Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum

Abstract

Arterial calcification is a common feature of pseudoxanthoma elasticum (PXE), a disease characterized by ABCC6 mutations, inducing a deficiency in pyrophosphate, a key inhibitor of calcium phosphate crystallization in arteries.

Methods: we analyzed whether long-term exposure of Abcc6^-/- mice (a murine model of PXE) to a mild vitamin D supplementation, with or without calcium, would impact the development of vascular calcification. Eight groups of mice (including Abcc6^-/- and wild-type) received vitamin D supplementation every 2 weeks, a calcium-enriched diet alone (calcium in drinking water), both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Aorta and kidney artery calcification was assessed by 3D-micro-computed tomography, Optical PhotoThermal IR (OPTIR) spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and Yasue staining.

Results: at 6 months, although vitamin D and/or calcium did not significantly increase serum calcium levels, vitamin D and calcium supplementation significantly worsened aorta and renal artery calcification in Abcc6^-/- mice.

Conclusions: vitamin D and/or calcium supplementation accelerate vascular calcification in a murine model of PXE. These results sound a warning regarding the use of these supplementations in PXE patients and, to a larger extent, patients with low systemic pyrophosphate levels.

Keywords: ABCC6; calcification; calcium; pseudoxanthoma elasticum; pyrophosphate; vascular; vitamin D.

Figure 1

(A). Representative 3D reconstructions of micro-CT analyses in aorta of two Abcc6^−/− mice exposed to, respectively, high-calcium diet and high-calcium + vitamin D for 6 months. Calcifications (high-density areas) appear in red. (B). Volume of aorta calcification quantified by CT-scan in WT and Abcc6^−/− mice from all groups. * p < 0.05 vs. all other groups, Mann-Whitney test.

Figure 2

(A). Yasue staining of an aorta (Abcc6^−/− mouse exposed to high-calcium + vitamin D) showing that incipient calcification, stai ned in black, seem localized in elastic fibers. (B,C). Scanning electron microscopy coupled with EDS evidencing the presence of high amounts of calcium and phosphate in specific areas, at the surface of elastic fibers, corresponding to arrows (B) and blue and green peaks (C). EDX spectra corresponding to area 16,17,18, and 19 including the different contributions coming from the support and the sample namely O (K_α = 0.524 KeV), Na (K_α = 1.041 KeV), Si (K_α = 1.740 KeV), P (K_α = 2.013 KeV), S (K_α = 2.307 KeV), Ag (L_α=2.985 KeV, L_β = 3.150 KeV), K (K_α=3.310 KeV) and Ca (K_α=3.690 KeV, K_β = 4.010 KeV). Spectra collected for the support show that Ca and P belong to the calcifications while other elements belong to the support (D,E). mIRage technique characterizing incipient calcifications localized in the close vicinity of elastic fibers. The spectra reveal the presence of different absorption bands of the apatite including the ν₃ P-O stretching vibration mode measured at 1035–1045 cm⁻¹. Carbonate ions are detected together with apatite by their ν₃ C-O stretching vibration mode around 1420 cm⁻¹. Each dot in the upper panel corresponds to one spectrum.

Figure 3

Representative 3D reconstruction (micro-CT) of one kidney from an Abcc6^−/− mouse exposed to high-calcium + vitamin D for 6 months. Calcifications (high-density areas) were stained in blue in the papilla (Randall’s plaque) and in red in the cortical and medullary areas.

Figure 4

Representative kidney calcifications stained with Yasue method, evidencing arterial calcifications (in the vascular wall).

Figure 5

Volume of kidney vascular calcification quantified by CT-scan in WT and Abcc6^−/− mice from all groups. * p < 0.05 vs. Abcc6^−/− control (untreated) mice. # p < 0.05 vs. WT control or WT Ca2⁺⁺ vitamin D mice.