Table 1.
Primers used for quantitative RT-PCR analyses.
Figure 1.
The effect of magnesium on calcification and osteoblast-like transformation in VSMC in vitro.
VSMC were cultured with 3.3 mM P, and increasing concentrations of magnesium (0.8 mM/1.4 mM/2.6 mM); control group with 0.9 mM P+0.8 mM Mg was also included. (A), calcium content of VSMC cultured for 9 days. Relative mRNA expression of Cbfa-1 (B) and osterix (C); a, P<0.001 vs Control; b, P<0.001 vs VSMC with 3.3 mM P+0.8 mM Mg. Bars are the mean±SE of three experiments in quadruplicate.
Figure 2.
Inhibition of TRPM7 abrogates protective effect of magnesium on calcification and gene expression on VSMC.
VSMC were cultured with 3.3+1.4 mM Mg with the TRPM7 inhibitor 2-APB (10 µM). VSMC with 3.3 mM P+0.8 mM Mg were also included. The graphics are (A) calcium content, and the relative mRNA expression of (B) Cbfa-1, (C) MGP, and (D) OPG. a, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg; b, P<0.05 vs VSMC with 3.3 mM P+1.4 mM Mg. Bars are the mean±SE of three experiments in quadruplicate.
Figure 3.
Magnesium inhibits β-catenin translocation into the nucleus in VSMC.
VSMC cultured with 3.3+1.4 mM Mg, and 3.3 mM P+1.4 mM Mg+10 µM 2-APB. Control group with 0.9 mM P+0.8 mM Mg was also included. Intracellular localization of β-catenin was visualized by immunofluorescence using confocal microscopy. (A) For each group, β-catenin (green immunofluorescence) is shown left; in the middle the same sample is counterstained with DAPI (blue) for nuclear stain; the merged image is shown right. Images represent four different experiments. (B) Quantification of nuclear β-catenin staining was performed by the Mander's coefficient (M2 plugin: DAPI vs. green). a, P<0.005 vs controls; b, P<0.005 vs VSMC with 3.3 mM P+0.8 mM Mg; and c, P<0.05 vs VSMC with 3.3 mM P+1.4 mM Mg. Bars are the mean±SE of four experiments.
Figure 4.
Magnesium inactivates Wnt/β-catenin signaling pathway in VSMC.
Relative mRNA expression on VSMC cultured with 3.3+1.4 mM Mg, and 3.3 mM P+1.4 mM Mg+10 µM 2-APB. Control group with 0.9 mM P+0.8 mM Mg was also included. (A) Frizzled 3, (B) DKK-1, (C) VCAN/versican, (D) cyclin D1, and (E) c-Myc. a, P<0.05 vs controls; b, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg; and c, P<0.05 vs VSMC with 3.3 mM P+1.4 mM Mg. Bars are the mean±SE of three experiments in triplicate.
Figure 5.
siRNA inhibition of TRPM7 activates Wnt/β-catenin signaling pathway in VSMC.
Relative mRNA expression of VSMC transfected with siRNA of TRPM7 24-type VSMC were included as controls. (A) TRPM7, (B) Frizzled 3, (C) VCAN/versican, (D) DKK-1. a, P<0.05 vs controls. Bars are the mean±SE of two experiments in triplicate.
Figure 6.
Magnesium halts VSMC calcification induced by high phosphate.
VSMC calcification was induced with 3.3+0.8 mM Mg for 5 days. At the 5th day, the medium was switched to 3.3 mM P+1.4 mM Mg, 3.3 mM P+1.4 mM Mg+10 µM 2-APB or 0.9 mM P+0.8 mM Mg and was maintained until day 9; a, P<0.01 vs 0 days; b, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg for 5 days; c, P<0.001 vs VSMC with 3.3 mM P+0.8 mM Mg for 9 days. Values are the mean±SE of four experiments in quadruplicate.
Figure 7.
Magnesium halts Cbfa-1 expression and up-regulates MGP and OPG when added to VSMC with already existing calcification.
VSMC calcification was induced with 3.3+0.8 mM Mg for 5 days. At the 5th day, the medium was switched to 3.3 mM P+1.4 mM Mg, 3.3 mM P+1.4 mM Mg+10 µM 2-APB or 0.9 mM P+0.8 mM Mg and was maintained until day 9. The graphics are the relative mRNA expression of (A) Cbfa-1, (B) osterix, (C) MGP, and (D) OPG. a, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg for 5 days 0 days; b, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg for 9 days; and c, P<0.01 vs VSMC with 3.3 mM P+1.4 mM Mg for 5 to 9 days. Values are the mean±SE of three experiments in triplicate.