The effects of folic acid on endothelial dysfunction

Research has shown that folic acid can have a beneficial effect on the vascular endothelium by reducing plasma homocysteine levels⁽Reference Wilmink, Stroes and Erkelens¹³⁾. This effect is a result of the role of folic acid during the methylation of homocysteine to methionine⁽Reference Malinow, Bostom and Krauss²⁰⁾. Homocysteine has several detrimental effects on the vascular endothelium, which include: increasing platelet aggregation and thrombosis⁽Reference Brown and Hu⁴⁾; increasing oxidative stress by increasing superoxide production⁽Reference McDowell and Lang²¹⁾; down regulating NO production⁽Reference Pruefer, Scalia and Lefer²²⁾; increasing vascular smooth muscle proliferation⁽Reference Tsai, Perrella and Yoshizumi²³⁾; increasing leucocyte–endothelium interactions⁽Reference Dudman, Temple and Guo²⁴⁾.

There have been several clinical studies that have investigated the effects of folic acid supplementation on endothelial function. It has been reported that when subjects who are mildly hyperhomocysteinaemic are supplemented with folic acid (5 mg/d) and pyridoxine (250 mg/d) over a 12-week period a decrease is found in both plasma von Willebrand factor and thrombomodulin⁽Reference Van den Berg, Boers and Franken²⁵⁾. Another study in which subjects received folic acid (5 mg/d) chose to investigate forty subjects with familial hypercholesterolaemia⁽Reference Verhaar, Wever and Kastelein²⁶⁾. After 4 weeks it was found that folic acid supplementation restores endothelium-dependent vasodilation, as assessed by serotonin-induced forearm blood flow measurement. In a further intervention study eighteen subjects with hyperhomocysteinaemia were given 5 mg folic acid daily during a 6-week intervention period⁽Reference Bellamy, McDowell and Ramsey²⁷⁾. Flow-mediated vasodilation of the brachial artery was measured and it was found that supplementation improves endothelium-dependent vasodilation.

Two intervention studies of folic acid supplementation have been reported that used healthy subjects⁽Reference Wilmink, Stroes and Erkelens^13, Reference Woo, Chook and Lolin²⁸⁾. Both studies used 10 mg folic acid/d or placebo and measured the outcome by flow-mediated vasodilation of the brachial artery. One study comprising seventeen subjects with an intervention period of 8 weeks has shown an improvement in endothelium-dependent vasodilation⁽Reference Woo, Chook and Lolin²⁸⁾. The other study of twenty subjects who received the supplement for a 2-week period carried out vasomotion measurements on each subject before and 4 h after an oral fat load (50 g, in the form of whipped cream)⁽Reference Wilmink, Stroes and Erkelens¹³⁾. It was found that folic acid supplementation prevents vasodilation impairment after a fat load.

These supplementation studies suggest that folic acid has a beneficial effect on endothelial function, as measured by haemostatic markers or flow-mediated vasodilation. Although it is suggested that the observations are a result of reduced homocysteine concentrations, other mechanisms may also contribute to the effects.

The effects of vitamins C and E on endothelial dysfunction

Endothelial function may be modulated by antioxidant vitamins such as vitamin C and vitamin E. Animal experiments have shown that one form of vitamin E, α-tocopherol, preserves NO-mediated vascular relaxation in subjects with hypercholesterolaemia⁽Reference Andersson, Matz and Ferns^29, Reference Stewartlee, Forster and Nouroozzadeh³⁰⁾. In vitro studies with antioxidant vitamins have shown that vitamin E reduces cell adhesion molecule expression⁽Reference Wu, Koga and Martin³¹⁾ and monocyte adhesion to the endothelium⁽Reference Faruqi, Delamotte and Dicorleto³²⁾ and vitamin C leads to improvement in endothelium-dependent vasodilation⁽Reference Fontana, Mcneill and Ritter³³⁾.

