The role of inorganic pyrophosphate in aortic valve calcification.

BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) calcification is a major cause of morbidity and mortality, yet the molecular mechanisms involved are poorly understood. Hence, an ex vivo model of calcification in intact AVs was developed in order to test the role of orthophosphate and pyrophosphate (PPi), both of which factors are known to influence vascular calcification. METHODS: Porcine AV leaflets were cultured in serum-free medium under static conditions for eight days, over which time leaflet architecture and viability were preserved. Calcification was measured as the incorporation of 45Ca, with confirmation by Alizarin Red staining. RESULTS: Calcification required both a high phosphate concentration (3.8 mM) and removal of PPi with alkaline phosphatase or inorganic pyrophosphatase. Calcification occurred predominantly on the fibrosa and was arrested by the bisphosphonate etidronate, a non-hydrolyzable analog of PPi. Leaflets released PPi into the medium, and this was enhanced by MLS38949, a specific inhibitor of tissue non-specific alkaline phosphatase (TNAP). Furthermore, leaflets synthesized PPi from extracellular ATP, which was reduced by ß,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1). CONCLUSION: The ex vivo AV calcification model developed in the present study showed that extracellular PPi, produced by valvular tissue, is a potent inhibitor of valvular calcification. In addition to synthesis, hydrolysis by TNAP also controls PPi levels and calcification. The results suggest that a decreased synthesis or increased hydrolysis of pyrophosphate may contribute to valvular calcification, and that bisphosphonates or inhibitors of TNAP are potential preventive strategies of the process. TNAP are potential preventive strategies.

Autosomal recessive mental retardation, deafness, ankylosis, and mild hypophosphatemia associated with a novel ANKH mutation in a consanguineous family.

CONTEXT: Mutations in ANKH cause the highly divergent conditions familial chondrocalcinosis and craniometaphyseal dysplasia. The gene product ANK is supposed to regulate tissue mineralization by transporting pyrophosphate to the extracellular space. OBJECTIVE: We evaluated several family members of a large consanguineous family with mental retardation, deafness, and ankylosis. We compared their skeletal, metabolic, and serological parameters to that of the autosomal recessive progressive ankylosis (ank) mouse mutant, caused by a loss-of-function mutation in the murine ortholog Ank. PARTICIPANTS: The studied patients had painful small joint soft-tissue calcifications, progressive spondylarthropathy, osteopenia, mild hypophosphatemia, mixed hearing loss, and mental retardation. RESULTS: After mapping the disease gene to 5p15, we identified the novel homozygous ANK missense mutation L244S in all patients. Although L244 is a highly conserved amino acid, the mutated ANK protein was detected at normal levels at the plasma membrane in primary patient fibroblasts. The phenotype was highly congruent with the autosomal recessive progressive ankylosis (ank) mouse mutant. This indicates a loss-of-function effect of the L244S mutation despite normal ANK protein expression. Interestingly, our analyses revealed that the primary step of joint degeneration is fibrosis and mineralization of articular soft tissues. Moreover, heterozygous carriers of the L244S mutation showed mild osteoarthritis without metabolic alterations, pathological calcifications, or central nervous system involvement. CONCLUSION: Beyond the description of the first human progressive ankylosis phenotype, our results indicate that ANK influences articular soft tissues commonly involved in degenerative joint disorders. Furthermore, this human disorder provides the first direct evidence for a role of ANK in the central nervous system.