The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PPi). The importance of PPi for inhibiting arterial calcification has been reinforced by the protective effects of PPi in various mouse models displaying ectopic calcifications. Besides PPi, Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp-deficient mice develop spontaneous calcification of the arterial media. Whereas PPi and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.

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A novel γ-carboxyglutamate (Gla)-containing protein, named Gla-rich protein (GRP) after its high content in Gla residues or upper zone of growth plate and cartilage matrix associated protein after its preferential expression by cartilage chondrocyte, was recently identified in sturgeon, mice, and humans through independent studies. GRP is the most densely γ-carboxylated protein identified to date and its structure has been remarkably conserved throughout vertebrate evolution but is apparently absent from bird genomes. Several transcript and genomic variants affecting key protein features or regulatory elements were described and 2 paralogs were identified in the teleost fish genome. In the skeleton, most relevant levels of GRP gene expression were observed in cartilaginous tissues and associated with chondrocytes, suggesting a role in chondrogenesis. But GRP expression was also detected in bone cells, indicative of a more widespread role for the protein throughout skeletal formation. Although the molecular function of GRP is yet unknown, the high content of Gla residues and its accumulation at sites of pathological calcification in different human pathologies affecting skin or the vascular system and in breast cancer tumors suggest that GRP may function as a modulator of calcium availability. Because of its association with fibrillar collagens, GRP could also be involved in the organization and/or stabilization of cartilage matrix. Although transgenic mice did not reveal obvious phenotypic alterations in skeletal development or structure, zebrafish morphants lack craniofacial cartilage and exhibit limited calcification, suggesting a role for GRP during skeletal development, but additional functional data are required to understand its function.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.