Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6 (-/-) mice. Abcc6 (-/-) mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.

Figures

Figure 1

Total calcium content in the muzzle skin of aging WT and Abcc6-/- mice. Calcium deposition in the Abcc6-/- mice was quantified by a colorimetric assay described in the Material and Methods section. Calcification initially increases with age with the significant increases occurring after 3 months of age. The largest increase in mineralization occurs between 3 weeks and 6 months. At around 12 months of age there is a gentle plateau in the rate of mineralization with no statistical increase measured throughout the rest of the ∼28–30 months life span of the mice.

Figure 2

Calcification in the whole muzzle skin of mice fed diets enriched with 5 or 100 mg/kg of vitamin K1. No significant difference in calcium deposition was observed between the Abcc6-/- mice on normal or vitamin K1 enriched diets, with all Abcc6-/- mice having significantly more mineralization than WT controls. Mouse diets are described as ‘pre-onset’ of phenotype consist of mice fed on the diet from ∼day 21 for 16 weeks. ‘Post-onset’ corresponds to mice fed from ∼3 months of age (already possessing the calcification phenotype) for 12 weeks. In utero mouse samples were taken from mice which, when in utero, had mothers fed on the relevant diet and continued with the diet, through breast feeding and weaning up until 5 months of age.

Figure 3

Muzzle skin calcification of mice fed diets enriched with 5 or 100 mg/kg of vitamin MK4. No significant difference in calcium deposition was observed between the Abcc6-/- mice on normal or vitamin MK4 enriched diets with all Abcc6-/- mice having significantly more mineralization than WT controls. Mouse diets are described as ‘pre-onset’ of phenotype consist of mice fed on the diet from ∼day 21 for 16 weeks. ‘Post-onset’ corresponds to mice fed from ∼3 months of age (already possessing the calcification phenotype) for 12 weeks. In utero mouse samples were taken from mice which, when in utero, had mothers fed on the relevant diet and continued with the diet, through breast feeding and weaning up until 5 months of age.

Figure 4

Histological evaluation of calcification in the vibrissae of WT and Abcc6-/- mice. Connective tissue calcium deposition was observed with Alizarin S Red staining in mice fed with either normal diet, or diet enriched in vitamin K1 or vitamin MK4 at 5 or 100 mg/kg. Sections of the muzzle skin were taken from each mouse from all mouse diet treatment groups. Mouse diets are described as ‘pre-onset’ of phenotype consist of mice fed with the diet from ∼day 21 for 16 weeks. ‘Post-onset’ corresponds to mice fed from ∼3 months of age (already possessing the calcification phenotype) for 12 weeks. In utero mouse samples were taken from mice which, when in utero, had mothers fed with the relevant diet and continued with the diet, through breast feeding and weaning up until 5 months of age. Size of hair follicle and depth of sections cut into the tissue were used as a guide to match relevant WT and Abcc6-/- sections. We observed no difference in placement or extent of calcium deposition. The same amount of staining occurred in the capsule surrounding the vibrissae in the whiskers in the Abcc6-/- mice fed control or either of the two vitamin K enriched diets.

Figure 5

HPLC-MS analysis of serum levels of vitamin K1 from mice fed diets enriched with 5 or 100 mg/kg of vitamin K1. We measured a general, non-significant increase in circulating vitamin K1 levels in the serum of both WT and Abcc6-/- mice fed on a diet enriched with 5 mg/kg of vitamin K1. When fed with a diet enriched with 100 mg/kg vitamin K1, WT mice had significantly more circulating vitamin K1 than the Abcc6-/- mice, with no significant increase in circulating vitamin K1 measured in Abcc6-/- mice fed with this diet.

Figure 6

HPLC-MS analysis of serum levels of vitamin MK4 from mice fed diets enriched with 5 or 100 mg/kg of vitamin K1. When fed with a normal diet the serum taken from Abcc6-/- mice had significantly less circulating MK4 than WT controls. We measured a general, non-significant increase in circulating vitamin MK4 levels in the serum of WT mice fed on a diet enriched with 100 mg/kg of vitamin K1. WT mice fed with 100 mg/kg of vitamin K1 from pre-onset had significantly more circulating MK4 than Abcc6-/- mice. There was no increase in circulating MK4 levels in the serum of Abcc6-/- mice fed on any of the vitamin K1 diets.

Figure 7

HPLC-MS analysis of serum levels of vitamin MK4 from mice fed diets enriched with 5 or 100 mg/kg of vitamin MK4. There was a significant increase in the levels of circulating MK4 in WT mice fed with a diet enriched with either a 5 mg/kg or 100 mg/mg of MK4. Additionally, we measured significant increases in circulating MK4 in Abcc6-/- mice fed with 100 mg/kg of MK4 in both pre- and post-onset groups. There was no significant increase in serum levels of MK4 in Abcc6-/- mice fed with the 5 mg/kg MK4 diet in either the pre- or post onset groups or in either of the in utero groups.

Figure 8

Immunofluorescent detection of MGP in the vibrissae's capsule of mice fed diets enriched with 100 mg/kg of vitamin K1 and MK4. Frozen serial sections from the muzzle tissue were used for both immunofluorescent and Alizarin S Red staining. We observed large positive staining of both ucMGP and tMGP (green) in the calcified regions of the vibrissae's capsule of Abcc6-/- mice fed with normal or vitamin K-enriched diets (Arrows), suggesting that the carboxylation status of MGP in these tissues is unaffected by increased dietary vitamin K1 or MK4. The counterstaining on immunofluorescent images was obtained with propidium iodide. The scale bar represents 200 µm.