Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

Aims
Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.
Methods
Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18F-fluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia.
Results
The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.
Conclusion
Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.

研究目的：炎症及潜在的缺氧状态可显著影响动脉粥样硬化血管的葡萄糖利用，进而改变诸多代谢通路。但目前动脉粥样硬化斑块内的代谢变化仍不明确。本研究旨在明确兔动脉粥样硬化血管与体外培养巨噬细胞中，与葡萄糖摄取及缺氧相关的代谢系统变化。

研究方法：本研究通过球囊损伤法，在饲喂0.5%胆固醇饲料或常规饲料的兔髂股动脉中构建富含巨噬细胞或平滑肌细胞（smooth muscle cell, SMC）的新生内膜模型。将经脂多糖（lipopolysaccharides, LPS）与干扰素-γ（INFγ）刺激的THP-1巨噬细胞，分别置于常氧与缺氧环境下培养。采用毛细管电泳-飞行时间质谱（capillary electrophoresis-time of flight mass spectrometry）进行代谢组学分析，以全面评估动脉与巨噬细胞的代谢状态。同时使用18F-氟代脱氧葡萄糖（18F-fluorodeoxyglucose, 18F-FDG）与缺氧标志物匹莫尼达唑（pimonidazole），评估葡萄糖摄取情况及其与血管缺氧的相关性。

研究结果：与未损伤动脉及富含SMC的新生内膜动脉相比，富含巨噬细胞的新生内膜髂股动脉中多种代谢物水平显著升高（糖酵解通路：9种代谢物中4种上调；磷酸戊糖通路：6种代谢物中4种上调；三羧酸循环：6种代谢物中4种上调；核苷酸类：20种代谢物中10种上调）。通过放射自显影技术检测的动脉壁18F-FDG摄取量，与巨噬细胞免疫阳性区域及匹莫尼达唑免疫阳性区域均呈显著正相关（相关系数r分别为0.76与0.59；两组样本量n=69；p<0.0001）。在富含巨噬细胞的新生内膜中，匹莫尼达唑免疫反应活性与缺氧诱导因子-1α（hypoxia inducible factor-1α）的核转位及己糖激酶II（hexokinase II）的表达紧密共定位。在缺氧而非常氧条件下，经LPS与INFγ刺激的巨噬细胞中，糖酵解通路（8种代谢物全部上调）与磷酸戊糖通路（6种代谢物中4种上调）的代谢物水平显著升高。细胞上清液中的纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor-1）蛋白水平，与巨噬细胞的代谢通路密切相关。

研究结论：动脉粥样硬化血管中的浸润性巨噬细胞可能调控代谢系统，而缺氧（而非经典活化通路）可增强巨噬细胞的糖酵解与磷酸戊糖通路活性。