Vitamin D receptor activators induce an anticalcific paracrine program in macrophages: requirement of osteopontin

Abstract

Objective: Vascular calcification is highly correlated with morbidity and mortality, and it is often associated with inflammation. Vitamin D may regulate vascular calcification and has been associated with cardiovascular survival benefits.

Methods and results: We developed a macrophage/smooth muscle cell (SMC) coculture system and examined the effects of vitamin D receptor activators (VDRA), calcitriol and paricalcitol, on SMC matrix calcification. We found that treatment of SMC alone with VDRA had little effect on phosphate-induced SMC calcification in vitro. However, coculture with macrophages promoted SMC calcification, and this was strikingly inhibited by VDRA treatment. Several VDRA-induced genes, including bone morphogenetic protein-2 (BMP2), tumor necrosis factor-alpha, and osteopontin, were identified as candidate paracrine factors for the protective effect of VDRA. Of these, osteopontin was further investigated and found to contribute significantly to the inhibitory actions of VDRA on calcification in macrophage/SMC cocultures.

Conclusions: The ability of VDRA to direct a switch in the paracrine phenotype of macrophages from procalcific to anticalcific may contribute to their observed cardiovascular survival benefits.

Figure 1

Macrophage coculture promotes SMC calcification in vitro. THP-1 macrophage/SMC cocultures were treated with GM or CM for 10 days. Calcium content of the SMC cultures was measured and presented as mean ± S.D. (n = 3). * Significant increase compared with SMC cultured alone (P <0.05).

Figure 2

VDRAs inhibit SMC calcification in macrophage/SMC coculture. THP-1 macrophage/SMC cocultures were treated with various concentrations of calcitriol or paricalcitol in GM or CM for 10 days. Calcium content of the SMC cultures was measured and presented as mean ± S.D. (n = 3). * Significant decrease compared with vehicle (P <0.05).

Figure 3

VDRAs inhibit BMP2 and TNFα mRNA levels. Total RNA was obtained from THP-1 macrophages treated with 50 nM calcitriol or paricalcitol for 3 and 6 days for BMP2 (A) and 3 days for TNFα (B) and analyzed by QPCR. Results were normalized to 18s rRNA and are presented as mean ± S.D. (n = 3). * Significant decrease compared with vehicle (P <0.05).

Figure 4

VDRAs induce OPN expression in macrophages. (A) Total RNA was obtained from THP-1 macrophages treated with 50 nM calcitriol or paricalcitol for 3 and 6 days respectively. The levels of OPN mRNA were determined by QPCR and normalized to 18s rRNA. (B) Conditioned media were collected from THP-1 macrophages treated with 50 nM of calcitriol or paricalcitol for 6 days. OPN protein levels were determined by ELISA. Data are presented as mean ± S.D. (n = 3).

Figure 5

Downregulation of OPN production in P388D1 macrophages prevents VDRA-mediated inhibition of SMC calcification in coculture. Cocultures of SMC with either CT siRNA or OPN siRNA macrophages were treated with 50 nM calcitriol or paricalcitol in CM for 10 days. Calcium content of the SMC culture was measured and presented as mean ± S.D. (n = 3). * Significant decrease compared with vehicle (P <0.05).