Fig. 2. Calcitriol þ DM and paricalcitol þ DM, but not R-568 þ DM, dose dependently increased calciﬁcation of human CASMC. A: CASMC were culture expanded for 5 days and exposed to DM supplemented with 10 11–10 7 M calcitriol, paricalcitol or R-568 for an additional 7 days. Calcium was determined and normalized to protein as in the Materials and Methods Section. P < 0.05 versus DM. Results represent the average of two independent experiments. B–D: Representative von Kossa stained CASMC cultures showing enhanced calciﬁcation by vitamin D sterol þ DM compared with DM. CASMC were culture expanded for 5 days and then exposed for 7 days to basic medium þ vehicle (B), vehicle þ DM (C), or calcitriol (10 7 M) þ DM (D) (20 magniﬁcation). E: Ribonuclease protection assay demonstrated an increase in alkaline phosphatase mRNA in 7-day cultures exposed to calcitriol þ DM (10 8 M) and DMþ paricalcitol (10 8 M). The protected fragment was 235 nt. E: Ribonuclease protection assay demonstrated Runx2 mRNA expression in 7-day CASMC cultures. The protected fragment was 192 nt. MICROARRAY ANALYSES To test the hypothesis that the enhanced CASMC calciﬁcation was associated with altered expression of genes we sought to identify genes differentially regulated by vitamin D sterols or R-568. Microarray analyses were performed with total RNA isolated from cultures exposed to vehicle þ DM or vitamin D sterol (10 8 or 10 11 M) þ DM or R-568 (10 7 or 10 11 M) þ DM for 4 h and 1, 3, and 7 days. The concentrations of vitamin D sterols were based JOURNAL OF CELLULAR BIOCHEMISTRY VITAMIN D STEROLS AFFECT CORONARY ASMC GENES 915