Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI

Abstract

The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal fin

“…Such analysis provided further insights into the mineralogenic effects related to bone density or ray thickness and is, therefore, a complement to other techniques that infer on the organization of the regenerated mineral, such as birefringence analysis1358, for the identification of pro- or anti-mineralogenic agents with effects not as evident as those of RA, for example. Our data confirmed the role of WARF in negatively regulating mineral density in zebrafish lepidotrichia, in a way contrasting to that described for the abcc6a mutant57. This can be simply explained by the fact that those are two distinct mineralizing systems.…”

“…Interestingly, in vitro studies reported an inhibition of osteoblast differentiation upon WARF exposure54, while VK media supplementation promoted osteoblast-to-osteocyte transition and decreased osteoclast differentiation5556. Exposure of zebrafish to WARF was found to induce pathological calcification of soft tissues and altered skeletal development during larval stages27, and to promote axial skeleton mineralization in an abcc6a mutant, while VK inhibited bone matrix mineralization57.…”

Retinoic acid-induced premature osteoblast-to-preosteocyte transitioning has multiple effects on calvarial development

“…1C-E), appearing like a subtle craniosynostosis. However, this ectopic mineralization does not seem to occur in intercalvarial regions, but primarily in the adjacent dermis, as described for diseases such as pseudoxanthoma elasticum (PXE) (Apschner et al, 2014;Mackay et al, 2015;Walker et al, 1989). Indeed, both dermal fibroblasts and basal keratinocytes express col1a2 (Fig.…”

Pseudoxanthoma elasticum

“…On the basis of animal model experiments, it has also been hypothesized that low vitamin K export from the liver would decrease the gamma-carboxylation of anti-mineralization proteins [76, 77]. Furthermore, MGP is not carboxylated in the elastic fibers of PXE patients [78], and PXE-like calcification of elastic fibers is observed in patients with mutation in the GGCX gene [78].…”

“…Most of the detailed experimental data on the pathophysiology of PXE comes from Abcc6 -deficient models in the zebrafish [77, 96, 97] and in the mouse [98–102]. The zebrafish model is a useful tool for testing potential therapies, such as premature termination codon read-through [103].…”