Li Xiao-Fei, Wang Yan, Zheng Dong-Dong, Xu Hai-Xia, Wang Teng, Pan Min, Shi Jia-Hai, Zhu Jian-Hua
Department of Cardiology, Affiliated Hospital of Nantong University Nantong 226001, China.
Department of Cardio-Thoracic Surgery, Affiliated Hospital of Nantong University Nantong 226001, China.
Am J Transl Res. 2016 Dec 15;8(12):5773-5783. eCollection 2016.
The identification of the biological function of M1 macrophages and the mechanism underlying their role in valvular interstitial cell (VIC) calcification may provide therapeutic targets for the prevention of aortic valve calcification (AVC). This study investigated the mechanism by which M1 macrophages and macrophage-derived microvesicles (MVs) affected the calcification of VICs. An additional aim was to investigate the involvement of the miR-214 pathway in this process.
The M1 or M2 macrophage phenotype in human calcific aortic valve was confirmed by gene expression analysis of M1 or M2 macrophage markers. Two macrophage cell lines (BMDMs and RAW 264.7 macrophages) were transformed into M1 macrophages by lipopolysaccharide (LPS) stimulation. To investigate the mechanism by which M1 macrophages promoted VIC calcification, the generated M1 macrophages and macrophage-derived MVs were co-cultured with VICs and VICs were then used for calcification or signals analysis. In addition, a hypercholesterolemic apoE AVC murine model was used to evaluate the therapeutic efficacy of miR-214 specific-siRNA (miR-214 inhibitor).
Macrophages in calcific aortic valves showed M1-directed polarization. In the VICs co-cultured with LPS-stimulated M1 macrophages and macrophage-derived MVs, VIC calcification was enhanced, and the expression of TWIST1, a direct target of miR-214, was downregulated. We showed that knockdown of TWIST1 serves as a responding molecule for miR-214 and reversed the anti-calcification action of miR-214 inhibitor, mediating signal delivery by the M1 macrophage-derived MVs to VICs and promoting VIC calcification. When M1 macrophages co-cultured with VICs, TWIST1 overexpression in M1 macrophages had no effect on the expression of TWIST1 in VICs. As shown by intravenous therapy, knockdown of miR-214 in mice seemed to improve AVC in apoE mice with high-cholesterol (HC)-diet induced AVC.
These findings suggested that M1 macrophages promoted AVC by the delivery of miR-214 to valvular interstitial cells via macrophage-derived MVs and subsequent downregulation of TWIST1 of valvular interstitial cells.
确定M1巨噬细胞的生物学功能及其在瓣膜间质细胞（VIC）钙化中作用的潜在机制，可能为预防主动脉瓣钙化（AVC）提供治疗靶点。本研究探讨了M1巨噬细胞和巨噬细胞衍生的微泡（MVs）影响VICs钙化的机制。另一个目的是研究miR-214通路在此过程中的作用。
通过对M1或M2巨噬细胞标志物的基因表达分析，确认人钙化主动脉瓣中的M1或M2巨噬细胞表型。通过脂多糖（LPS）刺激将两种巨噬细胞系（骨髓来源的巨噬细胞（BMDMs）和RAW 264.7巨噬细胞）转化为M1巨噬细胞。为了研究M1巨噬细胞促进VIC钙化的机制，将生成的M1巨噬细胞和巨噬细胞衍生的MVs与VICs共培养，然后将VICs用于钙化或信号分析。此外，使用高胆固醇血症载脂蛋白E AVC小鼠模型评估miR-214特异性小干扰RNA（miR-214抑制剂）的治疗效果。
钙化主动脉瓣中的巨噬细胞表现出M1极化。在与LPS刺激的M1巨噬细胞和巨噬细胞衍生的MVs共培养的VICs中，VIC钙化增强，并且miR-214的直接靶标TWIST1的表达下调。我们表明，TWIST1的敲低作为miR-214的反应分子，逆转了miR-214抑制剂的抗钙化作用，介导M1巨噬细胞衍生的MVs向VICs的信号传递并促进VIC钙化。当M1巨噬细胞与VICs共培养时，M1巨噬细胞中TWIST1的过表达对VICs中TWIST1的表达没有影响。如静脉内治疗所示，小鼠中miR-214的敲低似乎改善了高胆固醇（HC）饮食诱导的apoE小鼠的AVC。
这些发现表明，M1巨噬细胞通过经由巨噬细胞衍生的MVs将miR-214递送至瓣膜间质细胞并随后下调瓣膜间质细胞的TWIST1来促进AVC。
