Abstract 5868: Increased Expression Of Ectonucleotide Pyrophosphatase/phosphodiesterase-1 (enpp-1) In Aortic Stenosis: Implication For The Calcification Of The Aortic Valve
Introduction: Ectonucleotidases are a group consisting of 7 transmembrane proteins which catalyze the breakdown of nucleotides, and are thought to be of crucial importance in controlling mineralization process. Ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) generates pyrophosphate (PPi), an inhibitor of calcification. On the other hand, PPi can be metabolized by alkaline phosphatase (ALP) into Pi, which is a promoter of calcification. Thus a balance between ectonucleotidase and ALP might be crucial in controlling calcification process. Aortic stenosis (AS) is an active disorder characterized by abnormal ectopic mineralization. We have thus hypothesized that abnormal expression of ectonucleotidase(s) might be involved in the pathological calcification of the aortic valve.
Methods: In a first set of experiments, expression of ectonucleotidases (ENPP-1, ENPP-2, ENPP-3, ENTPD-1, ENTPD-2, ENTPD-3 and NT5E) has been compared between 5 controls and 5 AS valves using microarray (Affymetrix GeneChip). Then in 80 patients with AS and 14 control subjects, valve expression of ectonucleotidase was assessed by immunohistochemistry, enzyme activity along with q-PCR. Finally, in vitro studies with isolated valve interstitial cells (VICs) were performed in order to document the role of ectonucleotidase after treatment with a calcifying medium.
Results: Gene expression profile demonstrated that among the 7 ectonucleotidases, only ENPP-1 was significantly up-regulated in AS valves. When compared to control valves, q-PCR analyses confirmed that ENPP-1 expression was increased by 3-fold in AS valves. Immunohistochemistry and phosphodiesterase activity confirmed respectively higher protein expression and enzyme activity of ENPP-1 in AS compared to normal valves. In addition, phosphodiesterase activity in AS valves correlated significantly with calcium level (r=0.49; p=0.03). In vitro, ENPP-1 mRNA and protein levels were increased after treatment with the calcifying medium, whereas calcification was abrogated with ARL 67156, a specific inhibitor of ectonuclotidase.
Conclusion: The present results support that ENPP-1 is a critical regulator of calcification of aortic valve and may be involved in the development of AS.