Fig. 2.
Calcitriol + DM and paricalcitol + DM, but not R-568 + 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 + DM compared with DM. CASMC were culture expanded for 5 days and then exposed for 7 days to basic medium + vehicle (B), vehicle + DM (C), or calcitriol (10−7 M) + DM (D) (20× magnification). E: Ribonuclease protection assay demonstrated an increase in alkaline phosphatase mRNA in 7-day cultures exposed to calcitriol + DM (10−8 M) and DM+ 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.