Plasma PPi Deficiency Is the Major, but Not the Exclusive, Cause of Ectopic Mineralization in an Abcc6-/- Mouse Model of PXE

Abstract

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused in most cases by inactivating mutations in the ABCC6 gene. It was recently discovered that absence of ABCC6-mediated adenosine triphosphate release from the liver and consequently reduced plasma inorganic pyrophosphate (PPi) levels underlie PXE. This study examined whether reduced levels of circulating PPi, an antimineralization factor, is the sole mechanism of PXE. The Abcc6^-/- and Enpp1^asj mice were crossed with transgenic mice expressing human ENPP1, an ectonucleotidase that generates PPi from adenosine triphosphate. We generated Abcc6^-/- and Enpp1^asj mice, either wild-type or hemizygous for human ENPP1. Plasma levels of PPi and the degree of ectopic mineralization were determined. Overexpression of human ENPP1 in Enpp1^asj mice normalized plasma PPi levels to that of wild-type mice and, consequently, completely prevented ectopic mineralization. These changes were accompanied by restoration of their bone microarchitecture. In contrast, although significantly reduced mineralization was noted in Abcc6^-/- mice expressing human ENPP1, small mineralization foci were still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of ectopic mineralization in PXE, but there might be an alternative, as yet unknown mechanism, independent of PPi, by which ABCC6 prevents ectopic mineralization under physiologic conditions.

Figure 1.. Histopathologic examination demonstrates that overexpression of human ENPP1 completely prevents ectopic connective tissue mineralization in Enpp1^asj mice but not in Abcc6^−/− mice.

Tissues were collected in mice on the same diet at 12 weeks of age and analyzed by histopathology with Alizarin red stains. The Enpp1^asj mice developed ectopic mineralization of the dermal sheath of vibrissae in the muzzle skin, kidney, pinna and aorta (Fig. 1a, upper row). The Enpp1^asj;hENPP1^Tg/0 mice were entirely negative for mineralization in these tissues (Fig. 1a, lower row). The Abcc6^−/− mice also developed ectopic mineralization of the dermal sheath of vibrissae in the muzzle skin and in the kidney, although at a lesser extent (Fig. 1b, upper row). The corresponding Abcc6^−/−;hENPP1^Tg/0 mice demonstrated significantly reduced mineralization as compared to Abcc6^−/−;hENPP1^0/0 mice; however, distinct mineralization foci were still evident (Fig. 1b, lower row). Ectopic mineralization is indicated by arrows. Scale bar, 0.4 mm.

Figure 2.. The chemical assay of calcium demonstrates that overexpression of human ENPP1 reduces ectopic connective tissue mineralization in Enpp1^asj mice and Abcc6^−/− mice.

The calcium content was quantitatively determined in the muzzle skin biopsies containing the dermal sheath of vibrissae, kidney, pinna and aorta in mice on the same diet. Note the significantly elevated calcium content in Abcc6^−/−;hENPP1^0/0 and Enpp1^asj;hENPP1^0/0 mice as compared with the WT mice without mineralization. Overexpression of human ENPP1 in Enpp1^asj mice reduced the calcium content in tissues to that of WT mice (a). The calcium content was significantly reduced in Abcc6^−/− mice overexpressing human ENPP1, however, the amount of calcium in the muzzle skin and kidney was still significantly higher than WT mice (b). Values were expressed as mean ± SE; n = 11 – 17 mice per group. WT, wild type. ^*p < 0.05, ^**p < 0.01, compared with WT mice; ⁺p < 0.05, ⁺⁺p < 0.01, compared with either Abcc6^−/−;hENPP1^0/0 or Enpp1^asj;hENPP1^0/0 mice.

Figure 3.. MicroCT analysis reveals changes in bone microarchitecture in Enpp1^asj mice with overexpression of human ENPP1.

(a) Trabecular bone; (b) cortical bone. The left femurs in the Enpp1^asj;hENPP1^0/0 and Enpp1^asj;hENPP1^Tg/0 mice were compared with WT mice on the same diet. Note the distinct differences between male (top row in each panel) and female (bottom row in each panel) mice. Overexpression of human ENPP1 in Enpp1^asj mice restored the femoral microarchitecture similar to WT mice, as quantitatively detailed in Table 3 (n = 4–5 per sex in each group). WT, wild type.