THE FOLIC ACID STUDY · Effects of folic acid and its active metabolite 5-methyl-tetrahydrofolic acid on endothelial function, superoxide production and gene expression in human atherosclerosis

Final Activity Report Summary - THE FOLIC ACID STUDY (Effects of folic acid and its active metabolite 5-methyl-tetrahydrofolic acid on endothelial function ... in human atherosclerosis)

In this project, we first examined the effect of 5-methyl-tetrahydrofolate (5-MTHF), the circulating form of folic acid, on nitric oxide (NO) bioavailability and vascular redox state in internal mammary arteries (IMA) and human saphenous veins (SV) obtained from patients with coronary artery disease during coronary artery bypass grafting operations (CABG). We used an ex-vivo system to examine the direct impact of 5-MTHF on vascular function, and we observed that indeed, 45 minutes of incubation with 5-MTHF improved NO bioavailability in SVs and IMAs. We also observed that 5-MTHF improved endothelial nitric oxide synthase (eNOS) 'coupling', and reduced the eNOS-derived superoxide radical generation. We also observed that 5-MTHF is a strong scavenger of peroxynitrite radicals, which are responsible for the oxidation of eNOS co-factor tetrahydrobiopterin (BH4), in human vascular endothelium, and it increases in this way vascular BH4, improving eNOS coupling. To examine the in-vivo effect of 5-MTHF, we randomised 56 patients to receive intravenous infusion of 5-MTHF or placebo before CABG. Intravenous infusion of 5-MTHF improved NO bioavailability, improved eNOS coupling and reduced vascular superoxide in SVs and IMAs. This effect was via improvement of vascular BH4 bioavailability (published in Circulation 2006; 114:1193-1201 (Impact factor = 10.9)). To further examine the real clinical impact of folates on vascular function we randomised 56 patients to receive folic acid 5 mg/d or 400 µg/d (Recommended Daily Allowance-RDA) or placebo for 6 weeks before CABG. We observed that both high- and low- dose folic acid equally improved BH4 bioavailability in SVs and IMAs, and similarly improved vascular NO, decreased superoxide generation and improved eNOS coupling. Despite the greater circulating plasma 5-MTHF in the 5 mg/d-treated compared to 400 µg/d group, vascular 5-MTHF was similar in both groups. These findings suggested that the RDA of folic acid induces the maximum benefit on vascular function, reaching its maximum antiatherogenic capacity, while any additional treatment on top of that offers no additional benefit (published in Circulation 2007; 115(17):2262-70 (Impact factor = 10.9)). In this project, we also examined whether vascular BH4 is a determinant of eNOS coupling, superoxide generation and NO bioavailability in human arteries and veins. In 219 patients undergoing CABG, we determined plasma and vascular BH4 (and total biopterins), and we examined their impact on vascular function. We observed that vascular BH4 was a key determinant of eNOS coupling in human vessels, being positively correlated with vascular NO bioavailability and inverselly associated with vascular superoxide generation. On the contrary, plasma total biopterins were inversely associated with vascular biopterins and vascular NO bioavailability and positively associated with superoxide generation. In addition, plasma (but not vascular) biopterins were positively associated with plasma C-reactive protein levels (a marker of systemic inflammation), suggesting that plasma biopterins are a marker or inflammation but not a marker of vascular health (published in Circulation 2007; 2007;116: (in press) (Impact factor = 10.9). In addition, we examined the role of eNOS inhibitor asymmetrical dimethylarginine (ADMA) on vascular NO bioavailability and vascular redox state. We observed that plasma ADMA measured in 201 patients undergoing CABG, was inversely associated with eNOS coupling and NO bioavailability in SVs and IMAs, while it was positively associated with vascular superoxide generation. These findings suggests that ADMA may induce eNOS uncoupling in human vessels, and it could be used as a possible therapeutic target against atherosclerosis (This study arm received the first prize in the Young Investigators Award competition of European Society of Cardiology 2007, and it will be submitted for publication in European Heart Journal, as an invited manuscript).

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