on the physiological concentration of 1,25 dihydroxyvitamin D3 (10 10–10 11 M) and the supraphysiological concentration (10 8 M) commonly used in vitro to examine gene expression that effectively enhanced DM-induced calciﬁcation. In dialysis patients, calcitriol (5–35 mg/dialysis session) is administered to achieve normal blood concentrations of 40–80 pg/ml (¼1–2 10 10 M); and paricalcitol is administered at ﬁvefold higher doses. The R-568 concentrations were based on those found in patient sera (Cmax of 7.7 ng/ml, 2.3 10 8 M R-568) [Goodman et al., 2000a; Shalhoub et al., 2006]. The genes identiﬁed with signiﬁcantly ( P < 0.05) altered expression in medium containing AA þ Dex (BM) compared with EM, and AA þ Dex þ BGP (DM) compared with BM after 7 days of treatment, are reported in Supplemental Tables I–III. The effect of Dex in the absence of AA was not assessed. This analysis identiﬁed 53 genes that were signiﬁcantly ( P < 0.05 as compared to DM alone) upregulated by a 7-day exposure to calcitriol (10 8 M) þ DM exposure with fold changes ranging from 1.06 to 10.1 (Supplemental Table I). There were 35 genes signiﬁcantly downregulated by 1.07- to 1.84-fold following 7 days of calcitriol þ DM (Supplemental Table II). For most genes, induction and repression patterns with calcitriol þ DM and paricalcitol þ DM were virtually identical for signiﬁcantly regulated genes (Tables I–IV; Supplemental data, Tables I–III). When assessing the signiﬁcantly altered genes using a 1.3-fold change threshold for any one time point and any one treatment, 31 genes were upregulated with fold changes ranging from 1.3 to 10.1 and 14 genes were downregulated with fold changes of 1.3 to 1.84 (Supplemental Tables I and II). For Tables I and II in Results Section, treatment-induced gene expression alterations were identiﬁed as those genes that were signiﬁcantly ( P < 0.05) and substantially (fold change >1.3) altered at any time point following any treatment. Thus, if a gene was found signiﬁcantly elevated at day 7 for calcitriol þ DM the corresponding value for the same time Calcitriol fold change Paricalcitol fold change Gene BMP4 (7d) BMP6 (7d) TNFRSF11B (OPG) (7d) CILP (7d) IL-6 (3d) KCNK3 (7d) DSP (7d) THBD (1d) ENPP1 (7d) TNC (7d) GCLC (3d) ASPN (7d) HGF (7d) S100A4 (3d) CLEC3B (7d) R1.3 R1.3 R1.3 R1.5 R1.3 R1.5 R1.3 R1.4 1.3 1.3 1.4 1.4 1.8 1.5 1.7 þ1.1 R1.1 R1.3 R1.2 þ1.1 þ1.2 R1.2 R1.3 1.2 1.1 1.2 1.3 1.4 1.4 1.4 point for paricalcitol þ DM (even though not signiﬁcant) was included in Tables I and II for comparison. Supporting the validity for a 1.3-fold cut-off, the majority of genes identiﬁed with altered expression by 7 days of exposure had expression changes in the same direction at one or more of the earlier time points (Supplemental Tables I and II). In contrast to the signiﬁcant effects observed with the supraphysiological concentrations of vitamin D sterols, the 10 11 M concentration had no effect on calciﬁcation or gene expression in vitro (data not shown). In general, when administered at the same concentration (10 8 M), calcitriol exerted a more pronounced effect on CASMC gene regulation than paricalcitol (e.g., CYP24A1 mRNA increased 11.1- and 10.1-fold with calcitriol and 5.5- and 3.9-fold with paricalcitol at 3 and 7 days, respectively). In pre-clinical studies, calcitriol was about threefold more potent compared with paricalcitol to decrease PTH secretion from parathyroid glands [Slatopolsky, 1998]; and, clinically, a three to ﬁvefold higher dose of paricalcitol is administered to achieve the same lowering effect on PTH secretion. In contrast to the altered gene expression proﬁles in response to vitamin D sterols (10 8 M), no signiﬁcant changes were observed with R-568 (10 7 M) þ DM. ALP expression was signiﬁcantly elevated on microarrays for both vitamin D sterols þ DM ( P < 0.05) (Supplemental Table I). Genes identiﬁed as being modulated by Vitamin D sterol þ DM are involved in multiple cellular processes, including vitamin D metabolism, apoptosis, cell cycle, mineralization and cardiovascular pathways (Supplemental Tables I and II). Focusing on genes with a high probable involvement in VC, we identiﬁed the time point of maximal fold change (calcitriol þ DM or paricalcitol þ DM as compared to DM alone) for the signiﬁcantly regulated genes with known skeletal or cardiovascular function in human (Table I) and mouse (Table II). As a positive control for vitamin D sterol effect we used the vitamin D metabolizing enzyme, cytochrome P450, family 24, subfamily A (CYP24A1), which is upregulated in most vitamin D Skeletal disorder Polymorphisms associated with hip BMD Rheumatoid arthritis Idiopathic hyperphosphatasia (Juvenile Paget’s disease) SNIP associated with lumbar disc; crystal deposition in joint disease Periarticular calciﬁcation Polymorphism in Achilles tendon injury Susceptibility to arthritis Arthritis Rheumatoid arthritis Cardiovascular disorder Found in intimal plaque SMCs Elevated in serum of CKD patients and in calciﬁed vessels Cardiovascular disease Arrhythmia Arrhythmogenic ventricular cardiomyopathy Coronary heart disease Spontaneous infantile arterial calciﬁcation Hypertension. Left ventricular remodeling. Endothelial vasomotor dysfunction; myocardial infarction Hypertension/atherosclerosis/cardiovascular disease Plexogenic arteriopathy in pulmonary hypertension TABLE I. Calcitriol (10 8 M) þ DM and Paricalcitol (10 8 M) þ DM Signiﬁcantly Modulated Genes Involved in Human Cardiovascular and Skeletal Function, Compared With DM Affymetrix probe set numbers can be found in Supplemental data, Tables I and II. BMP, bone morphogenetic protein; OPG, osteoprotegerin; CILP, cartilage intermediate layer protein nucleotide pyrophosphohydrolase; IL-6, interleukin-6; KCNK3, potassium channel, subfamily K, member 3; DSP, desmoplakin; THBD, thrombomodulin; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; TNC, tenascin C; ASPN, asporin; TGFBR2, transforming growth factor beta receptor 2; HGF, hepatocyte growth factor; S100A4, S100 calcium binding protein A4; CLEC3B, C-type lectin domain family, member B; GCLC, glutamate-cysteine ligase, catalytic subunit. Values are for the measurement of maximal change (4 h, 1, 3, or 7 days) for calcitriol (10 8 M) þ DM. Bold: P < 0.001; italics: P < 0.05. 916 VITAMIN D STEROLS AFFECT CORONARY ASMC GENES JOURNAL OF CELLULAR BIOCHEMISTRY