Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder in which calcification of connective tissue leads to pathology in skin, eye and blood vessels. PXE is caused by mutations in ABCC6. High expression of this transporter in the basolateral hepatocyte membrane suggests that it secretes an as-yet elusive factor into the circulation which prevents ectopic calcification. Utilizing our Abcc6 (-/-) mouse model for PXE, we tested the hypothesis that this factor is vitamin K (precursor) (Borst et al. 2008, Cell Cycle). For 3 months, Abcc6 (-/-) and wild-type mice were put on diets containing either the minimum dose of vitamin K required for normal blood coagulation or a dose that was 100 times higher. Vitamin K was supplied as menaquinone-7 (MK-7). Ectopic calcification was monitored in vivo by monthly micro-CT scans of the snout, as the PXE mouse model develops a characteristic connective tissue mineralization at the base of the whiskers. In addition, calcification of kidney arteries was measured by histology. Results show that supplemental MK-7 had no effect on ectopic calcification in Abcc6 ( -/- ) mice. MK-7 supplementation increased vitamin K levels (in skin, heart and brain) in wild-type and in Abcc6 (-/-) mice. Vitamin K tissue levels did not depend on Abcc6 genotype. In conclusion, dietary MK-7 supplementation increased vitamin K tissue levels in the PXE mouse model but failed to counteract ectopic calcification. Hence, we obtained no support for the hypothesis that Abcc6 transports vitamin K and that PXE can be cured by increasing tissue levels of vitamin K.

Figures

Fig. 1

In vivo micro-CT imaging of calcification in the muzzle. X-ray image and cross sections of the 3D reconstructed micro-CT image of the rostral part of the head of a wt (left) and Abcc6−/− (right) mouse. Skull and teeth are clearly visible, both in wt and Abcc6−/− mouse. In addition, the Abcc6−/− mouse has shell-shaped calcified profiles (arrows) in the muzzles as illustrated by the cross-sections. Coloured lines indicate positions of the cross-sectional planes

Fig. 2

a Micro-CT scan of an Abcc6−/− mouse muzzle showing shell-shaped calcifications. b Paraffin section of the muzzle of an Abcc6−/− mouse (aged 9 months; 1×MK-7diet) stained with von Kossa localizes these calcifications (black) to the connective tissue capsule of the blood sinus of the whisker hair follicle. c Whisker follicle of a wt mouse (aged 8 months; 1×MK-7 diet) without calcification. Scale bar is 200 μm

Fig. 3

Cross sections from micro-CT scans of an Abcc6−/− mouse (on 1×MK-7 diet) showing the in vivo progression in calcification of whisker sinuses, from the start of the diet (0 months) until the end (3 months). Arrows point at whisker sinuses that are calcified after 3 months, whereas they were not calcified at the start of the diet

Fig. 4

Progression of calcification in the muzzles of Abcc6−/− mice from the start of the diet (time = 0) to the end, after 90 days. Coloured lines in the two graphs on the left depict the mineralization process in the individual mice. The graph on the right shows the averages per diet group (with missing values imputed). The 1×MK-7 diet group had, by chance, a slightly higher calcification at the start of the diet. Importantly, calcification progressed in all mice and no difference in the pace of calcification was noticed between the diets (for statistics, see Results)

Fig. 5

Effect of vitamin K supplementation on calcification in arteries in the kidney cortex. a–d Kidney sections of wt (a, c) and Abcc6−/− mice (b, d) on 1×MK-7 (a, b) or 100×MK-7 diet (c, d). Von Kossa staining shows calcification (black deposits) in walls of arteries in the cortex of Abcc6−/− mice on either diet. No calcifications were observed in wt mice. Cortex is on the right, medulla on the left. Bar is 200 μm. e Effect of diet on calcifications in arteries in the kidney cortex in Abcc6−/− mice, assessed by histology. Histograms represent mean number ± standard deviation (s.d.) of calcified profiles per sagittal kidney section. Diet did not cause a significant difference in calcification (p = 0.47, Student’s t test). f Effect of diet on calcifications in arteries in the kidney cortex in Abcc6−/− mice, assessed by micro-CT. Histograms represent average volume (±s.d.) of calcifications per kidney. Diet did not cause a significant difference in calcification (p = 0.31, Student’s t test)

Fig. 6

Vitamin K levels (±s.d.) in the liver as function of diet and Abcc6 genotype. Please note that some data points (mainly in the 1×Mk-7 diet group) were missing (as indicated by an absence of an error bar) presumably because the levels were below detection threshold

Fig. 7

MK-4 levels (±s.d.) in liver and extra-hepatic tissues as function of diet and genotype. The 100×MK-7 diet increased MK-4 tissue levels both in wt and Abcc6−/− mice (for statistics, see Table 1)

Fig. 8

MK-7 levels (±s.d.) in liver and in extra-hepatic tissues as function of diet and genotype. MK-7 levels were highly variable. No differences were observed between the genotypes. A statistically significant effect of diet was found for skin and liver (for statistics, see Table 1)