Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol

Abstract

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required.

Fig. 1

Elevated extracellular calcium and/or phosphate enhanced CASMC mineralization. CASMC were culture expanded for 5 days before exposure to different phosphorus (beta-glycerol-phosphate) and total calcium concentrations. Synergistic increase in calcification with combination of calcium and phosphorus. ^aP < 0.0001 versus 1.8/1.9 mM Ca/P; ^bP < 0.0001 and ^cP < 0.001 versus 2.8/0.9 mM Ca/P; ^dP < 0.0001 versus 3.6/0.9 mM Ca/P.

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.

Fig. 3

Protein analysis in culture medium of human CASMCs after 7 days of DM, calcitriol + DM or paricalcitol + DM exposure confirms microarray profile. Upper three panels: HGF after calcitriol, paricalcitol and R-568 exposure, respectively. Lower three panels: TGFβ2 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 = 2 wells/plate/concentration); thus n = 4 wells total/concentration. Each concentration was assayed in duplicate wells (after n = 4 wells were pooled into n = 2 wells), hence the two points per concentration on graphs. M + AA + DEX + BGP = DM.

Fig. 4

Human CASMCs express vitamin D receptor (VDR), but not calcium sensing receptor (CaSR), mRNA by RNase protection assays. A: The 282 nt VDR probe protected a 184 nucleotide (nt) fragment. B: The 540 nt CaR probe protected a 448 nt fragment. The 138 nt cyclophilin (cyclo) probe protected a 103 nt fragment. Lane 1: Probe; lanes 2 and 3: CASMC lot number 3F0246; lane 4: CASMC lot number 00077; lane 5: yeast (negative control); lane 6: kidney (positive control).