Original article Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis Rihab Bouchareb a , Nancy Côté a , Marie-Chloé-Boulanger a , Khai Le Quang d , Diala El Husseini a , Jérémie Asselin b , Fayez Hadji a , Dominic Lachance d , Elnur Elyar Shayhidin a , Ablajan Mahmut a , Philippe Pibarot d , Yohan Bossé c , Younes Messaddeq b , Denis Boudreau b , André Marette d , Patrick Mathieu a, ⁎ a Laboratoire d'Études Moléculaires des Valvulopathies (LEMV), Groupe de Recherche en Valvulopathies (GRV), Quebec Heart and Lung Institute/Research Center, Department of Surgery, Laval University, Québec, Canada b The Center for Optics, Photonics and Lasers (COPL), Department of Physics, Laval University, Québec, Canada c Department of Molecular Medicine, Laval University, Québec, Canada d Department of Medicine, Laval University, Québec, Canada abstract article info Article history: Received 12 December 2014 Received in revised form 20 February 2015 Accepted 2 March 2015 Available online 11 March 2015 Keywords: Calcific aortic valve disease Calcific aortic stenosis Carbonic anhydrase XII P2Y 2 receptor Mineral resorption Mineral regression Aims: Calcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. Methods and results: By using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R -/- mice. Measurements of extracellular pH (pHe) by using core–shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR -/- /ApoB 100/100 /IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2 mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR -/- /ApoB 100/100 /IGF2 mice. Conclusion: P2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Calcific aortic valve stenosis (CAVS) is a chronic disorder character- ized by an abnormal mineralization of aortic leaflets [1]. Although CAVS can be diagnosed in its early stage with echocardiographic exam- ination, there is no medical treatment that can prevent the progressive calcification of leaflets, which ultimately results in a severe stenosis [2]. Hence, the only treatment of CAVS is thus a surgical intervention, which consists in the replacement of the aortic valve by using either an open heart surgery or a percutaneous intervention. However, these interventions are invasive and are associated with a significant morbidity/mortality as well as with elevated cost. Bone mineralization is the result of a delicate balance between deposition and resorption of minerals [3]. If applied to pathologic mineralization this concept suggests that it could be possible to promote the resorption of ectopic calcium deposit. Emerging evidence suggests that the expression of carbonic anhydrase (CA) during vascular calcification may promote the resorption of minerals [4]. In the reversa LDLR -/- ApoB 100/100 mice the normalization of cholesterol level after 6 months of western type diet led to a significant reduction of mineral content in the aortic valve [5]. These data suggest that ectopic mineral- ization of the aortic valve is potentially reversible. However, the molecular mechanisms that may promote the regression of aortic valve mineralization are presently unknown. The purinergic system exerts an important control over pathologic mineralization of the aortic valve [6]. In CAVS, the overexpression of ectonucleotidase enzymes, Journal of Molecular and Cellular Cardiology 82 (2015) 104–115 ⁎ Corresponding author at: Institut de Cardiologie et de Pneumologie de Québec/ Quebec Heart and Lung Institute, 2725 Chemin Ste-Foy Québec, Québec G1V 4G5, Canada. Tel.: +1 418 656 4717; fax: +1 418 656 4707. E-mail address: patrick.mathieu@chg.ulaval.ca (P. Mathieu). http://dx.doi.org/10.1016/j.yjmcc.2015.03.002 0022-2828/© 2015 Elsevier Ltd. 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