A number of clinical studies have investigated the effects of antioxidant supplementation, in the form of vitamins C or E, on markers of endothelial activation or endothelium-dependent vasodilation. These studies have found a decrease in monocyte adhesion with both vitamin C⁽Reference Weber, Erl and Weber³⁴⁾ and vitamin E⁽Reference Devaraj, Li and Jialal¹⁴⁾, and plasma P-selectin is reduced in subjects with hypercholesterolaemia following α-tocopherol supplementation⁽Reference Davi, Romano and Mezzetti³⁵⁾. It has been reported that infusion with vitamin C leads to an increase in forearm blood flow of 1·4 ml/100 ml per min in subjects after a 6 h hyperglycaemic clamp⁽Reference Beckman, Goldfine and Gordon³⁶⁾. Vitamin C supplementation has been shown to improve endothelium-dependent vasodilation, measured by flow-mediated dilation, from 4·2 (se 0·7) % to 9·1 (se 1·3) % in subjects with chronic heart failure⁽Reference Hornig, Arakawa and Kohler³⁷⁾ and from 6·6 (sd 3·5) % to 10·1 (sd 5·2) % in subjects with coronary artery disease⁽Reference Gokce, Keaney and Frei³⁸⁾. In healthy subjects in whom hyperhomocysteinaemia has been induced with an oral methionine load the reduction in flow-mediated dilation can be ameliorated by pre-treatment with vitamin C supplementation of 1000 mg/d for 1 week⁽Reference Chambers, McGregor and Jean-Marie¹⁵⁾. In smokers supplemented with vitamin E (545 mg all-racemic α-tocopherol/d) there is an improvement in flow-mediated vasodilation of 5·8 (sd 3·2) % for the supplemented group v. 2·7 (sd 2·8) % for the placebo group⁽Reference Neunteufl, Priglinger and Heher³⁹⁾.

Dilation of blood vessels occurs when endothelial cells generate NO⁽Reference Brown and Hu⁴⁾. This molecule can react with superoxide generated by endothelial cells during cytoplasmic and mitochondrial metabolism to form peroxynitrite, which is destructive to cells⁽Reference Ischiropoulos⁴⁰⁾. Antioxidants could therefore decrease this consumption of NO and render it bioavailable for vasodilation⁽Reference Bendich, Machlin and Scandurra⁴¹⁾. However, it has been found that high physiological concentrations of vitamin C (>1 mM) are needed to effectively scavenge superoxide and enhance endothelium-dependent vasodilation⁽Reference Jackson, Xu and Vita⁴²⁾. Vitamin C could augment delivery of NO from the plasma to the vascular cells⁽Reference May⁴³⁾. NO can be transported as S-nitrosothiols on albumin or free cysteine⁽Reference Keaney, Simon and Stamler⁴⁴⁾ and vitamin C can release NO from S-nitrosothiols and S-nitrosoalbumin⁽Reference Scorza, Pietraforte and Minetti⁴⁵⁾. However, as most of the free NO is likely to be scavenged by Hb and erythrocytes⁽Reference Jia, Bonaventura and Bonaventura^46, Reference Patel, Hogg and Spencer⁴⁷⁾ further investigations are required to determine whether sufficient NO can be released by this mechanism to promote vasodilation. Another mechanism whereby vitamin C could increase vasodilation is by regulating endothelial NO synthase (eNOS) by increasing the affinity of eNOS for tetrahydrobiopterin⁽Reference Heller, Munscher-Paulig and Grabner⁴⁸⁾. This outcome could be achieved by preserving crucial thiol groups on eNOS, which are needed in order to bind tetrahydrobiopterin⁽Reference Hofmann and Schmidt⁴⁹⁾, or by reducing the redox cycling of tetrahydrobiopterin by decreasing cellular superoxide or peroxynitrite⁽Reference Kojima, Ona and Iizuka⁵⁰⁾.

Overall, the studies generally show a supportive role for antioxidant vitamins C and E in preserving endothelium-dependent vasodilation. This effect is particularly evident in subjects with cardiovascular risk factors such as hyperlipidaemia or in patients with diabetes or established CVD.

The effects of carotenoids on endothelial dysfunction

The addition of tomato to the diet has been reported to have a protective effect on acetylcholine-induced vasorelaxation in hypercholesterolaemic mice fed on an atherogenic diet⁽Reference Suganuma and Inakuma¹⁸⁾. Mice fed a diet high in saturated fat and cholesterol for 4 months were found to have an increased plasma lipid peroxide level and a decreased vasorelaxing activity in the aorta induced by acetylcholine compared with mice on a commercial diet. However, when 20% (w/w) powdered tomato was added to the atherogenic diet a lesser increase in the plasma lipid peroxide level was found and acetylcholine-induced vasorelaxation was maintained at the same level as that for normal mice. Although it was concluded that tomato has a preventive effect on atherosclerosis by protecting plasma lipids from oxidation, the nature of the protective factor in the tomato could not be determined. However, it was noted that tomatoes contain a relatively large amount of the carotenoid lycopene and that the tomato powder used in the study contained 1·6 mg lycopene/g. Based on a report that indicates that lycopene supplementation inhibits singlet-oxygen-mediated oxidation of human plasma LDL⁽Reference Oshima, Ojima and Sakamoto⁵¹⁾ and the finding that the uptake of tomato reduces the increase in plasma lipid peroxide in hypercholesterolaemic mice, it was suggested that lycopene might act as an antioxidant in plasma⁽Reference Suganuma and Inakuma¹⁸⁾. However, plasma lycopene was not measured in the mouse study and it was noted that carotenoids are thought to be less absorbed by rodents than by human subjects. Thus, it was concluded that the findings strongly suggest that dietary ingestion of tomato inhibits the oxidative modification of serum lipids and subsequently reduces endothelial dysfunction⁽Reference Suganuma and Inakuma¹⁸⁾.

