TABLE IV. Branched DNA (bDNA) Analysis of Human CASMC Gene Expression After 7 days of DM, Calcitriol þ DM or Paricalcitol þ DM Exposure Gene Alkaline phosphatase (ALP) Matrix gla protein (MGP) Fibrillin 1 (FBN1) Osteoprotegerin (TNFRSF11B) Secreted frizzled related protein-3 (FRZB) Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) Runt related transcription factor 2 (RUNX2) Fold change Cal þ DM vs. DM Par þ DM vs. DM 1.47 1.08 1.04 2.13 1.25 1.52 1.04 1.31 1.10 1.08 1.82 1.08 1.40 1.09 Cal, calcitriol; Par, paricalcitol; DM, differentiation medium. The data were normalized to cyclophilin levels and then were transformed using log2. The values displayed here are fold change (or negative reciprocal if fc < 1) of the treatment 1–treatment 2, where treatments 1 and 2 are given as column names. Bold values indicate fold changes are not equal to 1 ( P < 0.05). phosphate-containing DM was enhanced in a dose-dependent manner by calcitriol and paricalcitol over 7 days. This vitamin D sterol-enhanced calciﬁcation was correlated with CASMC gene expression changes which began by 4 h after initial exposure, conﬁrming that vitamin D sterols have direct effects on SMC. Although most fold changes in gene expression obtained after exposure to vitamin D sterols þ DM, compared with DM, were modest ( P < 0.001; Tables I–III), they were signiﬁcant at more than one time point (Supplemental Tables I and II). The modest fold changes were not unexpected given the vitamin D concentration (10 8 M) administered in vitro in this study. The gene changes may have been greater with higher vitamin D sterol doses than used here [Wu-Wong et al., 2007a] or in the absence of dexamethasone, which is known to antagonize vitamin D effects in some systems. Although the fold change was generally not greater than 2, it is likely that the totality of gene changes induced by vitamin D sterol þ DM treatment, tipped the balance towards the calciﬁcation phenotype, with some protective against calciﬁcation, and others supporting calciﬁcation. The present study is consistent with some, but not all, past reports of increased phosphate-enhanced calciﬁcation of bovine ASMC (BASMC) by vitamin D sterols. In bovine SMC, calcitriol [Shalhoub et al., 2006] and paricalcitol [Shalhoub et al., 2006] increased phosphate-induced calciﬁcation and chondro/osteoblastic gene Fig. 3. Protein analysis in culture medium of human CASMCs after 7 days of DM, calcitriol þ DM or paricalcitol þ DM exposure conﬁrms microarray proﬁle. Upper three panels: HGF after calcitriol, paricalcitol and R-568 exposure, respectively. Lower three panels: TGFb2 after calcitriol, paricalcitol and R-568 exposure, respectively. Each point on the dose–response curves represents results from one out of two experiments. Each experiment was performed on duplicate plates (n ¼ 2 wells/plate/concentration); thus n ¼ 4 wells total/concentration. Each concentration was assayed in duplicate wells (after n ¼ 4 wells were pooled into n ¼ 2 wells), hence the two points per concentration on graphs. M þ AA þ DEX þ BGP ¼ DM. 918 VITAMIN D STEROLS AFFECT CORONARY ASMC GENES JOURNAL OF CELLULAR BIOCHEMISTRY