Abstract

BackgroundExtracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors.ResultsNucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm2). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = − 0.30, p < 0.05; vs. AMP hydrolysis: r = − 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = − 0.26, p < 0.05; vs. AMP hydrolysis: r = − 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = − 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = − 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD.ConclusionsThis work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy.Graphic abstract.

Highlights

  • Calcific aortic valve disease (CAVD) is a slowly progressive disorder related to the mineralization of aortic valve leaflets [1]

  • Microphotographs of histological stainings for representative aortic valves (Fig. 3a) had been complied with immunofluorescence analysis (Fig. 3b). These results indicated that in both stenotic and non-stenotic aortic valves are enzymes that can be engaged in nucleotide and adenosine metabolism, including ectonucleoside triphosphate diphosphohydrolase 1, ecto-nucleotide pyrophosphatase/phosphodiesterase 1, ecto-5′-nucleotidase (e5NT, CD73), alkaline phosphatase (ALP) and adenosine deaminase (ADA)

  • Since, increased expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) and ALP in stenotic aortic valves have been previously described [22, 45], in this study we focused on CD73, CD39 and ADA mRNA expressions that were measured in aortic valve sections free of calcifications

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Summary

Introduction

Calcific aortic valve disease (CAVD) is a slowly progressive disorder related to the mineralization of aortic valve leaflets [1]. CAVD is an active cell-regulated process initiated by endothelial disruption with macrophages and T cell infiltration with accumulation and oxidation of lipoproteins [4, 5] These factors activate quiescent valvular interstitial cells (qVIC) to activated VICs (aVIC), which are characterized by the expression of smooth muscle cells alpha actin (α-SMA). Results Nucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs 0.67 ± 0.11 nmol/min/cm).

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