The evidence in support of a preventative function of lycopene in CVD primarily originates from epidemiological observations in normal and at-risk populations. The most powerful population-based evidence has been derived from a European multicentre case–control study⁽Reference Kohlmeier, Kark and Gomezgracia⁵²⁾. This study investigated the relationship between acute myocardial infarction and adipose tissue antioxidant status. Levels of α- and β-carotenes, lycopene and α-tocopherol were measured in adipose tissue collected shortly after the infarction. However, after adjustment for age, BMI, socio-economic status, smoking, hypertension and family history of the disease, only lycopene levels were found to be protective. The results show a dose–response relationship between each quintile of adipose tissue lycopene content and the risk of myocardial infarction.

Although the European multicentre case–control study observed no protective effect of β-carotene or α-tocopherol on myocardial infarction⁽Reference Kohlmeier, Kark and Gomezgracia⁵²⁾, other research has shown that these lipophilic antioxidants preserve endothelium-dependent relaxation in cholesterol-fed rabbits⁽Reference Keaney, Gaziano and Xu⁵³⁾. Male rabbits were divided into four groups; one group was fed a commercial diet, another group received the commercial diet with 1% (w/w) cholesterol added and the final two groups received the 1% (w/w) cholesterol diet with either β-carotene (0·6 g/kg chow) or α-tocopherol (1000 mg α-tocopheryl acetate/kg chow). After 28 d on the diets thoracic aortas were collected and analysed for vascular function and tissue antioxidant levels. Acetylcholine-induced vasorelaxation was found to be significantly impaired in vessels from the cholesterol group (P<0·001), whereas vessels from the animals supplemented with β-carotene or α-tocopherol displayed normal arterial relaxation. Preservation of vasorelaxation was found to be associated with vascular incorporation of β-carotene and α-tocopherol, but unrelated to plasma lipoprotein levels, smooth muscle cell function or the extent of atherosclerosis. Increased LDL resistance to ex vivo Cu-mediated oxidation was only found in the α-tocopherol group. As dysfunction in endothelium-dependent vasorelaxation is associated with risk of CVD⁽Reference Anderson, Uehata and Gerhard¹⁰⁾, this research may suggest that the benefit of diets containing carotenoids on this risk factor may be through vascular tissue antioxidant content and not associated with the resistance of LDL to ex vivo oxidation.

The pro-vitamin A activity of β-carotene is well known⁽Reference Dimascio, Murphy and Sies⁵⁴⁾ and vitamin A derivatives, particularly all-trans-retinoic acid (atRA), have been shown to inhibit cellular proliferation and promote cellular differentiation⁽Reference Achan, Tran and Arrigoni⁵⁵⁾. In the latter study it was found that atRA increases NO synthesis by endothelial cells. Murine endothelioma cells treated with or without atRA were found to show a maximum increase in nitrite production 24 h after stimulation with atRA. eNOS mRNA expression was not found to increase after treatment with atRA and there was no detectable eNOS or inducible NO synthase mRNA in the cells. The effect of atRA on the induction of the dimethylarginine dimethylaminohydrolase enzymes that regulate asymmetric dimethylarginine metabolism was also investigated. Dimethylarginine is an endogenous competitive inhibitor of NOS and dimethylarginine dimethylaminohydrolase II has been shown to be highly expressed in cardiovascular tissues and to have a distribution similar to eNOS⁽Reference Leiper, Maria and Chubb⁵⁶⁾. It was found that dimethylarginine dimethylaminohydrolase II expression is induced by atRA, with the maximum increase occurring 12 h after stimulation⁽Reference Achan, Tran and Arrigoni⁵⁵⁾. This effect was also observed in primary porcine aortic endothelial cells and the human cell lines SGHEC-7 (SV40 transfected human umbilical vein endothelial cells) and ECV304. As raised levels of dimethylarginine have been shown to be associated with endothelial dysfunction and atherosclerosis⁽Reference Miyazaki, Matsuoka and Cooke^57, Reference Boger, Bode-Boger and Szuba⁵⁸⁾ it was suggested that induction of dimethylarginine dimethylaminohydrolase II by atRA may lower dimethylarginine levels and consequently restore NO production.

