The Effect of Osteoprotectin (OPG)/Receptor Activator of Nuclear Factor-κB Ligand (RANKL)/Receptor Activator of Nuclear Factor-κB (RANK) Gene Methylation on Aortic Valve Calcified

Abstract

To evaluate the effect of the methylation of osteoprotectin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK) pathway on aortic valve calcification, the aortic valve tissue was collected from 38 aortic stenosis (AS) patients who underwent valve replacement. OPG and RANKL gene methylation, RT-PCR, and ELISA were performed. Hematoxylin-eosin staining (HE), alizarin red-S staining, and immunohistochemically staining of OPG, RANKL, and CD68 were simultaneously performed. The patients were divided into noncalcified group ( $n=21$ ) and calcified group ( $n=17$ ). The methylation rate of OPG gene in noncalcified group was higher than that in calcified group ( $P=0.027$ ). The methylation degree of RANKL gene was generally lower, but the noncalcified group was still higher than that in the calcified group ( $P=0.025$ ). RT-PCR analysis showed that the mRNA expression of OPG and RANKL was higher in calcified group than in noncalcified group ( $P=0.007$ and $P=0.036$ , respectively), and the mRNA expression was negatively correlated with the gene methylation rate. The protein expression of OPG and RANKL was detected by immunohistochemistry and ELISA, showing significantly increased in calcified group ( $P=0.004$ and $P=0.042$ , respectively). Soluble RANKL (sRANKL) in CD68-positive group was significantly different from that in negative group ( $0.1243\pm 0.0321$ vs $0.0984\pm 0.0218$ pg/mL, $P=0.007$ ). There was no significant difference in OPG value between positive group ( $1.9411\pm 0.4554$ ng/mL) and negative group ( $1.8422\pm 0.5218$ ng/mL, $P=0.587$ ). In conclusion, the degree of methylation of OPG and RANKL genes may play an important role in regulating valve calcification in AS patients.