Lerman Daniel Alejandro, Prasad Sai, Alotti Nasri
Department of Cardiothoracic Surgery, Royal Infirmary Hospital of Edinburgh (NHS Lothian) The University of Edinburgh, United Kingdom.
Department of Cardiothoracic Surgery, Teaching Hospital of Zala County, Pécs University, Hungary.
Int J Cardiovasc Res. 2016 Jan 3;5(1). doi: 10.4172/2324-8602.1000249.
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 porcine valvular interstitial cells (VICs) model.
VICs were extracted from fresh porcine hearts by serial collagenase digestion. Spontaneous calcification of VICs was increased by adding NaPO (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.
Porcine aortic VICs were induced to calcify by the addition of either 3 mM NaPO, showing a 5.2 fold increase by 14 days (P<0.001), or 3 mM NaPO + 10 ng/ml of TGFβ, showing a 7 fold increase by Day 14 (P<0.001). Denosumab inhibited induced calcification by 3 mM NaPO and 3 mM NaPO 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).
This study has proved that Denosumab could be a potential inhibitor of the calcification of VICs . A fuller understanding of the actions of Denosumab may identify a novel therapeutic strategy for clinical intervention against aortic valve calcification and aortic stenosis.
地诺单抗是一种全人源单克隆抗体和新型抗吸收剂，其作用机制是结合核因子κ-β受体激活剂配体（RANKL）并抑制导致破骨细胞成熟、活性和存活的信号级联反应。我们旨在阐明地诺单抗在猪瓣膜间质细胞（VICs）模型中自发和诱导钙化过程中的作用。
通过连续胶原酶消化从新鲜猪心脏中提取VICs。通过添加NaPO（3 mM，pH 7.4）和不同浓度（0.1、1和10 ng/ml）的转化生长因子β（TGFβ）来增加VICs的自发钙化。用地诺单抗处理前后的钙化程度通过茜素红染色检测钙沉积，天狼星红染色检测胶原蛋白来评估。采用比色技术定量测定钙和胶原蛋白沉积。统计分析使用SPSS和Microsoft Office Excel 2013。
添加3 mM NaPO可诱导猪主动脉VICs钙化，14天时增加5.2倍（P<0.001）；添加3 mM NaPO + 10 ng/ml TGFβ，14天时增加7倍（P<0.001）。地诺单抗仅在浓度为50 μg/ml时才能将3 mM NaPO以及添加了0.1、1或10 ng/ml TGFβ的3 mM NaPO诱导的钙化抑制至基础水平（P<0.001）。
本研究证明地诺单抗可能是VICs钙化的潜在抑制剂。更全面地了解地诺单抗的作用可能会为临床干预主动脉瓣钙化和主动脉狭窄确定一种新的治疗策略。
