Impact of calcium on ATP hydrolysis.

<p>Rat aortic rings were incubated <i>ex vivo</i> in sHBSS (supplemented with 1 mmol/L magnesium) and with 1 mmol/L calcium (<b>A</b>) or without calcium (<b>B</b>). Hydrolysis of 1 μmol/L ATP (and 10 μCi/mL [α³²P]ATP as radiotracer) showed ADP, AMP and α-Pi production in the indicated times, following separation by thin layer chromatography as described in the methods section. Results are means ± SEM of two independent experiments, with a total of 6 rings per condition (6 different rats). The same ring was used to analyze the hydrolysis of ATP in the absence or presence of calcium.</p

Impact of magnesium:calcium ratio on calcification of the aortic wall

<div><p>Objective</p><p>An inverse relationship between serum magnesium concentration and vascular calcification has been reported following observational clinical studies. Moreover, several studies have been suggesting a protective effect of magnesium on the vascular calcification. However, the exact mechanism remains elusive, and investigators have speculated among a myriad of potential actions. The effect of magnesium on calcification of the aortic wall is yet to be investigated. In the present study, the effects of magnesium and calcium on the metabolism of extracellular PPi, the main endogenous inhibitor of vascular calcification, were investigated in the rat aorta.</p><p>Approach and results</p><p>Calcium and magnesium have antagonist effects on PPi hydrolysis in the aortic wall. <i>K</i>_<i>m</i> and <i>K</i>_<i>i</i> values for PPi hydrolysis in rat aortic rings were 1.1 mmol/L magnesium and 32 μmol/L calcium, respectively, but ATP hydrolysis was not affected with calcium. Calcium deposition in the rat aortic wall dramatically increased when the magnesium concentration was increased (ratio of Mg:Ca = 1:1; 1.5 mmol/L calcium and 1.5 mmol/L magnesium) respect to low magnesium concentration (ratio Mg:Ca = 1:3, 1.5 mmol/L calcium and 0.75 mmol/L magnesium).</p><p>Conclusion</p><p>Data from observational clinical studies showing that the serum magnesium concentration is inversely correlated with vascular calcification could be reinterpreted as a compensatory regulatory mechanism that reduces both PPi hydrolysis and vascular calcification. The impact of magnesium in vascular calcification in humans could be studied in association with calcium levels, for example, as the magnesium:calcium ratio.</p></div

Kinetic characterisation of calcium and magnesium on pyrophosphate hydrolysis.

<p>Rat aortic rings were incubated <i>ex vivo</i> in HBSS containing the indicated amount of magnesium and calcium. (<b>A</b>) Michaelian saturation curves were plotted to determinate the <i>K</i>_<i>m</i> value of magnesium for PPi hydrolysis. HBSS contained 5 μmol/L PPi and 10 μCi/mL ³²PPi as radiotracer. (<b>B</b>) Competitive inhibition of PPi hydrolysis by calcium; inset, logarithmic transformation of calcium concentration. HBSS contained 1.1 mmol/L magnesium, 5 μmol/L PPi and 10 μCi/mL ³²PPi as radiotracer. PPi hydrolysis was measured by Pi release. Pi was separated from PPi using the molybdate method as explained in the methods section. Results are represented as means ± SEM of three independent experiments with a total of 6, 7 or 13 rings per point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178872#pone.0178872.s002" target="_blank">S2 Table</a>) and 26 rats in total (A) and two independent experiments with a total of 6 rings per point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178872#pone.0178872.s003" target="_blank">S3 Table</a>) and 12 rats in total (B).</p

Impact of calcium and magnesium on aortic wall calcification.

<p>Rat aortic rings were incubated <i>ex vivo</i> in DMEM supplemented with the indicated concentration of magnesium and calcium. The medium was replaced every day and contained 45-calcium as a radiotracer. After 7 days of incubation, aortic rings were dried and radioactivity was measured by liquid scintillation counting. Results are represented as means ± SEM of two independent experiments, with a total of 14 rings per condition and 11 rats in total (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178872#pone.0178872.s005" target="_blank">S5 Table</a>). One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis. <b><i>*p</i> <0.05</b>; <b><i>***p</i> <0.001</b>.</p

MPiφs show elevated antioxidant synthesis.

<p>(<b>A</b>) RNAseq data summarizing fold mRNA downregulation or upregulation in MPiφs of enzymes involved in glutathione metabolism. (<b>B</b>) Total antioxidant capacity. (<b>C</b>) GSH/GSSG ratio. (<b>D</b>) Total GSH. Statistical significance was determined Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

MPiφs degrade arginine via Arg1.

