Internationale Zeitschrift für Herz-Kreislauf-Forschung

Denosumab could be a Potential Inhibitor of Valvular Interstitial Cells Calcification in vitro

Daniel Alejandro Lerman, Sai Prasad and Nasri Alotti

Denosumab could be a Potential Inhibitor of Valvular Interstitial Cells Calcification in vitro

Objective: Denosumab is a fully human monoclonal antibody and novel antiresorptive agent that works by binding receptor activator of nuclear factor kappa-β ligand (RANKL) and inhibiting the signaling cascade that causes osteoclast maturation, activity, and survival. We aimed to elucidate the effect of Denosumab in the process of spontaneous and induced calcification in an in vitro porcine valvular interstitial cells (VICs) model.

Materials and Methods: VICs were extracted from fresh porcine hearts by serial collagenase digestion. Spontaneous calcification of VICs was increased in vitro by adding Na₃PO₄ (3 mM, pH 7.4) and different concentrations (0.1, 1 and 10 ng/ml) of transforming growth factor beta (TGFß). The degree of calcification before and after treatment with Denosumab was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. For statistical analysis we used SPSS and Microsoft Office Excel 2013.

Results: Porcine aortic VICs in vitro were induced to calcify by the addition of either 3 mM Na₃PO₄, showing a 5.2 fold increase by 14 days (P<0.001), or 3 mM Na₃PO₄ + 10 ng/ml of TGFβ, showing a 7 fold increase by Day 14 (P<0.001). Denosumab inhibited induced calcification by 3 mM Na₃PO₄ and 3 mM Na₃PO₄ with the addition of TGFß at either 0.1, 1 or 10 ng/ml to basal levels only at a concentration of 50 μg/ml (P<0.001).

Conclusion: This study has proved that Denosumab could be a potential inhibitor of the calcification of VICs in vitro. A fuller understanding of the actions of Denosumab may identify a novel therapeutic strategy for clinical intervention against aortic valve calcification and aortic stenosis.