pyrophosphate and Aortic-Valve-Stenosis

Pyrophosphate (PPi) is a potent inhibitor of ectopic mineralization but its role during aortic valve calcification is not known.. Anti-calcific effect of PPi was investigated by using an in vitro model of serum-driven calcification of collagen sponges and decellularized porcine aortic valve leaflets. Bovine interstitial valve cells (VIC), seeded either within the collagen matrices or in transwell chambers, were used to test cellular ability to inhibit serum-induced calcification. PPi metabolism was investigated in clonal VIC harboring different calcifying potential.. In a cell-free system, high serum levels induced a dose-dependent calcification of type I collagen matrices which was prevented by PPi and ATP supplementation. Blockade of serum-driven calcification by PPi and ATP was also observed when using decellularized porcine aortic valve leaflets. A similar anti-calcific effect was also seen for bovine VIC, either statically seeded into the collagen matrices or co-cultured by using a transwell system. However, when we performed co-culture experiments by using clonal VIC harboring different calcifying potential, we observed that the subset of cells acquiring a pro-calcific profile lost the ability to protect the collagen from serum-driven calcification. Pro-calcific differentiation of the clonal VIC was accompanied by increase in ALP along with significant reduction in NPP activity and ATP/PPi extracellular accumulation. These changes were not observed in the clonal subtype with lower propensity towards calcification.. We showed that PPi and ATP are potent inhibitors of serum-driven calcification of collagen matrix and that their extracellular accumulation is reduced in calcifying VIC.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Calcium; Cattle; Cell Differentiation; Cell-Free System; Cloning, Molecular; Collagen; Diphosphates; Microscopy, Electron, Scanning; Nucleotides; Swine; X-Ray Diffraction

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.. 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.. 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 β,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1).. 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.

Topics: Alkaline Phosphatase; Animals; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Diphosphates; Etidronic Acid; Female; Phosphates; Pyrophosphatases; Swine; Tissue Culture Techniques