Thornton C C, Al-Rashed F, Calay D, Birdsey G M, Bauer A, Mylroie H, Morley B J, Randi A M, Haskard D O, Boyle J J, Mason J C
Vascular Sciences, Imperial Centre for Translational and Experimental Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Hospital, London, UK.
Vascular Sciences, Imperial Centre for Translational and Experimental Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Hospital, London, UK King Fahad Cardiac Centre, King Saud University, Riyadh, Saudi Arabia.
Ann Rheum Dis. 2016 Feb;75(2):439-48. doi: 10.1136/annrheumdis-2014-206305. Epub 2015 Jan 9.
Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes.
METHODS/RESULTS: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKα(Thr172), and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)(Ser133) phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKα(Thr172) phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression.
These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.
过早发生的心血管事件使慢性炎症性疾病复杂化。低剂量每周一次甲氨蝶呤（MTX）是类风湿关节炎（RA）中使用最广泛的改善病情药物，可降低疾病相关的心血管死亡率。MTX增加细胞内单磷酸腺苷（AMP）和5-氨基咪唑-4-甲酰胺核苷酸的积累，从而激活AMP激活的蛋白激酶（AMPK）。我们假设MTX通过诱导AMPK调节的保护基因来特异性保护血管内皮免受炎症损伤。
方法/结果：在炎症性血管病的（NZW×BXSB）F1小鼠模型中，每周1 mg/kg的MTX显著减少心肌内血管病并减轻终末器官损伤。对人脐静脉内皮细胞（HUVEC）和动脉内皮细胞（HAEC）的研究表明，治疗相关浓度的MTX使AMPKα（Thr172）磷酸化，并诱导包括锰超氧化物歧化酶（MnSOD）和血红素加氧酶-1（HO-1）在内的细胞保护基因。当用肿瘤坏死因子-α预处理HUVEC以模拟功能失调的内皮时，这些反应得以保留。此外，MTX可防止葡萄糖剥夺诱导的内皮细胞凋亡。从机制上讲，MTX处理导致环磷酸腺苷反应元件结合蛋白（CREB）（Ser133）磷酸化，而AMPK耗竭减弱了这种反应以及MnSOD和HO-1的诱导。CREB siRNA抑制MTX对两种细胞保护基因的上调，而染色质免疫沉淀表明MTX处理的内皮细胞中CREB与MnSOD启动子结合。同样，用MTX处理（NZW×BXSB）F1小鼠可增强AMPKα（Thr172）磷酸化和MnSOD，并降低主动脉细胞间黏附分子-1的表达。
这些数据表明MTX通过激活AMPK-CREB来治疗性调节血管内皮。我们认为这种机制有助于保护接受MTX治疗的RA患者免受心血管事件的影响。
