Mechanistic insights into folic acid-dependent vascular protection: Dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: A novel HPLC-based fluorescent assay for DHFR activity
ABSTRACT 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 μmol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 μmol/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 H4B and NO bioavailability while decreasing superoxide (O2−) 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 H4B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O2− 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.
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ABSTRACT: Hyperhomocysteinemia is an independent risk factor for coronary heart disease (CHD). Dietary supplementation with B vitamins lowers plasma homocysteine by up to 30%. However, little is known about the potential beneficial effects of homocysteine lowering on vascular function in patients with CHD. We investigated 89 men with CHD (aged 56 [range 39 to 67] years). Brachial artery flow-mediated dilatation (endothelium dependent) and nitroglycerin-induced dilatation (endothelium independent) were measured before and 8 weeks after treatment with either (1) folic acid (5 mg) and vitamin B(12) (1 mg) daily (n=59) or (2) placebo (n=30). Total, protein-bound, and free plasma homocysteine, serum folate, and vitamin B(12) were measured at baseline and at 8 weeks. Flow-mediated dilatation improved after treatment with B vitamins (2.5+/-3.2% to 4.0+/-3.7%, P:=0.002) but not placebo (2.3+/-2.6% to 1.9+/-2.6%, P:=0.5). Vitamin therapy lowered plasma concentrations of total homocysteine (from 13.0+/-3.4 to 9.3+/-1.9 micromol/L, P:<0.001), protein-bound homocysteine (from 8.7+/-2.8 to 6.2+/-1.4 micromol/L, P:<0.001), and free homocysteine (from 4.3+/-1.2 to 3.0+/-0.6 micromol/L, P:<0.001) and raised concentrations of serum folate (from 10.3+/-4.3 to 31.2+/-10.8 ng/mL, P:<0.001) and vitamin B(12) (from 314+/-102 to 661+/-297 pg/mL, P:<0.001). In regression analysis, improved flow-mediated dilatation correlated closely with the reduction in free plasma homocysteine (r=-0.26, P:=0.001), independent of changes in protein-bound homocysteine, folate, and vitamin B(12). Nitroglycerin-induced dilatation was unchanged after both B vitamins and placebo. Folic acid and vitamin B(12) supplementation improves vascular endothelial function in patients with CHD, and this effect is likely to be mediated through reduced concentrations of free plasma homocysteine concentrations. Our data support the view that lowering homocysteine, through B vitamin supplementation, may reduce cardiovascular risk.Circulation 11/2000; 102(20):2479-83. · 15.20 Impact Factor
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ABSTRACT: We conducted a systematic review of studies designed to increase awareness of knowledge about, and consumption of folic acid before and during pregnancy. Studies were identified from Cochrane Library, Medline, and the references of primary studies and reviews. Studies included randomized controlled trials, quasi-experimental interrupted time series studies, follow-up studies, case-control studies, and before-and-after studies, all of which were conducted between 1992 and 2005 on women ages 15 to 49 years and/or health professionals, evaluating awareness and/or knowledge and/or consumption of folic acid both before and after intervention. Studies were excluded if data were not presented both before and after intervention or were other outcomes than those mentioned here. Data were extracted in relation to characteristics of studies, participants, interventions, and outcomes. Because of heterogeneity, we performed a narrative synthesis describing the direction and the size of effects. On average, women's awareness increased from 60% to 72%, knowledge from 21% to 45%, and consumption from 14% to 23%. Interventions had a positive effect on folic acid intakes before and during pregnancy, although the average usage reached less than 25%.American journal of health promotion: AJHP 01/2007; 22(4):237-45. · 2.37 Impact Factor
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ABSTRACT: Previous in vitro experiments have shown that hyperhomocysteinemia leads to oxidative inactivation of nitric oxide, in part by inhibiting the expression of cellular glutathione peroxidase (GPx-1). To elucidate the role of intracellular redox status on homocysteine-induced endothelial dysfunction and oxidant stress, heterozygous cystathionine beta-synthase-deficient (CBS(-/+)) and wild-type (CBS(+/+)) mice were treated with the cysteine donor L-2-oxothiazolidine-4-carboxylic acid (OTC). CBS(-/+) mice had significantly lower GPx-1 activity compared with their CBS(+/+) littermates, and OTC treatment led to a modest increase in tissue GPx-1 activity and significant increases in total thiols and in reduced glutathione levels in both CBS(+/+) and CBS(-/+) mice. Superfusion of the mesentery with beta-methacholine or bradykinin produced dose-dependent vasodilation of mesenteric arterioles in CBS(+/+) mice and in CBS(+/+) mice treated with OTC. In contrast, mesenteric arterioles from CBS(-/+) mice manifested dose-dependent vasoconstriction in response to both agonists. OTC treatment of CBS(-/+) mice restored normal microvascular vasodilator reactivity to beta-methacholine and bradykinin. These findings demonstrate that mild hyperhomocysteinemia leads to endothelial dysfunction in association with decreased bioavailable nitric oxide. Increasing the cellular thiol and reduced glutathione pools and increasing GPx-1 activity restores endothelial function. These findings emphasize the importance of intracellular redox balance for nitric oxide bioactivity and endothelial function.Arteriosclerosis Thrombosis and Vascular Biology 02/2002; 22(1):34-41. · 6.34 Impact Factor