These studies support the epidemiological findings that carotenoid-containing foods may be beneficial in reducing the risk of CVD⁽Reference Cherubini, Vigna and Zuliani⁵⁹⁾. However, they suggest that their mode of action may not be entirely a result of their antioxidant properties.

The effects of polyphenols on endothelial dysfunction

In 1993 it was reported that grape juice and grape-skin extracts cause endothelium-dependent vasorelaxation⁽Reference Fitzpatrick, Hirschfield and Coffey⁶⁰⁾. The study focused on the effect of certain wines, grape juices and grape-skin extracts on precontracted smooth muscle of rat aortic rings. Relaxation was found in intact aortic rings, but no effect was observed on aortas in which the endothelium had been removed. The polyphenolic compounds quercetin and tannic acid were found to produce endothelium-dependent relaxation. The grape extracts were found to increase cGMP levels in the intact vascular tissue. Both relaxation and the increase in cGMP were reversed by NG-monomethyl-l-arginine and NG-nitro-l-arginine, which are competitive inhibitors of NO synthesis. These findings suggest that grape-product-induced vasorelaxation is mediated by the NO–cGMP pathway. To further investigate the effect of various plant extracts on endothelium-dependent vasorelaxation aqueous extracts of numerous fruits, vegetables, nuts, herbs, spices and teas were studied⁽Reference Fitzpatrick, Hirschfield and Ricci⁶¹⁾. It was found that many of, but not all, these extracts exhibit endothelium-dependent relaxation that is again reversed in the presence of a NO synthase inhibitor.

Following on from the initial work with grape products performed on rat aorta⁽Reference Fitzpatrick, Hirschfield and Coffey^60, Reference Fitzpatrick, Hirschfield and Ricci⁶¹⁾, other studies on various isolated animal and human blood vessels have reached similar conclusions⁽Reference Stoclet, Chataigneau and Ndiaye⁶²⁾. These studies used a variety of plant polyphenol sources such as cocoa, tea, hawthorn (Crataegus monogyna and Crataegus oxyacantha fruit), honey and pine (Pinus maritima) bark. All these studies have found that plant polyphenols produce endothelium-dependent vasorelaxation that is associated with increased cGMP formation and reduced by NO synthase inhibitors.

Structure–activity (endothelial NO release and relaxation of isolated blood vessels) relationships for different classes of polyphenols have been examined⁽Reference Andriambeloson, Magnier and Haan-Archipoff^16, Reference Taubert, Berkels and Klaus¹⁷⁾. The most active fractions in red wine were found to be flavan-3-ol-enriched oligomeric condensed tannins, particularly dimers and trimers, and the most active monomer was the anthocyanin delphinidin⁽Reference Andriambeloson, Magnier and Haan-Archipoff¹⁶⁾. Surprisingly, structurally-similar anthocyanins, such as malvidin and cyanidin, were not found to be active. However, substitution of the flavan moiety with free hydroxyl residues at precise locations has been found to be important to induce endothelial NO release⁽Reference Taubert, Berkels and Klaus¹⁷⁾.

The mechanisms of eNOS activation in response to circulating hormones, local autacoids and substances released by platelets, by the coagulation cascade and by the autonomic nervous system involve intracellular Ca^{2+ (}Reference Dimmeler, Fleming and Fisslthaler⁶³⁾. This role of Ca²⁺ is supported by the relaxation of rat aorta⁽Reference Andriambeloson, Stoclet and Andriantsitohaina⁶⁴⁾ and the increase in Ca²⁺ concentration in bovine aortic endothelial cells⁽Reference Stoclet, Kleschyov and Andriambeloson⁶⁵⁾ caused by a polyphenolic extract from alcohol-free red wine and by delphinidin, which are both Ca²⁺ dependent effects. Furthermore, wine polyphenols are unable to increase Ca²⁺ concentration in smooth muscle cells, indicating the selectivity of this effect⁽Reference Stoclet, Kleschyov and Andriambeloson⁶⁵⁾.

