The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the <i>ABCC6</i> gene

<p>Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the <i>ABCC6</i> gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by <i>ABCC6</i> mutations by feeding <i>Abcc6^−/−</i> mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the <i>ABCC6</i> gene, when combined with careful monitoring of efficacy and potential side-effects.</p

Analysis of <i>ABCC6</i> mRNA levels in the liver.

<p>(A) Relative mRNA levels in the mice with the A allele (KK/HlJ, DBA/2J, C3H/HeJ and 129S1/SvImJ), in comparison to the mouse with the G allele, C57BL/6J. Note that the mRNA level is essentially undetectable in <i>Abcc6^−/−</i> mice; (n = 4–5; *p<0.05 in comparison to other groups; Student’s t-test). (B) Separation of the G and A alleles by fine resolution gel electrophoresis, represented by 144 and 139 bands, respectively.</p

Western blot analysis of ABCC6 protein isolated from the liver of mice.

<p>(A) The blot was stained with a mouse anti-ABCC6 antibody S20 (upper panel). The same blot was rehybridized with an antibody for sodium potassium ATPase as a control of loading of plasma membrane proteins (lower panel). (B) The relative concentration of the protein, as determined from the Western blots in A by scanning densitometry, normalizing the ABCC6 protein levels by those with sodium potassium ATPase (n = 4–5; p<0.05 in comparison to other groups; Student’s t-test). (C) Lack of conservation of the arginine (R) in position p.621 of ABCC6 in different vertebrates.</p

Immunofluorescence staining of Abcc6 in the liver of mice.

<p>Note positive staining associated with the plasma membranes in C57BL/6J mice (arrowheads) and complete absence of the staining in <i>Abcc6^−/−</i> mice. A low, yet detectable level of immunofluorescence was noted in the four other strains which harbor the A allele of the <i>Abcc6</i> gene.</p

Vibrissae dermal sheath mineralization in different mouse strains examined in this study.

<p>The table shows the frequency and severity of vibrissae dermal sheath mineralization in different strains: −, not present; +, mild; ++++, severe. Mice were placed on either normal diet or acceleration diet at 4 weeks of age and tissues were collected for histopathology. The frequency values represent the percent of mice affected by vibrissae dermal sheath mineralization, as examined by Hematoxylin-Eosin stain on one section.</p

Mineralization of vibrissae dermal sheath in mice placed on experimental “acceleration diet” at the age of 4 weeks and sacrificed at 5 months of age.

<p>Note the absence of mineralization in the C57BL/6J mice and extensive mineralization in <i>Abcc6^−/−</i> mice when examined by hematoxylin-eosin (HE) and by Alizarin red (AR) stains. Note ectopic mineralization in the four mouse strains with the A allele of the <i>Abcc6</i> gene (arrows).</p

Non-invasive demonstration of ectopic mineralization in <i>asj-2J</i> mice by micro CT.

<p>Note the extensive mineralization in the muzzle skin containing the dermal sheath of vibrissae and in the ears (A, arrows); in the juxta-articular connective tissue in the elbows (B, arrows); axial view of dorsal artery (C, arrow); spinal and intercostal tissues (D, arrows); and in the right rib fusion leading to scoliosis as well as in the ears (E, arrows).</p

Calcium, phosphorus and pyrophosphate concentrations in serum/plasma of mice maintained on a regular rodent diet.

<p>Blood samples were collected by cardiac puncture, Ca and P concentrations were determined in serum, and PP_i levels were measured in heparinized plasma after removal of platelets. Statistical significance in comparison to <i>Enpp1^+/+</i> mice: *<i>P</i><0.01.</p><p>Calcium, phosphorus and pyrophosphate concentrations in serum/plasma of mice maintained on a regular rodent diet.</p

Aberrant tissue mineralization in <i>Enpp1^+/+</i>, <i>Enpp1^+/asj-2J</i> and <i>Enpp1^asj-2J</i> mice on normal and acceleration diet compared with <i>Enpp1^asj</i> mice on acceleration diet.

<p>Mice were placed on either normal diet or acceleration diet at 4 weeks of age, and tissues were collected for histopathology at 3 months of age. The values represent the number of mice, as percent of all animals examined, affected by mineralization as examined by hematoxylin and eosin stain on one section. Statistical analyses were performed with Fisher’s Exact test. Comparison to <i>Enpp1^asj-2J</i> mice on normal diet: *<i>P</i><0.05; **<i>P</i><0.01. Comparison to <i>Enpp1^asj-2J</i> mice on acceleration diet: ⁺<i>P</i><0.05.</p><p>Aberrant tissue mineralization in <i>Enpp1^+/+</i>, <i>Enpp1^+/asj-2J</i> and <i>Enpp1^asj-2J</i> mice on normal and acceleration diet compared with <i>Enpp1^asj</i> mice on acceleration diet.</p

Enhanced mineralization in <i>asj-2J</i> mice placed on “acceleration diet”, rich in phosphate and low in magnesium.

<p>The mice were placed on this diet at 4 weeks of age and necropsy was performed at 12 weeks. Note extensive mineralization as visualized by special stain (Alizarin Red). Assessment of the histopathology suggested that the mineralization was more extensive in mice kept on acceleration diet compared to the same mice kept on control diet (compare mineralization in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113542#pone-0113542-g001" target="_blank">Fig. 1B</a>).</p