Magnesium inactivates Wnt/β-catenin signaling pathway in VSMC.

<p>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. <b>(A)</b> Frizzled 3, <b>(B)</b> DKK-1, <b>(C)</b> VCAN/versican, <b>(D)</b> cyclin D1, and <b>(E)</b> c-Myc. <b>a</b>, P<0.05 vs controls; <b>b</b>, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg; and <b>c</b>, P<0.05 vs VSMC with 3.3 mM P+1.4 mM Mg. Bars are the mean±SE of three experiments in triplicate.</p

siRNA inhibition of TRPM7 activates Wnt/β-catenin signaling pathway in VSMC.

<p>Relative mRNA expression of VSMC transfected with siRNA of TRPM7 24-type VSMC were included as controls. <b>(A)</b> TRPM7, <b>(B)</b> Frizzled 3, <b>(C)</b> VCAN/versican, <b>(D)</b> DKK-1. <b>a</b>, P<0.05 vs controls. Bars are the mean±SE of two experiments in triplicate.</p

Magnesium inhibits β-catenin translocation into the nucleus in VSMC.

<p>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. <b>(A)</b> 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>(B)</b> Quantification of nuclear β-catenin staining was performed by the Mander's coefficient (M2 plugin: DAPI vs. green). <b>a</b>, P<0.005 vs controls; <b>b</b>, P<0.005 vs VSMC with 3.3 mM P+0.8 mM Mg; and <b>c</b>, P<0.05 vs VSMC with 3.3 mM P+1.4 mM Mg. Bars are the mean±SE of four experiments.</p

Inhibition of TRPM7 abrogates protective effect of magnesium on calcification and gene expression on VSMC.

<p>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 <b>(A)</b> calcium content, and the relative mRNA expression of <b>(B)</b> Cbfa-1, <b>(C)</b> MGP, and <b>(D)</b> OPG. <b>a</b>, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg; <b>b</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.</p

Magnesium halts Cbfa-1 expression and up-regulates MGP and OPG when added to VSMC with already existing calcification.

<p>VSMC calcification was induced with 3.3+0.8 mM Mg for 5 days. At the 5^th 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 <b>(A)</b> Cbfa-1, <b>(B)</b> osterix, <b>(C)</b> MGP, and <b>(D)</b> OPG. <b>a</b>, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg for 5 days 0 days; <b>b</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.</p

Primers used for quantitative RT-PCR analyses.

<p>Primers used for quantitative RT-PCR analyses.</p

The effect of magnesium on calcification and osteoblast-like transformation in VSMC <i>in vitro</i>.

<p>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. (<b>A)</b>, calcium content of VSMC cultured for 9 days. Relative mRNA expression of Cbfa-1 <b>(B)</b> and osterix <b>(C)</b>; <b>a</b>, P<0.001 vs Control; <b>b</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.</p

Magnesium halts VSMC calcification induced by high phosphate.

<p>VSMC calcification was induced with 3.3+0.8 mM Mg for 5 days. At the 5^th 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; <b>a</b>, P<0.01 vs 0 days; <b>b</b>, P<0.05 vs VSMC with 3.3 mM P+0.8 mM Mg for 5 days; <b>c</b>, 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.</p