Calciprotein particle inhibition explains magnesium-mediated protection against vascular calcification

Abstract

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.

Keywords: calcification propensity; calciprotein particle; chronic kidney disease; magnesium; vascular calcification.

FIGURE 1

Mg²⁺ prevents Pi-induced calcification in hVSMCs. (A) Alizarin red staining was negative in Mg²⁺-supplemented Pi cultures, indicating the absence of calcification after 2, 4 and 8 days. (B) Ca²⁺ deposition on VSMCs was increased in Pi-treated cells compared with controls, but was absent after 2.0 mmol/L Mg²⁺ supplementation. (C) Pi-induced calcification decreased ALP and non-significantly decreased calcification inhibitor MGP and VSMC contractile genes SM22α and CNN1 after 6 days (black bars) and 8 days (white bars) of incubation, which was prevented by Mg²⁺. (D) Protein expression of OPN, SM22α and MGP was measured and quantified. Data are shown as fold changes compared with the day-matched controls and presented as the mean of three separate experiments, consisting of three replicate cultures ± SD. *P < 0.05 versus day-matched control.

FIGURE 2

Mg²⁺ does not prevent CPP2-induced calcification in hVSMCs. (A) Alizarin red staining and (B) Ca²⁺ deposition quantification show that CPP2-induced calcification is not prevented by 2.0 mmol/L Mg²⁺ after 24 h and 4 days. (C) Gene expression related to calcification after CPP2 treatment was measured after 2 days (black bars) and 4 days (white bars) and remained unchanged by Mg²⁺. (D) Protein expression of OPN, SM22α and MGP was measured and quantified. Data are shown as fold changes compared with the day-matched controls and presented as the mean of three separate experiments, consisting of three replicate cultures ± SD. *P < 0.05.

FIGURE 3

High Mg²⁺ concentration stimulates CPP2-induced calcification. Ca²⁺ deposition was measured after incubation of VSMCs for 24 h with varying CPP2 concentrations in the presence of 0.8 (control), 2 and 5 mmol/L Mg²⁺. Mg²⁺ did not prevent CPP2-induced calcification at any CPP concentration. At 5 mmol/L, Mg²⁺ increased cellular Ca²⁺ deposition at CPP2 concentrations of 25, 50 and 75 μg/mL Ca²⁺ versus CPP2 treatment without additional Mg²⁺. Data are shown as fold changes compared with 100 CPP2 (set at 100%). Presented data are the mean of three separate experiments consisting of three replicate cultures ± SD. P < 0.05 for CPP2 incubated with *0.8, ^#2 and ^$5 mmol/L Mg²⁺ versus CPP2 concentration-matched formalin-fixed cultures.

FIGURE 4

Mg²⁺ dose-dependently inhibits the transition from CPP1 to CPP2 and does not affect CPP2 morphology after nucleation. (A) Absorbance of the CPP mixture containing different Mg²⁺ concentrations was measured at 570 nm as a readout for CPP1 (∼0.05 AU) to CPP2 (∼0.15 AU) transition. This graph is representative of five independently executed experiments each consisting of three replicates. Data are expressed as mean ± SD. A final concentration of 2.0 mmol/L Mg²⁺ after CPP2 (E–G) formation did not alter morphology (SEM) or composition (EDX) compared with control CPP2 (B–D). A TEM picture was inserted in B to confirm CPP2. Scale bars correspond to 300 nm (white) and 500 nm (black).

FIGURE 5

Mg²⁺ dose-dependently increases T₅₀ in CKD patient serum. Serum T₅₀ is increased upon Mg²⁺ supplementation of serum from both healthy controls (solid line) and CKD patients (dotted line, A). (B) Change in T₅₀ similar in CKD patients after each addition of Mg²⁺ compared with healthy controls. Black and white bars represent healthy controls and CKD patients, respectively. Data are shown as the mean of 10 patients in each group and are expressed as mean ± SD. P < 0.05 *for healthy controls versus baseline; ^$for CKD patients versus baseline and ^#for CKD patients versus healthy controls at specific Mg²⁺ concentration.