Elevated dietary magnesium prevents connective tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6(-/-))

Abstract

Pseudoxanthoma elasticum (PXE) is an autosomal recessive multisystem disorder characterized by ectopic connective tissue mineralization, with clinical manifestations primarily in the skin, eyes, and cardiovascular system. There is considerable, both intra- and interfamilial, variability in the spectrum of phenotypic presentation. Previous studies have suggested that mineral content of the diet may modify the severity of the clinical phenotype in PXE. In this study, we utilized a targeted mutant mouse (Abcc6(-/-)) as a model system for PXE. We examined the effects of changes in dietary phosphate and magnesium on the mineralization process using calcification of the connective tissue capsule surrounding the vibrissae as an early phenotypic biomarker. Mice placed on custom-designed diets either high or low in phosphate did not show changes in mineralization, which was similar to that noted in Abcc6(-/-) mice on control diet. However, mice placed on diet enriched in magnesium (fivefold) showed no evidence of connective tissue mineralization in this mouse model of PXE. The inhibitory capacity of magnesium was confirmed in a cell-based mineralization assay system in vitro. Collectively, our observations suggest that assessment of dietary magnesium in patients with PXE may be warranted.

Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1

Mineralization of the connective tissue capsule surrounding the bulb of vibrissae of Abcc6−/− mice fed either control diet or diet with mineral modifications. The mice were placed on special diets at 4 weeks of age, and the muzzle skin was biopsied and histopathologic sections were stained with Hematoxylin-Eosin at 12 weeks. For comparison, a wild-type (Abcc6+/+) mouse on control diet at 12 weeks of age is included (left panel). The Abcc6−/− mice on control diet showed distinct foci of mineralization at 12 weeks of age (arrows), and mice on high phosphate, low phosphate or low magnesium diet showed similar degree of mineralization. However, in mice fed high magnesium containing diet (Group C), no evidence of mineralization was noted, similar to the Abcc6+/+ control mice. Scale bar = 0.1 mm.

Figure 2

Quantitation of connective tissue mineralization in the vibrissae of the wild-type (WT) and knock-out (KO) mice fed control or experimental diet, as indicated in parenthesis, was accomplished either by computerized morphometric analysis (upper panel, a) or by chemical assay for calcium and phosphate (lower panel, b) in the muzzle skin biopsies. The bars represent mean ± S.E., n = 5 in each group. The statistical significance between different groups is indicated by an asterisk (p

Figure 3

Demonstration that magnesium in a dose dependent manner inhibits mineralization and calcium deposition in an in vitro culture system. Human aortic smooth muscle cells were cultured on tissue culture plastic flasks in a medium containing 10% FBS or serum from WT or KO mice (10%). After the cultures reached ~80% of confluence, 2 mM inorganic phosphate (+Pi) was added and the degree of mineralization was assessed either by phase contrast light microscopy (panel a) or by chemical assay of calcium deposition in the cell layer (panel b). A dramatic, about 60-fold increase in the degree of mineralization was noted in the presence of FBS and WT serum, and even higher, ~90-fold increase, was noted in the presence of KO mouse serum upon addition of Pi (b, left panel). Addition of magnesium in concentrations indicated either into WT or KO mouse serum-containing culture media just prior to mineralization prevented the calcium deposition in a dose-dependent manner (b, right panel). Mean ± S.E.; n = 3. Scale bar = 0.1 mm.

Figure 4

Cell-based mineralization assay, similar to that shown in Fig. 3, demonstrates that addition of magnesium (0.1–1.0 mg/ml) into the culture medium 14 days after the mineralization had taken place does not reverse the process. (a) Phase contrast light microscopy; (b) Calcium deposition determined by a chemical assay. Mean ± S.E.; n = 3. Scale bar = 0.1 mm.