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（vascular calcification）与心血管疾病的发病率及死亡率密切相关。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。载脂蛋白E缺陷型（apolipoprotein E-deficient，ApoE-/-）小鼠中OPG的失活会加剧病变体积与钙化程度。OPG发挥抗动脉粥样硬化与抗钙化作用的具体机制尚未完全阐明。本研究旨在探究OPG缺陷型血管平滑肌细胞（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>研究结果：</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，呈现骨软骨源性因子的表达特征，且平滑肌标志物水平显著降低。在成骨诱导条件下，从ApoE-/-OPG-/-基因敲除小鼠（KO-VSMC）中分离得到的血管平滑肌细胞，其钙沉积量显著高于从ApoE-/-OPG+/+野生型（WT-VSMC）小鼠中分离得到的细胞。基因表达与生化分析结果显示，KO-VSMC的骨软骨源性分化进程被加速。在KO-VSMC中阻断RANKL信号通路可逆转这一加速的钙化表型。野生型VSMC对RANKL处理无明显响应，而KO-VSMC则表现为钙化程度增强，且骨软骨源性基因的表达水平上调。RANKL可强烈诱导白细胞介素6（interleukin 6，IL-6）的表达，而IL-6可部分介导KO-VSMC中RANKL依赖的钙化进程与基因表达变化。<b><i>研究结论：</i></b> 骨保护素（OPG）可通过调控RANKL对血管平滑肌细胞的促钙化作用，从而抑制血管钙化，因此可作为心血管钙化相关疾病治疗干预的潜在靶点。