title: Denosumab could be a Potential Inhibitor of Valvular Interstitial Cells Calcificationin vitro.
abstract: 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 anin vitroporcine valvular interstitial cells (VICs) model.
  
  
  METHODS
  VICs were extracted from fresh porcine hearts by serial collagenase digestion. Spontaneous calcification of VICs was increasedin vitroby 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 VICsin vitrowere 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 Na3PO4and 3 mM Na3PO4with 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).
  
  
  CONCLUSIONS
  This study has proved that Denosumab could be a potential inhibitor of the calcification of VICsin 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.
authors.affiliation: Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian) The University of Edinburgh, United Kingdom.
authors.forename: Daniel Alejandro
authors.initials: DA
authors.lastname: Lerman
authors.affiliation: Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian) The University of Edinburgh, United Kingdom.
authors.forename: Sai
authors.initials: S
authors.lastname: Prasad
authors.affiliation: Department of Cardiothoracic Surgery, Teaching Hospital of Zala County, Pécs University, Hungary.
authors.forename: Nasri
authors.initials: N
authors.lastname: Alotti
keywords: Aortic valve calcification disease; Aortic valve interstitial cells; Calcification; Denosumab; Porcine VICs model; RANK; RANKL