 mM Ca/P; bP < 0.0001 and cP < 0.001 versus 2.8/0.9 mM Ca/P; dP < 0.0001 versus 3.6/0.9 mM Ca/P. CKD-MBD patients [Reynolds et al., 2004; Yang et al., 2004], calciﬁcation in response to increased calcium and phosphorus concentrations was monitored. A 7-day exposure to BM with elevated concentrations of total calcium and phosphorus increased the calciﬁcation of CASMCs (Fig. 1). This Ca- and P-induced calciﬁcation was dose-dependent and synergistic (Fig. 1). In contrast to the lack of calciﬁcation in human CASMC cultured in BM containing AA (50 mg/ml), Dex (10 9 M), Ca (1.8 mM), and P (0.9 mM) (BM) for 7 days (Fig. 2A,B), there was pronounced black von Kossa stained mineral deposits in the extracellular matrix of CASMC exposed to 10 mM BGP (DM) (Fig. 2C). Calciﬁcation in the presence of DM was quantiﬁed in Figure 2A. CALCIFICATION OF CASMCS IN THE PRESENCE OF PHOSPHATE AND VITAMIN D The addition of vitamin D sterols (calcitriol or paricalcitol) enhanced the calciﬁcation of CASMCs induced by 7 days of DM [AA (50 mg/ ml), Dex (10 9 M), Ca (1.8 mM), and BGP (10 mM)] (Fig. 2A). The von Kossa mineral staining induced by DM was enhanced by exposure to calcitriol (10 7 M) þ DM (Fig. 2D) or paricalcitol (10 7 M) þ DM (not shown) for 7 days. This enhanced calcium accumulation was dose- dependent over the range of 10 11–10 7 M vitamin D sterol (calcitriol EC50 9.9 0.3 nM, paricalcitol EC50 8.7 0.3 nM) (Fig. 2A). There was no effect on calcium accumulation in response to the calcimimetic R-568 (10 11–10 7 M; 7 days) þ DM (Fig. 2A). To further conﬁrm the utility of the model cell system used, we sought to establish the expression and vitamin D sterol-induced regulation of two well-characterized mineralization genes in the CASMCs. CASMCS cultures expressed both ALP and runt related transcription factor 2 (Runx2) mRNA (Fig. 2). Whereas ALP mRNA expression was increased by calcitriol þ DM (Figure 2E) and paricalcitol þ DM (Fig. 2E), Runx2 mRNA expression was unchanged (Fig. 2F). 914 VITAMIN D STEROLS AFFECT CORONARY ASMC GENES JOURNAL OF CELLULAR BIOCHEMISTRY