动脉粥样硬化中fractalkine/CX3CR1促进VSMC向成骨表型转化的作用及机制

Vascular smooth muscle cells (VSMC) differentiation to an osteogenic phenotype plays a critical role in the pathophysiology of atherosclerotic intimal calcification. However, its mechanism is not clear. The chemokine fractalkine/CX3CR1 mediates the interaction between the inflammatory cells and vascular wall cells, which plays an important role in the AS course. Recent studies have found that fractalkine/CX3CR1 can promote the maturation and differentiation of osteocyte precursor cells. We hypothesize that fractalkine/CX3CR1 promote the transformation of VSMC into osteogenic phenotype through ①activating JAK-STAT signaling pathway and up-regulating pro-calcification factor Runx2 expression, ②inhibiting anti-calcification factor OPG expression. The purpose of this research is to verify the effects of fractalkine/CX3CR1 in atherosclerotic intimal calcification and VSMC osteogenic phenotype transformation by gene engineering technology and ApoE-/-/CX3CR1-/- gene knockout mice; clarify the expressions of Runx2 and OPG in VSMC as well as their effect on VSMC osteogenic phenotype transformation under the action of fractalkine/CX3CR1; investigate the specific downstream signal transduction pathway under the action of fractalkine/CX3CR1 through inhibiting the expressions of CX3CR1, JAK and STAT. We intended to investigate the regulatory mechanism of VSMC osteogenic phenotype transformation in AS lesions and to seek a new target for AS prevention and treatment.

血管平滑肌细胞(VSMC)转化为成骨表型是动脉粥样硬化(AS)内膜钙化的重要环节，其具体机制不清。fractalkine及其受体CX3CR1诱导炎症细胞迁移，促进细胞因子释放，介导VSMC迁移及增殖，在AS病程中发挥重要作用。最近研究发现fractalkine/CX3CR1可促进骨前体细胞成熟分化。我们推测fractalkine/CX3CR1可能通过①激活JAK/STAT信号通路上调促钙化因子Runx2表达，②抑制抗钙化因子OPG表达，促进VSMC向成骨表型转化。为此本研究拟应用基因工程技术和ApoE-/-/CX3CR1-/-基因敲除鼠验证fractalkine/CX3CR1在VSMC成骨表型转化和AS内膜钙化中的作用，明确fractalkine/CX3CR1作用下上述分子表达变化及对VSMC表型转化的影响。本研究旨在探讨AS中VSMC成骨表型转化的调控机制，寻找新的AS防治干预靶点。

Fractalkine（FKN）是趋化因子CX3C亚类中的唯一成员，通过与受体CX3CR1特异性结合，募集炎症细胞、促进血管平滑肌细胞（VSMC）增殖迁移在动脉粥样硬化的病理过程中发挥重要作用，FKN/CX3CR1还可促进成骨前体细胞或成骨细胞成熟分化，但是其是否参与调节动脉粥样硬化性钙化尚不明确。我们的研究第一次观察了FKN/CX3CR1在VMSC成骨转化及动脉粥样硬化性钙化过程中的作用，并探讨了FKN/CX3CR1在VSMC成骨表型转化中的分子信号通路。本研究（1）构建ApoE和CX3CR1双基因缺陷小鼠（ApoE-/-/CX3CR1-/-），观察CX3CR1缺陷对高脂饮食诱导的动脉粥样硬化性钙化斑块形成的影响；检测小鼠主动脉VSMCs中FKN/CX3CR1的表达和成骨转化标志蛋白【骨桥蛋白（OPN）、骨形态发生蛋白（BMP-2）、碱性磷酸酶（ALP）】以及VSMCs标志蛋白【平滑肌α-肌动蛋白（α-SMA）、平滑肌22α蛋白（SM22α）】的表达变化，明确FKN/CX3CR1对VSMC成骨转化和动脉粥样硬化性钙化的影响。（2）体外培养VSMCs，观察FKN/CX3CR1孵育后，VSMCs中促钙化因子Runt相关转录因子2（RUNX2）以及抑钙化因子骨保护素（OPG）的表达情况；应用慢病毒转染的方法敲低或过表达VSMCs中CX3CR1水平，检测RUNX2、OPG以及成骨转化标志蛋白（OPN、BMP-2、ALP）的表达变化，探讨FKN/CX3CR1促进VSMC成骨转化的作用机制。（3）检测FKN/CX3CR1作用后VSMCs中Jak/Stat磷酸化水平，并检测Jak2抑制剂AG490作用后，相关钙化因子及骨转化标志蛋白表达变化情况，探讨FKN/CX3CR1促进VSMC成骨转化的信号通路。本研究结果发现FKN及其特异性受体CX3CR1在高脂诱导的动脉粥样硬化性钙化和VSMC成骨转化中起重要作用，其分子机制为激活Jak2/Stat3信号通路，上调促钙化因子RUNX2，同时抑制抗钙化蛋白OPG，继而促进RANK/RANKL及其他成骨标志蛋白的表达，最终促进VSMCs转化为成骨表型，促进动脉粥样硬化性钙化病灶的形成。FKN/CX3CR1作为动脉粥样硬化性钙化的关键调控分子，对其进行有效干预可能同时起到抑制炎症反应和动脉粥样硬化性钙化的作用，从而为防治动脉粥样硬化性钙化以及