Magnesium reduces calcification in bovine vascular smooth muscle cells in a dose-dependent manner

Abstract

Background: Vascular calcification (VC), mainly due to elevated phosphate levels, is one major problem in patients suffering from chronic kidney disease. In clinical studies, an inverse relationship between serum magnesium and VC has been reported. However, there is only few information about the influence of magnesium on calcification on a cellular level available. Therefore, we investigated the effect of magnesium on calcification induced by β-glycerophosphate (BGP) in bovine vascular smooth muscle cells (BVSMCs).

Methods: BVSMCs were incubated with calcification media for 14 days while simultaneously increasing the magnesium concentration. Calcium deposition, transdifferentiation of cells and apoptosis were measured applying quantification of calcium, von Kossa and Alizarin red staining, real-time reverse transcription-polymerase chain reaction and annexin V staining, respectively.

Results: Calcium deposition in the cells dramatically increased with addition of BGP and could be mostly prevented by co-incubation with magnesium. Higher magnesium levels led to inhibition of BGP-induced alkaline phosphatase activity as well as to a decreased expression of genes associated with the process of transdifferentiation of BVSMCs into osteoblast-like cells. Furthermore, estimated calcium entry into the cells decreased with increasing magnesium concentrations in the media. In addition, higher magnesium concentrations prevented cell damage (apoptosis) induced by BGP as well as progression of already established calcification.

Conclusions: Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification. Thus, magnesium is influencing molecular processes associated with VC and may have the potential to play a role for VC also in clinical situations.

Fig. 1.

Effect of magnesium on BGP-induced deposition of calcium in BVSMCs. BVSMCs were incubated for 7 days (A) and 21 days (B) under calcifying conditions (+10 mM BGP) either in the presence of physiological (1.1 mM = control) or elevated extracellular magnesium (2 and 3 mM). Magnesium was added from start (A and B) or at the time points indicated (C). Dashed lines in graph C indicate the presence of magnesium. Data are given as means and SD of at least six replicates (a = difference versus BGP: P < 0.05, b = difference versus same condition Day 14: P < 0.05). In parallel, von Kossa (D) Alizarin red (E) staining was performed with cells treated for 14 days under the same conditions as mentioned above.

Fig. 2.

Effect of magnesium on BGP-induced expression of proteins involved in calcification in BVSMCs. BVSMCs were incubated for 14 days under calcifying conditions either in the presence of physiological or elevated extracellular magnesium. ALP activity was quantified in cell lysates (C) and normalized to the amount of protein. For western blot analysis equal amounts of proteins were stained for CBFA1> and ß-actin (D). (A, B and C) data shown are means and SD of eight replicates (a = difference versus BGP: P < 0.05).

Fig. 3.

Influence of magnesium on the secretion of proteins involved in calcification. Protein concentrations of MGP and BMP2 were quantified in the supernatants of BVSMCs by ELISA (A and B). Cells were incubated for 14 days under calcifying conditions either in the presence of physiological or elevated extracellular magnesium concentrations. Data shown are means and SD of eight replicates (a = difference versus BGP: P < 0.05; b = difference versus control: P < 0.05).

Fig. 4.

Effect of calcification on the induction of apoptosis and on cell viability. To induce calcification, BVSMCs were incubated in the presence of 10 mM BGP for 14 days. The inhibitory effect of magnesium was assessed by raising the physiological magnesium concentration in the media from 1.1 mM (control) to 2 and 3 mM. The percentage of apoptotic cells (A) and cell viability (B) was quantified. Data shown are mean and SD of 4 (A) and 3 (B) experiments (a = difference versus BGP: P < 0.05).