Etidronate prevents, but does not reverse, ectopic mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6-/- )

Abstract

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable disorders manifesting with ectopic tissue mineralization. Most cases of PXE and some cases of GACI are caused by mutations in the ABCC6 gene, resulting in reduced plasma pyrophosphate (PPi) levels. There is no effective treatment for these disorders. It has been suggested that administration of bisphosphonates, stable and non-hydrolyzable PPi analogs, could counteract ectopic mineralization in these disorders. In this study we tested the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6^-/- mice, a model of PXE. The Abcc6^-/- mice received subcutaneous injections of etidronate, 0.283 and 3.40 mg/kg per injection (0.01× and 0.12×), twice a week, in both prevention and reversal studies. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by micro-computed tomography. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as the diagnosis is made, with careful monitoring of potential side effects.

Keywords: bisphosphonates; ectopic mineralization; etidronate treatment; mouse model; pseudoxanthoma elasticum.

Figure 1. Histopathology with Alizarin red stain demonstrates that etidronate treatment prevents, but does not reverse, ectopic soft tissue mineralization in Abcc6^−/− mice

The Abcc6^−/− mice develop ectopic mineralization of the dermal sheath of vibrissae prior to 12 weeks of age, and this process progresses to 24 weeks of age. Subcutaneous injection of etidronate at 0.01× and 0.12× doses during 4–12 weeks markedly reduced the degree of mineralization in the dermal sheath of vibrissae, when compared to age-matched Abcc6^−/− mice receiving saline injections (left 3 panels). Etidronate treatment from 12 to 24 weeks of age prevented further mineralization but did not reverse mineralization that had developed prior to 12 weeks of age (right 3 panels). ETD, etidronate; KO, knockout. Scale bar = 400 μm.

Figure 2. Etidronate treatment prevents vibrissae mineralization as determined by the direct chemical assay of calcium

Note the significantly elevated calcium content in the muzzle skin of Abcc6^−/− mice, either NT or injected with saline, as compared with the WT mice (right panel). Injections of Abcc6^−/− mice subcutaneously with 0.01× and 0.12× ETD significantly reduced the calcium content of the muzzle skin as compared with age-matched mice injected with saline. Etidronate treated Abcc6^−/− mice at 24 weeks of age demonstrated mineralization similar to Abcc6^−/− mice at 12 weeks of age, indicating that ETD prevents further mineralization but does not reverse mineralization that had developed before initiation of treatment. ^*p < 0.05, ^**p < 0.01, compared to KO mice at 12 weeks of age; ⁺⁺p < 0.01, compared to KO mice at 24 weeks of age. Mean ± SE; n = 7–10 mice per group. ETD, etidronate; KO, knockout; WT, wild type; NT, not treated.

Figure 3. Etidronate treatment of Abcc6^−/− mice results in altered femur microarchitecture

Note the distinct difference between male (upper panel) and female (lower panel) mice, assessed by μCT scan. Treatment with etidronate caused changes in femur microarchitecture, as quantitatively detailed in Table 2. Three males and 3 females were examined in each group with similar findings. ETD, etidronate; KO, knockout; WT, wild type; NT, not treated.

Figure 4. The PPi and Pi generating pathway points to the critical role of components of the pro-mineralization/anti-mineralization network

Mutations in the ABCC6, ENPP1, and NT5E genes cause pseudoxanthoma elasticum (PXE), generalized arterial calcification of infancy (GACI), and arterial calcification due to CD73 deficiency (ACDC), respectively. ABCC6, a putative transmembrane transporter, mediates ATP release from hepatocytes to extracellular space where ATP is converted to PPi and AMP by ENPP1, a membrane-bound pyrophosphatase/phosphodiesterase. CD73 converts AMP to Pi and adenosine, the latter one being an inhibitor of tissue nonspecific alkaline phosphatase (TNAP), an extracellular, yet membrane-bound protein, that hydrolyze PPi to Pi. PPi is an anti-mineralization factor, and Pi is a pro-mineralization factor. Deficiencies in ABCC6, ENPP1 and CD73 proteins lead to reduced plasma PPi levels and PPi/Pi ratio, thereby promoting mineralization in peripheral tissues. EC, extracellular; IC, intracellular.