Supplementary Material for: Increased Calcification in Osteoprotegerin-Deficient Smooth Muscle Cells: Dependence on Receptor Activator of NF-κB Ligand and Interleukin 6

<b><i>Objective:</i></b> Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-κB ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are more susceptible to mineralization and whether RANKL mediates this process. <b><i>Results:</i></b> Lesion-free aortas from 12-week-old ApoE-/-OPG-/- mice had spotty calcification, an appearance of osteochondrogenic factors and a decrease of smooth muscle markers when compared to ApoE-/-OPG+/+ aortas. In osteogenic conditions, VSMCs isolated from ApoE-/-OPG-/- (KO-VSMC) mice deposited more calcium than VSMCs isolated from ApoE-/-OPG+/+ (WT-VSMC) mice. Gene expression and biochemical analysis indicated accelerated osteochondrogenic differentiation. Ablation of RANKL signaling in KO-VSMCs rescued the accelerated calcification. While WT-VSMCs did not respond to RANKL treatment, KO-VSMCs responded with enhanced calcification and the upregulation of osteochondrogenic genes. RANKL strongly induced interleukin 6 (IL-6), which partially mediated RANKL-dependent calcification and gene expression in KO-VSMCs. <b><i>Conclusions:</i></b> OPG inhibits vascular calcification by regulating the procalcific effects of RANKL on VSMCs and is thus a possible target for therapeutic intervention.

<b><i>研究目的：</i></b> 血管钙化与心血管疾病的发病率及死亡率高度相关。骨保护素（Osteoprotegerin, OPG）是核因子κB受体活化因子配体（Receptor Activator of NF-κB Ligand, RANKL）的分泌型诱饵受体。在载脂蛋白E缺陷（apolipoprotein E-deficient, ApoE-/-）小鼠中，OPG的缺失会增大动脉粥样硬化病变面积并加重钙化程度。目前OPG发挥抗动脉粥样硬化及抗钙化作用的具体机制尚未完全阐明。本研究旨在探究OPG缺陷的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）是否更易发生矿化，以及RANKL是否介导这一过程。<b><i>实验结果：</i></b> 与ApoE-/-OPG+/+小鼠的主动脉相比，12周龄ApoE-/-OPG-/-小鼠的无病变主动脉出现斑点状钙化，同时伴随骨软骨源性因子表达上调和平滑肌标志物水平下降。在成骨诱导培养条件下，从ApoE-/-OPG-/-小鼠（KO-VSMC）中分离得到的血管平滑肌细胞，其钙沉积量显著高于ApoE-/-OPG+/+小鼠（WT-VSMC）分离得到的细胞。基因表达与生化分析结果表明，KO-VSMC的骨软骨源性分化进程被显著加速。阻断KO-VSMC中的RANKL信号通路可逆转这种加速的钙化表型。WT-VSMC对RANKL处理无明显响应，而KO-VSMC则表现出钙化增强及骨软骨源性基因表达上调的现象。RANKL可强力诱导白细胞介素6（interleukin 6, IL-6）的表达，而IL-6可部分介导RANKL依赖的KO-VSMC钙化进程与基因表达变化。<b><i>研究结论：</i></b> OPG通过调控RANKL对血管平滑肌细胞的促钙化作用，从而抑制血管钙化，因此OPG可作为心血管钙化治疗干预的潜在靶点。