P6342RANKL expression is increased in peripheral mononuclear cells of patients with severe aortic valve stenosis and promotes pro-calcific differentiation of valve cells

Abstract

Background: Calcific aortic valve disease (CAVD) is now the leading valvular heart disease. Mediators of bone remodeling such as the OPG/RANK/RANKL triad, FGF‐23/Klotho axis, and pyrophosphate (PPi) have been recently involved in vascular calcification. It is unclear whether these molecules can play a role in the pathogenesis of CAVD and/or predict the disease progression. Methods: We enrolled 50 patients affected by CAVD and a comparable group of subjects without valve disease. Peripheral blood mononuclear cells (PBMC) were collected from each subjects and RNA was extracted to assess gene expression of OPG/RANK/RANKL. Serum levels of OPG, FGF‐23 and PPi were determined through dedicated ELISA assays. CT scan images of patients with CAVD have been obtained and used to quantify the extent of calcium deposits within the valves. Bovine interstitial aortic valve cells (BVIC) have been seeded in vitro on type‐I collagen matrix and treated for nine days with RANKL (100 ng/ml) and inorganic phosphate (Pi, 0.6 mmol/L) to study the induction of matrix mineralization. Results: The two groups were comparable in term of age, gender, lipid profile, kidney function, and blood pressure levels. A significant increase in PTH (43,3 ng/L ± 28,1 vs 30,2 ng/L ± 16,6, p=0,006) and Pi levels (1,07 mmol/L ± 0,33 vs 0,94 mmol/L ± 0,16, p=0,012) was found in patients affected by CAVD. The gene expression analysis showed significantly higher expression of RANKL in the PBMC from CVAD patients as compared to controls (fold change 5,62±1,76 vs 1,57±0,32, p=0,018). RANK expression was identical in the two groups and OPG was not detectable in the cells. No differences between cases and controls were found in serum levels of FGF‐23 and PPi levels whereas circulating OPG was significantly increased in subjects with CAVD (1754,40 pg/mL ± 108,5 vs 1323 pg/mL ± 90,8) (p=0,003). As for the image analysis of valve calcium accumulation, we found a direct significant correlation between expression levels of RANKL in PBMC and the degree of calcium deposition (r=0,358, p=0,032). Finally, in vitro studies showed that treatment of BVIC with RANKL and Pi was followed by significant increase in type‐I collagen matrix mineralization as compared to control and Pi alone (Pi 0,030±0,006 vs RANKL+Pi 0,223±0,092 mg/mg collagen weight, p=0,001). Conclusions: We observed that RANKL expression is increased in PBMC of patients with CVAD and it is directly correlated with the degree of valve calcification. In addition, RANKL is able to promote pro‐calcific differentiation of VIC, suggesting a direct pathogenic role during valve disease progression.