Abstract 11911: ATP Release Acts as Survival Signal and Prevents the Mineralization of Aortic Valve by P2Y2 and PI3K/AKT Signaling
Calcified aortic stenosis (AS) is the most common valvular heart disease in western societies and is the first cause for surgical valve replacment. Recent studies revealed similarities in the mechanisms of aortic valve (AV) calcification and process involved in bone mineralization. Like bone cells, valvular interstitial cells (VICs) can liberate ATP in the pericellular environment. We thus hypothesized that ATP could stimulate purinergic receptors (P2) and ectonucleotidases and control AV calcification.
Methods and results: A tissue-based microarray and real-time PCR (qPCR) analyses revealed that ectonucleotidase ENPP1 is strongly up-regulated in calcified AV and VICs. In cultured calcified VICs, ENPP1 inhibitior, ARL67156, reduced significantly mineralization induced by both NaH2PO4and ENPP1 transfection (p<0.0001). A treatment of VICs with a siRNA for ENPP1 reduced its enzymatic activity as well as mineralization of VICs. In VICs, qPCR analyses demonstrated the presence of P2X(1,4,5,6,7), P2Y(1,2) and P1 adenosine receptors(2a, 2b). Inhibition of P2X (isoPPADS) and adenosine receptors (CGS 15943) did not modify the mineralization of VICs, however, suramin, a general purinergic receptor antagonist, markedly increased calcification. Interestingly, treatment of cells with a P2Y2 agonist, 2-thioUTP, reduced significantly calcification (p<0.0001). In isolated VICs, LY294002, a PI3K inhibitor, significantly increased the calcification of VICs and antagonized the protective effect of P2Y2 agonist. Furthermore, Tunel staining showed reduced apoptotic cells with ARL 67156 and 2-thioUTP but LY294002 negated this effect (pANOVA<0.0001). Western-blot analyses showed reduced levels of Akt, BCL-2 and increased clivage of caspase-3, however, 2-thioUTP prevented this effect. Ultimately, rats treated with warfarin developed AS and a significant rise of transaortic velocity, whereas the treatment with ARL67156 prevented the appearance of a stenosis and reduced apoptotic cells in AV.
Conclusion: ATP is a crucial signal for survival or death of VICs and plays an important role in AS development via ENPP1 and P2Y2 signaling. Hence, present findings may help to identify new targets for the treatment of AS and cardiovascular calcification.
- © 2011 by American Heart Association, Inc.