Changes in dermal fibroblasts from Abcc6(-/-) mice are present before and after the onset of ectopic tissue mineralization

Abstract

Pseudoxanthoma elasticum (PXE), a rare genetic disease caused by mutations in the ABCC6 gene, is characterized by progressive calcification of elastic fibers in the skin, eyes, and the cardiovascular system. The pathomechanism of the mineralization is still obscure. Several hypotheses have been proposed, one of them suggesting a role for fibroblasts in controlling the amount and the quality of the calcified extracellular matrix. This hypothesis raises the question whether changes in mesenchymal cells are the cause and/or the consequences of the calcification process. In this study, fibroblasts were isolated and cultured from Abcc6(+/+) and Abcc6(-/-) mice of different ages to investigate parameters known to be associated with the phenotype of fibroblasts from PXE patients. Results demonstrate that a few changes (Ank and Opn downregulation) are already present before the occurrence of calcification. By contrast, a modification of other parameters (intracellular O2- content, Tnap activity, and Bmp2 upregulation) can be observed in Abcc6(-/-) mice after the onset of tissue mineralization. These data suggest that in the Abcc6(-/-) genotype, dermal fibroblasts actively contribute to changes that promote matrix calcification and that these cells can be further modulated with time by the calcified environment, thus contributing to the age-dependent progression of the disease.

Conflict of interest statement

Conflict of interests

Authors declare no conflict of interest

Figures

Figure 1

Dermal fibroblasts cultured from KO (Abcc6−/−) and WT (Abcc6+/+) mice of 0.5 and 12 months of age and observed by phase contrast microscopy (a). Differences in the proliferation capabilities are related to the age of the animals, but are independent of the genotype as clearly shown in graph (b). Bar = 300 μm.

Figure 2

Anion superoxide (O2−) and hydrogen peroxide (H2O2) content measured by flow cytometry in dermal fibroblasts cultured from WT (Abcc6+/+) and KO (Abcc6−/−) mice of 0.5 and 12 months of age. *p<0.05, KO vs WT of the same age; # p<0.05 0.5 vs 12 months of age within the same genotype.

Figure 3

Expression of Bmp2 measured by Western blot in dermal fibroblasts cultured from WT (Abcc6+/+) and KO (Abcc6−/−) mice of 0.5 and 12 months of age. Data are expressed as mean values ± SD of densitometric analyses were values in cells from WT animals 0.5 month-old were set at 1. A representative Western blot is shown. *p<0.05, KO vs WT of the same age; ## p<0.01, 0.5 vs 12 months within the same genotype.

Figure 4

Tnap expression and activity were evaluated in dermal fibroblasts cultured from WT (Abcc6+/+) and KO (Abcc6−/−) mice of 0.5 and 12 months of age. Data are expressed as mean values ± SD of different measurements were values in cells from WT animals 0.5 month-old were set at 1. A representative Western blot is shown. *p<0.05, KO vs WT of the same age; ## p<0.01 and ### p<0.001, 0.5 vs 12 months within the same genotype.

Figure 5

Enpp1 and Ank expression measured by Western blot in dermal fibroblasts cultured from WT (Abcc6+/+) and KO (Abcc6−/−) mice of 0.5 and 12 months of age. Data are expressed as mean values ± SD of densitometric analyses were values in cells from WT animals 0.5 month-old were set at 1. Representative Western blots are shown. **p<0.01 KO vs WT of the same age; ## p<0.01 0.5 vs 12 months within the same genotype.

Figure 6

Expression of Opn measured by Western blot in dermal fibroblasts cultured from WT (Abcc6+/+) and KO (Abcc6−/−) mice of 0.5 and 12 months of age. Data are expressed as mean values ± SD of densitometric analyses were values in cells from WT animals 0.5 month-old were set at 1. A representative Western blot is shown. *p<0.05 and **p<0.01 KO vs WT of the same age.