Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China.
J Thorac Cardiovasc Surg. 2017 Jun;153(6):1318-1327.e1. doi: 10.1016/j.jtcvs.2017.01.052. Epub 2017 Feb 9.
The purpose of the present study was to comprehensively compare the phenotype profile of infiltrated macrophages in human noncalcified and calcific aortic valves, and to determine whether the shift of macrophage polarization modulates valvular calcification in vitro.
Cell surface markers of macrophages and inflammatory cytokines expression in 90 cases of human noncalcified and calcific aortic valve leaflets were analyzed. The normal aortic valve interstitial cells were isolated and cultured in vitro. After incubation with nonconditioned medium and conditioned medium from unstimulated or lipopolysaccharide-stimulated U937 monocytes, valve interstitial cells were evaluated by osteogenic differentiation markers.
Infiltration of macrophages was enhanced in the calcific aortic valves, and M1 phenotype was the predominant macrophage subsets. In addition, both proinflammatory and anti-inflammatory cytokines were significantly upregulated in the calcific aortic valves. Furthermore, lipopolysaccharide-stimulated monocytes presented with increased expression of inducible nitric oxide synthase and high proportional CD11c-positive (M1) macrophages. Conditioned medium from unstimulated monocytes promoted the osteogenic differentiation of valve interstitial cells in vitro, as evidenced by increased markers such as bone morphogenetic protein 2, osteopontin, and alkaline phosphatase. Conditioned medium from M1 macrophages further enhanced valve interstitial cells calcification. Enzyme-linked immunosorbent assay showed that M1 phenotype macrophages secreted tumor necrosis factors α and interleukin 6, and neutralizing antibodies to these 2 proinflammatory cytokines attenuated induction of osteogenic differentiation and calcification by the conditioned media.
Both total numbers and polarization of macrophage influence the process of calcification in human aortic valve. The shift toward M1 phenotype might promote valve interstitial cell calcification.
本研究旨在全面比较人未钙化和钙化主动脉瓣浸润巨噬细胞的表型谱，并确定巨噬细胞极化状态的转变是否在体外调节瓣膜钙化。
分析 90 例人未钙化和钙化主动脉瓣叶中巨噬细胞的细胞表面标志物和炎症细胞因子表达。从体外分离和培养正常主动脉瓣膜间质细胞。用未条件培养基和未刺激或脂多糖刺激的 U937 单核细胞的条件培养基孵育后，用成骨分化标志物评估瓣膜间质细胞。
在钙化主动脉瓣中，巨噬细胞浸润增强，M1 表型是主要的巨噬细胞亚群。此外，在钙化主动脉瓣中，促炎和抗炎细胞因子均显著上调。此外，脂多糖刺激的单核细胞表现出诱导型一氧化氮合酶表达增加和高比例 CD11c 阳性（M1）巨噬细胞。未刺激单核细胞的条件培养基在体外促进瓣膜间质细胞的成骨分化，表现为骨形态发生蛋白 2、骨桥蛋白和碱性磷酸酶等标志物增加。M1 表型巨噬细胞的条件培养基进一步增强了瓣膜间质细胞的钙化。酶联免疫吸附试验显示，M1 表型巨噬细胞分泌肿瘤坏死因子 α 和白细胞介素 6，中和这 2 种促炎细胞因子的抗体可减弱条件培养基诱导的成骨分化和钙化。
巨噬细胞的总数和极化状态都影响人主动脉瓣的钙化过程。向 M1 表型的转变可能促进瓣膜间质细胞钙化。
