Warfarin accelerates ectopic mineralization in Abcc6(-/-) mice: clinical relevance to pseudoxanthoma elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by mutations in the ABCC6 gene. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis, we developed a model in which Abcc6(-/-) mice, which recapitulate features of PXE, were fed a diet supplemented with warfarin and vitamin K1. Warfarin action was confirmed by significantly increased serum levels of oxidized vitamin K. For mice placed on a warfarin-containing diet, quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits in a number of tissues. Mice fed a warfarin-containing diet were also shown to have abundant uncarboxylated form of matrix Gla protein, which allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, 1747 patients with PXE from the approximately 4000 patients in the PXE International database were surveyed about the use of warfarin. Of the 539 respondents, 2.6% reported past or present use of warfarin. Based on the prevalence of PXE (approximately 1:50,000), thousands of patients with PXE worldwide may be at risk for worsening of PXE as a result of warfarin therapy.

Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Enhanced mineralization in the dermal sheath of vibrissae and in the aorta, eyes, heart, and kidneys of Abcc6−/− mice fed the experimental W/K1 diet (group D), compared with Abcc6−/− mice kept on the control diet (group C). Mineral deposits (arrows) were visualized by H&E, Alizarin Red (AR), and von Kossa (VK) stains. For vibrissae and aorta, images in the right column correspond to the boxed regions in the center column, at higher magnification (×300). Original magnification: ×100 (vibrissae, aorta, heart); ×150 (eye, kidney).

Figure 2

Quantitation of the mineral deposits in the muzzle skin containing the dermal sheath of vibrissae by direct chemical assay of calcium and phosphorus (A) and by computerized morphometric analysis of histopathological sections (B) in groups A to D (both genotypes, control and W/K1 diets). Data are expressed as means ± SEM. n = 8 (control) or 9 (W/K1). ∗∗∗P < 0.001.

Figure 3

Noninvasive evaluation of the mineralization process in dermal sheath of vibrissae by micro-CT scan. The presence of mineral deposits (arrows) is revealed in single slices (bottom row). Three-dimensional reconstruction (top row) demonstrates the presence of mineral deposits in the vibrissae (arrowheads), particularly in Abcc6−/− mice fed the acceleration diet (group G) or the acceleration diet + W/K1 (group H). No mineralization of dermal sheath of vibrissae was noted in either wild-type or knockout mice kept on the control diet (groups A and E, respectively). The Abcc6−/− mice on the W/K1 diet (group F) showed extensive mineralization.

Figure 4

Quantitation of mineralization in dermal sheath of vibrissae of Abcc6−/− mice by direct chemical assay of calcium and phosphorous (A) and by computerized morphometric analysis of histopathological sections (B) in groups E to H. Data are expressed as means ± SEM. n = 5 (W/K1), 7 (acceleration), 8 (control), or 9 (acceleration + W/K1). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5

Analysis of the mineral content in deposits in dermal sheath of vibrissae of Abcc6−/− mice on the control, acceleration, or acceleration + W/K1 diet (groups E, G, and H, respectively). Mineral deposits identified in H&E-stained sections (arrows, top row) were subjected to EDAX analysis. The elemental spectrum (row 2) revealed the presence of calcium and phosphorus as the principal ions in approximately a 2.0:1 ratio in all samples. Topographic (RADAR) mapping identified calcium and phosphorous (rows 3 and 4) in a colocalization distribution, as shown by overlay maps (bottom row). Scale bar = 200 μm. Original magnification (H&E stain), ×100.

Figure 6

Immunological demonstration of osteopontin (top row) and α-fetuin (middle row) associated with mineral deposits (arrows) in the dermal sheath of vibrissae and aorta in Abcc6−/− mice fed the control diet (group C) or the W/K1 diet (group D). Mineral deposits (arrows, bottom row) were visualized by H&E. Original magnification, ×100.

Figure 7

Analysis of cMGP and ucMGP in aorta of Abcc6−/− mice kept on the control diet (group C) or the W/K1 diet (group D). Note mineralization in von Kossa (VK)–stained sections (arrows). There was a greater abundance of ucMGP (arrow) than cMGP in group D. Original magnification, ×300.