wells/plate/concentration; thus n ¼ 4 wells total/concentration [n ¼ 3 independent experiments]). Culture conditions were chosen based on those for optimal conversion of vascular SMC [Mori et al., 1999] or mesenchymal stem cell [Cheng et al., 1994] into osteoblastic cells. Ascorbic acid (AA) is a cofactor for collagenous extracellular matrix formation (not present in DMEM), while BGP provides a phosphate source after alkaline phosphatase (ALP) enzymatic phosphate release. The glucocorticoid dexamethasone (Dex) accelerates phosphate-induced CASMC calciﬁcation [Mori et al., 1999], therefore a concentration of 10 9 M Dex was included in differentiation medium (DM) comprised of expansion medium, AA (50 mg/ml), Dex (10 9 M), and BGP (10 mM) [Shioi et al., 1995; Mori et al., 1999]. Expansion medium with AA and Dex is referred to henceforth as ‘‘basic medium, BM.’’ For vitamin D sterol and calcimimetic exposure, DM was supplemented with vehicle (vehicle þ DM), calcitriol (10 11–10 7 M) (calcitriol þ DM), parical- citol (10 11–10 7 M) (paricalcitol þ DM), or R-568 (10 11–10 7 M) (R-568 þ DM). Although three different vehicles were used (100% ethanol [calcitriol], 30% polyethylene glycol/20% ethanol in water [paricalcitol], and distilled water [R-568]), no vehicle-induced differences in gene expression were observed. Cell medium was changed every 2–3 days for a total of three feedings over 7 days. TREATMENT PROTOCOL FOR CASMC CALCIFICATION WITH CALCIUM AND PHOSPHATE Cells were cultured in 24-well plates (3,500 cells/cm2, n ¼ 4) in EM. After 5 days CASMC were exposed to BM or BM adjusted with increasing concentrations of total calcium (in the form of CaCl2) and/or phosphate (in the form of BGP). The ﬁnal concentration in BM of total calcium was 1.8, 2.8, and 3.6 mM and phosphate was 0.9, 2, 4, 5 mM. Cell medium was changed every 2–3 days for a total of three feedings over 7 days. In DMEM calcium and phosphate concentrations are 1.8 and 0.9 mM, respectively. MINERALIZATION (VON KOSSA) STAINING Mineral deposits were detected with von Kossa staining by incubating cultures with 3% silver nitrate (Sigma) for 30 min under ultraviolet light as previously described [Shalhoub et al., 2006]. QUANTITATIVE ASSAY FOR CALCIUM ACCUMULATION Calcium was quantiﬁed as previously described [Mori et al., 1999]. Brieﬂy, cell layers were washed with Tris buffer (10 mM, pH 7.0) prior to Ca solubilization with 0.6 N HCl. Ca concentration was determined by cresolphthalein procedure (Stanbio Laboratory, Boerne, TX), normalized to protein content from the same well. Protein was solubilized with 0.1 N NaOH/0.1% SDS and quantiﬁed by the Coomassie Blue method (Pierce, Rockford, IL). Calcium is therefore expressed as micrograms Ca/mg protein. RNASE PROTECTION ASSAYS Total RNA was extracted from CASMC using STAT60 reagent (Tel- test, Friendswood, TX) and quantiﬁed as previously described [Shalhoub et al., 2006]. Radiolabeled antisense RNA probes were transcribed from linearized plasmid template using RNA polymerase and a 32P-uridine triphosphate (UTP). RNase protection assays were performed using the RPA II kit (Ambion, Austin, TX) and 5 mg (ALP, Runx2) or 10 mg (CaSR, VDR) of total cellular RNA. After overnight exposure, phosphor screens were scanned and densities of the protected bands were calculated with ImageQuant software (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The GenBank # and size of protected fragment for the examined genes were: CaSR (U20759 nts 1786–2234), VDR (J03258.1 nts 192–376), ALP (X14174 nts 450–685), Runx2 (NM_004348 nts 853–1045). MICROARRAY ASSAYS For microarray studies, CASMCs were cultured in parallel with dose– response experiments using the same batch of cells. Passage 7 cells grown in 6-well dishes at 3,500 cells/cm2 were exposed to vehicle þ DM alone, calcitriol (10 8 or 10 11 M) þ DM, paricalcitol (10 8 or 10 11 M) þ DM, or R-568 (10 7 or 10 11 M) þ DM for 4 h or 1, 3, or 7 days (n ¼ 1 well per treatment, three independent experiments). Results were the average of three independent experiments. Total RNA was isolated using Qiagen RNeasy Mini Kit (Valencia, CA) and processed following the protocols described in section 2 (Eukaryotic Sample and Array Processing; 701024 rev 1) of the Affymetrix Technical manual. Brieﬂy, 5 mg total RNA was used to synthesize cDNA (10 pmol of T7-(dT)24 primer, and Superscript II (Invitrogen, Carlsbad, CA). Puriﬁed double-stranded cDNA (MinElute Reaction Cleanup Kit (Qiagen, Valencia, CA)) was used to generate biotinylated cRNA (Bioarray High Yield RNA Transcript labeling Kit (Enzo Diagnostics, Farmingdale, NY); puriﬁed by Qiagen RNeasy Mini kit) for hybridization to the Affymetrix Human Genome U133 Plus 2.0 array. Arrays were washed on a GeneChip Fluidic Station 450 (EukGE_WS2v4_450 protocol) and scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). Data can be accessed at http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE11917. BRANCHED DNA (BDNA) ANALYSIS Changes in expression of select genes were conﬁrmed by bDNA analyses using the QuantiGene Screen Kit (Genospectra, Fremont, CA) according to the manufacturer’s instructions. Expression values normalized to cyclophilin levels were log2 transformed. PROTEIN ANALYSIS OF CULTURE MEDIUM After the last feeding, the 48-h conditioned culture medium (with different concentrations of vitamin D sterols or R-568) was transferred at harvest on day 7, and frozen at 808C for later protein determination. Transfer involved pooling each two of n ¼ 4 wells (1 ml DM/well) from duplicate plates. This resulted in two, 2 ml samples per treatment. Each point on dose–response curves represents results from one, 2 ml pooled sample, from one out of two experiments, each performed in duplicate. The medium was thawed on ice and TGFB2 and HGF levels were measured using the sensitive MILLIPLEXTM MAP assay, which is based on LuminexR xMAP technology (Millipore). Each kit contained two quality control samples, a low range (146–303 ng/ml) and high range (558– 1,158 ng/ml) for TGFB2; and a low range (546–1,595 pg/ml) and high range (2,896–7,888 pg/ml) for HGF. The quality control sample values for TGFB2 and HGF were veriﬁed within the expected ranges, validating assay reagents and results obtained with conditioned medium. JOURNAL OF CELLULAR BIOCHEMISTRY VITAMIN D STEROLS AFFECT CORONARY ASMC GENES 913