Magnesium has been shown to effectively prevent vascular calcification associated with chronic kidney disease. Magnesium has been hypothesized to prevent the upregulation of osteoblastic genes that potentially drives calcification. However, extracellular effects of magnesium on hydroxyapatite formation are largely neglected. This study investigated the effects of magnesium on intracellular changes associated with transdifferentiation and extracellular crystal formation. Bovine vascular smooth muscle cells were calcified using β-glycerophosphate. Transcriptional analysis, alkaline phosphatase activity and detection of apoptosis were used to identify transdifferentiation. Using X-ray diffraction and energy dispersive spectroscopy extracellular crystal composition was investigated. Magnesium prevented calcification in vascular smooth muscle cells. β-glycerophosphate increased expression of osteopontin but no other genes related to calcification. Alkaline phosphatase activity was stable and apoptosis was only detected after calcification independent of magnesium. Blocking of the magnesium channel TRPM7 using 2-APB did not abrogate the protective effects of magnesium. Magnesium prevented the formation of hydroxyapatite, which formed extensively during β-glycerophosphate treatment. Magnesium reduced calcium and phosphate fractions of 68% and 41% extracellular crystals, respectively, without affecting the fraction of magnesium. This study demonstrates that magnesium inhibits hydroxyapatite formation in the extracellular space, thereby preventing calcification of vascular smooth muscle cells.

Background Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation. Methods Vascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls. Results Mg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P < 0.05) and CKD patients by 44 ± 13 min (P < 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups. Conclusions Our results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification.

Over the last decade, an increasing number of studies report a close relationship between serum magnesium concentration and cardiovascular disease risk in the general population. In end-stage renal disease, an association was found between serum magnesium and survival. Hypomagnesemia was identified as a strong predictor for cardiovascular disease in these patients. A substantial body of in vitro and in vivo studies has identified a protective role for magnesium in vascular calcification. However, the precise mechanisms and its contribution to cardiovascular protection remain unclear. There are currently 2 leading hypotheses: first, magnesium may bind phosphate and delay calcium phosphate crystal growth in the circulation, thereby passively interfering with calcium phosphate deposition in the vessel wall. Second, magnesium may regulate vascular smooth muscle cell transdifferentiation toward an osteogenic phenotype by active cellular modulation of factors associated with calcification. Here, the data supporting these major hypotheses are reviewed. The literature supports both a passive inorganic phosphate-buffering role reducing hydroxyapatite formation and an active cell-mediated role, directly targeting vascular smooth muscle transdifferentiation. However, current evidence relies on basic experimental designs that are often insufficient to delineate the underlying mechanisms. The field requires more advanced experimental design, including determination of intracellular magnesium concentrations and the identification of the molecular players that regulate magnesium concentrations in vascular smooth muscle cells.

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Vascular calcification is a prognostic marker for cardiovascular mortality in chronic kidney disease (CKD) patients. In these patients, magnesium balance is disturbed, mainly due to limited ultrafiltration of this mineral, changes in dietary intake and the use of diuretics. Observational studies in dialysis patients report that a higher blood magnesium concentration is associated with reduced risk to develop vascular calcification. Magnesium prevents osteogenic vascular smooth muscle cell transdifferentiation in in vitro and in vivo models. In addition, recent studies show that magnesium prevents calciprotein particle maturation, which may be the mechanism underlying the anti-calcification properties of magnesium. Magnesium is an essential protective factor in the calcification milieu, which helps to restore the mineral-buffering system that is overwhelmed by phosphate in CKD patients. The recognition that magnesium is a modifier of calciprotein particle maturation and mineralization of the extracellular matrix renders it a promising novel clinical tool to treat vascular calcification in CKD. Consequently, the optimal serum magnesium concentration for patients with CKD may be higher than in the general population.