<p>(<b>A</b>) RNAseq data summarizing fold mRNA upregulation in MPiφs of collagen types and enzymes involved in the urea cycle. (<b>B</b>) <i>Arg1</i> and <i>iNOS</i> mRNA levels. (<b>C</b>) Arginase activity. Statistical significance was determined by Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. ***, <i>P</i><0.001.</p

Influence of plasma p<i>H</i> on alkaline phosphatase activity.

<p>(<b>A</b>) A p<i>H</i>-dependent curve using ten pools comprising four different post-hemodialysis plasma samples per pool at the p<i>H</i> indicated. Friedman’s one-way ANOVA test and Dunn’s post-test were used for statistical analysis. (<b>B</b>) Box and whiskers graph demonstrating pre- and post-hemodialysis plasma p<i>H</i> values (preHD and postHD, respectively; n = 10). (<b>C</b>) ALP activity in post-hemodialysis plasmas was quantified at the p<i>H</i> indicated. (<b>B, C</b>) Plasma samples were collected from all patients (n = 45), and results were analyzed using the Wilcoxon matched pairs test. (<b>D</b>) The scattergraph demonstrates a correlation between Δp<i>H</i> (the difference in post- and pre-hemodialysis p<i>H</i>) and Δ Relative ALP (the difference in post- and pre-hemodialysis relative ALP levels). <b>* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001</b>.</p

Plasma phosphate levels inhibit ALP activity.

<p>(<b>A</b>) PreHD (left) and postHD (right) levels of urea and phosphate in plasma. Results are represented as mean ± SEM (n = 45). Wilcoxon’s matched pairs test was used for statistical analysis. (<b>B</b>) ALP activities of post-hemodialysis samples (with the exception of the controls, which were pre-hemodialysis samples) without or with potential inhibitors: (<b>1</b>) 120 mg/mL (20 mmol/L) urea; (<b>2</b>) no addition; (<b>3</b>) 4.5 mg/dL (1.5 mmol/L) inorganic phosphate; (<b>4</b>) 9 mg/dL inorganic phosphate; (<b>5</b>) 20 mmol/L EDTA; and (<b>6</b>) 100 μmol/L levamisole. Results are represented as mean ± SEM (n = 45). For statistical analysis, Friedman’s one-way ANOVA test and Dunn’s post-test were used. (<b>C</b>) The scattergraph demonstrates a correlation between ΔPi (the difference in post- and pre-hemodialysis phosphate levels) and ΔALP (the difference in post- and pre-hemodialysis ALP levels). PreHD, pre-hemodialysis; PostHD, post-hemodialysis. <b>* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001</b>.</p

MPiφs prevent calcium-phosphate deposition by increasing extracellular ATP and PPi.

<p>Fixed VSMCs were incubated in pro-calcifying medium over 5 days with 0.4 μm polycarbonate transwells containing unpolarized M0φsor Pi-activated MPiφs. (<b>A, B</b>) Extracellular ATP and PPi released by the indicated macrophage types. (<b>C</b>) PPi/ATP ratio. Statistical significance was determined by unpaired Student’s <i>t</i>-test. Results are presented as mean ± SE of 3 independent experiments with 3 mice per experiment. (<b>D</b>) Calcium deposition on fixed VSMCs in absence (Control) or presence of M0φs or MPiφs. Experiments were performed in the presence or absence of exogenous alkaline phosphatase (-AP and +AP). Statistical significance was determined by one-way ANOVA analysis of variance followed by Tukey’s multicomparison test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

PPi synthesis remains unaltered post-hemodialysis.

<p>Thin layer chromatography. (<b>A</b>) Plasma hydrolysis of 1 μmol/L ATP showed PPi and Pi production at the indicated times. (<b>B</b>) PPi quantification at the indicated conditions and sample types following 1 hr of incubation with 1 μmol/L ATP and [γ³²P]ATP as a radiotracer. Pre-hemodialysis plasmas without levamisole were used as controls. Results are represented as mean ± SEM for all plasma samples (n = 45). There was a significant difference in post-hemodialysis (postHD) without interaction (two-way ANOVA; <i>P</i> = 0.017). The Bonferroni post-test was used for statistical analysis. PreHD, pre-hemodialysis. PPi, pyrophosphate; Pi, phosphate. <b>CPM</b>: counts per minute. <b>* <i>P</i> < 0.05</b>.</p