Ecto-Nucleotidase-Derived Adenosine Promotes Mineralization Through A2a Receptor in Calcified Aortic Valves Disease

Background: Mineralization plays a crucial role in the development of calcific aortic valve disease (CAVD). The expression of ecto-nucleotidases may promote the mineralization of valve interstitial cells (VICs). We hypothesized that the expression of NPP1, which generates adenosine monophosphate (AMP), and 5’nucleotidase, an enzyme using AMP as a substrate to produce adenosine, may co-regulate the mineralization of the aortic valve. Methods: We have investigated the expression of NPP1 and 5’nucleotidase in CAVD tissues and determined the role of these ecto-nucleotidases on the mineralization of isolated VICs. The signaling pathway involving adenosine receptors was also studied. Results: In CAVD tissues, we documented that NPP1 and 5’nucleotidase enzyme activities were increased. NPP1 and 5’nucleotidase were co-expressed by VICs. In cell culture we found that mineralization induced by ATP was decreased by silencing NPP1 and 5’nucleotidase, suggesting a role for adenosine. Adenosine and specific A2a receptor agonist increased the calcification of VICs. Silencing of A2a receptor and the use of A2a-/- receptor mouse VICs abrogated adenosine-induced mineralization. Also, A2a receptor-mediated mineralization of VICs was negated by the transfection of a mutant dominant negative Gαs vector. Inhibition of the protein kinase A (PKA) pathway prevented adenosine-induced mineralization of VICs and expression of NPP1. We next showed that activation of PKA promoted in luciferase assay the activity of the NPP1 promoter. By using chromatin immunoprecipitation assay (ChIP) we documented that the cAMP response element binding protein (CREB), downstream of PKA, physically interacted with the NPP1 promoter region. Furthermore, the transfection of a mutant dominant active CREBDIEDML in isolated VICs increased the NPP1 promoter activity by 6.5-fold. Conclusion: The overexpression of NPP1 and 5’nucleotidase in CAVD promotes the mineralization of the aortic valve through A2a adenosine receptor, which signals through Gαs and the cAMP/PKA/CREB pathway. CREB is a positive regulator of NPP1 promoter activity in a positive feedback loop. The ecto-nucleotidases and/or A2a adenosine receptor could represent potential novel pharmaceutical targets in CAVD.