The role of inorganic pyrophosphate in aortic valve calcification

Abstract

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 β,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.

Figure 1

Effect of phosphate and pyrophosphatase on calcification of aortic valve leaflets ex vivo. A). Incorporation of ⁴⁵Ca in valve tissues over eight days at different phosphate concentrations with or without inorganic pyrophosphatase (IP). # p <0.05 versus 0.8 mM phosphate alone; * p <0.05 versus 3.8 mM phosphate alone (n = 8). B) Corresponding alizarin red stains for valves cultured with IP. A freshly isolated leaflet is included as a negative control (far left). F: Fibrosa; V: Ventricularis. The arrows point to calcified areas. C) Time course of ⁴⁵Ca incorporation in valve tissues cultured with or without IP and 3.8mM PO₄³⁻; * p <0.05 versus 2, 4 days; & p <0.1 versus 4 days; # p <0.05 versus 4, 6, 8 days (n = 8). D) Corresponding alizarin red stains.

Figure 2

Effect of phosphatases and devitalization on calcification of AV leaflets ex vivo. A) Incorporation of ⁴⁵Ca into AV leaflets cultured in different conditions as indicated. Control refers to leaflets cultured with 3.8 mM PO₄³⁻ only. IP: Inorganic pyrophosphatase; ALP: Alkaline phosphatase; Leaflets were devitalized by freezing and thawing several times. * p <0.05 versus control (n = 8). B) Corresponding alizarin red stains. F: Fibrosa; V: Ventricularis. The arrows point to calcified areas.

Figure 3

Effect of etidronate on calcification of AV leaflets ex vivo. A) Incorporation of ⁴⁵Ca into valve leaflets over eight days at different concentrations of etidronate with inorganic pyrophosphatase (IP) and 3.8 mM PO₄³⁻; * p <0.05 versus all other groups; # p <0.05 versus 3.8 mM phosphates only and other concentrations of etidronate. B) Corresponding alizarin red stains. The arrows point to calcified areas. F: Fibrosa; V: Ventricularis.

Figure 4

AV structure and cell viability ex vivo. A) Verhoeff-Van Gieson elastin stain shows preserved AV structure under all culture conditions. The arrows point to elastin fibers oriented along the length of the tissue. B) MTT stain shows preserved viability of AV tissue under all culture conditions. Leaflets were devitalized by freezing and thawing several times; a negative control showed no staining. C) TUNEL stain for apoptosis. Apoptotic nuclei stain red superimposed on the blue DAPI counterstain. Positive control: After treatment with DNAse; Negative control: Without TMR red stain. The arrows point to apoptotic cells. Fresh and cultured leaflets each had less than 0.2% positive cells (of >500 counted). F: Fibrosa; V: Ventricularis; IP: Inorganic pyrophosphatase; ALP: Alkaline phosphatase; Et: Etidronate.

Figure 5

Synthesis of pyrophosphate from AV leaflets. A) Quantification of PPi released into the medium from AV leaflets incubated with or without 30 μM MLS38949 (an inhibitor of TNAP) as a function of time. * p <0.05 versus no MLS38949 at 5 h (n = 4). B) Autoradiogram of a representative thin-layer chromatogram after incubation of valve leaflets with [γ³²P]ATP in 3 nM ATP with or without 30 μM MLS38949 or 300 μM β, γ-methylene ATP, an inhibitor of NPP1. All of the exogenous ATP was consumed in 5 min. Control leaflets were incubated without any inhibitors. C) Quantification of PPi production by AV tissue from ATP. * p <0.05 versus control.