Division of Molecular Medicine, Cardiovascular Research Laboratories (CVRL), The Departments of Anesthesiology and Medicine, UCLA David Geffen School of Medicine, 650 Charles E Young Drive, BH550, Los Angeles, CA, 90095, USA.
J Mol Cell Cardiol. 2009 Dec;47(6):752-60. doi: 10.1016/j.yjmcc.2009.07.025. Epub 2009 Aug 3.
Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious. Expression of dihydrofolate reductase (DHFR) was markedly increased by folic acid (FA, 50 micromol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 micromol/L of substrate dihydrofolic acid. THF could then be detected and quantified by high performance liquid chromatography (HPLC) with a fluorescent detector (295/365 nm). Using this novel and sensitive assay, we found that DHFR activity was significantly increased by FA. Furthermore, FA improved redox status of Ang II treated cells by increasing H(4)B and NO() bioavailability while decreasing superoxide (O(2)(-)) production. It however failed to restore NO() levels in DHFR siRNA-transfected or methotrexate pre-treated cells, implicating a specific and intermediate role of DHFR. In mice orally administrated with FA (15 mg/kg/day, 16 days), endothelial upregulation of DHFR expression and activity occurred in correspondence to improved NO() and H(4)B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O(2)(-) production. 5'-methyltetrahydrofolate (5'-MTHF) levels, GTPCH1 expression and activity remained unchanged in response to FA or Ang II treatment in vitro and in vivo. FA supplementation improves endothelial NO() bioavailability via upregulation of DHFR expression and activity, and protects endothelial cells from Ang II-provoked oxidant stress both in vitro and in vivo. These observations likely represent a novel mechanism (intermediate role of DHFR) whereby FA induces vascular protection.
叶酸补充可改善高同型半胱氨酸血症患者的血管内皮功能。然而，一般人群中叶酸对血管功能潜在益处的机制仍不清楚。叶酸（FA，50μmol/L，24 小时）处理可显著增加内皮细胞中二氢叶酸还原酶（DHFR）的表达。将纯化的 DHFR 或细胞提取物与 50μmol/L 的底物二氢叶酸孵育后，可形成四氢叶酸（THF）。然后可以通过高效液相色谱（HPLC）和荧光检测器（295/365nm）检测和定量 THF。使用这种新颖而灵敏的测定法，我们发现 FA 可显著增加 DHFR 活性。此外，FA 通过增加 H（4）B 和 NO（）的生物利用度，同时减少超氧化物（O（2）（-））的产生，改善了 Ang II 处理细胞的氧化还原状态。然而，它未能恢复 DHFR siRNA 转染或甲氨蝶呤预处理细胞中的 NO（）水平，表明 DHFR 具有特定和中间作用。在口服给予 FA（15mg/kg/天，16 天）的小鼠中，DHFR 表达和活性的内皮上调与改善的 NO（）和 H（4）B 生物利用度相对应，这在很大程度上有效降低了 Ang II 输注（0.7mg/kg/天，14 天）刺激的主动脉 O（2）（-）产生。5'-甲基四氢叶酸（5'-MTHF）水平、GTPCH1 表达和活性在体外和体内对 FA 或 Ang II 处理均无反应。FA 补充通过上调 DHFR 表达和活性来改善内皮细胞的 NO（）生物利用度，并在体外和体内保护内皮细胞免受 Ang II 引起的氧化应激。这些观察结果可能代表了一种新的机制（DHFR 的中间作用），即 FA 诱导血管保护。
