Fractalkine (FKN) and its specific receptor CX3CR1 play a critical role in the pathogenesis of atherosclerosis including recruitment of vascular cells and the development of inflammation. However, its contribution to regulating the development of atherosclerotic calcification has not been well documented. Osteogenic transformation of vascular smooth muscle cells (VSMCs) is critical in the development of calcification in atherosclerotic lesions. In this study, for the first time, we evaluated the effect of FKN/CX3CR1 on the progression of VSMCs calcification and defined molecular signaling that is operative in the FKN/CX3CR1-induced osteogenic transformation of VSMCs. We found that high-fat diet induced atherosclerotic calcification in vivo was markedly inhibited in the Apolipoprotein E (ApoE) and CX3CR1 deficient (ApoE^−/−/CX3CR1^−/−) mice compared with their control littermates. FKN and CX3CR1 were both expressed in VSMCs and up-regulated by oxidized low-density lipoprotein (ox-LDL). FKN/CX3CR1 promoted the expression of osteogenic markers, including osteopontin (OPN), bone morphogenetic protein (BMP)-2 and alkaline phosphatase (ALP) and decreased VSMCs markers, including smooth muscle (SM) α-actin and SM22-α in a dose-dependent manner. The essential role of FKN/CX3CR1 in VSMCs calcification was further confirmed by lentivirus-mediated knockdown or overexpression of CX3CR1 blocked or accelerated osteogenic transformation of VSMCs. This response was associated with reciprocal up- and down-regulation of osteogenic factor, runt-related transcription factor 2 (RUNX2), transcription factors in osteoclast differentiation, receptor activator of nuclear factor-κB (RANK), RANK ligand (RNAKL) and osteoprotegerin (OPG), respectively. Inhibition of FKN/CX3CR1-activated Jak2/Stat3 signaling by the Jak/Stat inhibitor AG490 blocked osteogenic transformation of VSMCs and RUNX2 induction concurrently. Taken together, our data uncovered novel roles of FKN/CX3CR1 in promoting VSMC osteogenic transformation and atherosclerotic calcification by activating RUNX2 through Jak2/Stat3 signaling pathway and suppressing OPG. Our findings suggest that targeting FKN/CX3CR1 may provide new strategies for the prevention and treatment of atherosclerotic calcification.

Fractalkine (FKN) 及其特异性受体 CX3CR1 在动脉粥样硬化的发病机制中起关键作用，包括血管细胞的募集和炎症的发展。然而，它对调节动脉粥样硬化钙化发展的贡献尚未得到充分证明。血管平滑肌细胞 (VSMC) 的成骨转化对于动脉粥样硬化病变钙化的发展至关重要。在这项研究中，我们首次评估了 FKN/CX3CR1 对 VSMC 钙化进程的影响，并确定了在 FKN/CX3CR1 诱导的 VSMC 成骨转化中起作用的分子信号传导。我们发现高脂肪饮食诱导的体内动脉粥样硬化钙化在载脂蛋白 E (ApoE) 和 CX3CR1 缺陷 (ApoE ^-/-/CX3CR1 ^-/-) 小鼠与其对照同窝仔鼠进行比较。FKN 和 CX3CR1 均在 VSMC 中表达，并被氧化低密度脂蛋白 (ox-LDL) 上调。FKN/CX3CR1 促进成骨标志物的表达，包括骨桥蛋白 (OPN)、骨形态发生蛋白 (BMP)-2 和碱性磷酸酶 (ALP) 并降低 VSMC 标志物，包括平滑肌 (SM) α-肌动蛋白和 SM22-α剂量依赖性方式。FKN/CX3CR1 在 VSMCs 钙化中的重要作用通过慢病毒介导的敲除或 CX3CR1 的过表达阻止或加速 VSMCs 的成骨转化得到进一步证实。这种反应与成骨因子、矮小相关转录因子 2 (RUNX2)、破骨细胞分化中的转录因子、核因子-κB 受体激活剂 (RANK) 的相互上调和下调有关，RANK 配体 (RNAKL) 和骨保护素 (OPG)。Jak/Stat 抑制剂 AG490 对 FKN/CX3CR1 激活的 Jak2/Stat3 信号的抑制同时阻止了 VSMC 的成骨转化和 RUNX2 诱导。总之，我们的数据揭示了 FKN/CX3CR1 通过 Jak2/Stat3 信号通路激活 RUNX2 并抑制 OPG 在促进 VSMC 成骨转化和动脉粥样硬化钙化中的新作用。我们的研究结果表明，靶向 FKN/CX3CR1 可能为预防和治疗动脉粥样硬化钙化提供新的策略。我们的数据揭示了 FKN/CX3CR1 通过 Jak2/Stat3 信号通路激活 RUNX2 并抑制 OPG 在促进 VSMC 成骨转化和动脉粥样硬化钙化方面的新作用。我们的研究结果表明，靶向 FKN/CX3CR1 可能为预防和治疗动脉粥样硬化钙化提供新的策略。我们的数据揭示了 FKN/CX3CR1 通过 Jak2/Stat3 信号通路激活 RUNX2 并抑制 OPG 在促进 VSMC 成骨转化和动脉粥样硬化钙化方面的新作用。我们的研究结果表明，靶向 FKN/CX3CR1 可能为预防和治疗动脉粥样硬化钙化提供新的策略。