Long-term incubation of endothelial cells with red wine increases eNOS expression⁽Reference Wallerath, Deckert and Ternes^66, Reference Wallerath, Poleo and Li⁶⁷⁾. This effect is only achieved, however, with large volumes of red wine (1% (v/v) in culture medium for 10 d, 3% (v/v) for 24 h and 10% (v/v) for 12 h)⁽Reference Wallerath, Poleo and Li⁶⁷⁾. Resveratrol, also a polyphenolic component of red wine, stimulates eNOS expression in the concentration range of 10–100 μM, as well as increasing the activity of the eNOS promoter and stabilising eNOS mRNA⁽Reference Wallerath, Deckert and Ternes⁶⁶⁾.

In a study of the incorporation of elderberry (fruit of elder; Sambucus nigra) anthocyanins by bovine and human aortic endothelial cells treated with an elderberry extract (1 mg/ml) and incubated for 1, 2, 4, 6, 8, 16 and 24 h, maximum incorporation was found after 4 h⁽Reference Youdim, Martin and Joseph⁶⁸⁾. By separation of the cell membranes and cytosol it was demonstrated that the anthocyanins were incorporated into the plasma membrane as well as penetrating into the cell cytosol. Uptake into both regions was found to be structure dependent, with monoglycoside concentrations higher than those of the diglucosides. The incorporation of elderberry anthocyanins was found to be protective against H₂O₂, 2,2′-azobis(2-amidinopropane) dihydrochloride and FeSO₄–ascorbic acid.

A protective effect of blackberry (Rubus fruticosus) anthocyanins, notably cyanidin-3-O-glucoside, on endothelial cells has also been reported⁽Reference Serraino, Dugo and Dugo⁶⁹⁾. In this study the effect of peroxynitrite on human umbilical vein endothelial cells incubated with differing concentrations of blackberry juice was investigated. It was found that blackberry juice reduces the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and poly (ADP-ribose) synthetase activation in human umbilical vein endothelial cells. It was also reported that vascular rings exposed to peroxynitrite demonstrate reduced endothelium-dependent relaxant responses to acetylcholine, which is improved by the blackberry juice.

In addition to research with endothelial cells, polyphenols have also been incubated with platelets⁽Reference Freedman, Parker and Li⁷⁰⁾. Incubation of platelets with diluted purple-grape juice leads to inhibition of aggregation, enhanced release of platelet-derived NO and decreased superoxide production⁽Reference Freedman, Parker and Li⁷⁰⁾. In order to investigate these effects in vivo, an intervention trial was conducted in which twenty healthy subjects consumed 7 ml purple-grape juice/kg daily for 14 d. Following the intervention platelet aggregation was found to be inhibited, platelet-derived NO production increased from 3·5 pmol/10⁸ platelets to 6·0 pmol/10⁸ platelets and superoxide release decreased from 29·5 arbitrary units to 19·2 arbitrary units. It was also reported that α-tocopherol levels significantly increase and plasma protein-independent antioxidant activity increases by 50%.

Other intervention trials have investigated the effect of a range of polyphenols on endothelial dysfunction⁽Reference Whelan, Sutherland and Mccormick^71, Reference Duffy, Keaney and Holbrook⁷²⁾. In a study of the effects of white and red wines on the endothelial function of fourteen subjects with coronary artery disease 4 ml wine/kg was administered alongside a controlled meal low in antioxidants⁽Reference Whelan, Sutherland and Mccormick⁷¹⁾. Measurement of flow-mediated dilation of the brachial artery indicated an improvement in dilation after 360 min, although no difference was found between the two types of wine. Consumption of an isoenergetic cordial was not found to improve dilation. No difference in plasma polyphenols was observed in blood samples taken at baseline, 60 min and 360 min after consumption of either type of wine.

In another study of patients with coronary artery disease fifty patients with proven coronary artery disease underwent an intervention trial of black tea consumption⁽Reference Duffy, Keaney and Holbrook⁷²⁾. Short-term effects were measured by examining the subjects 2 h after consumption of 450 ml tea or water and long-term effects were measured after consumption of 900 ml tea or water daily for 4 weeks. Measurements of flow-mediated vasodilation of the brachial artery showed improvement with both the short- and long-term tea consumption, whereas water was found to have no effect. Administration of an equivalent oral dose of caffeine was not found to have any short-term effect on flow-mediated vasodilation. Plasma concentrations of flavonoids were shown to increase after both the short- and long-term tea consumption. Although tea contains antioxidant flavonoids, studies have shown that tea consumption has no effect on plasma antioxidant capacity, plasma concentrations of F₂-isoprostanes, markers of systemic lipid peroxidation or 8-hydroxydeoxyguanosine⁽Reference Vita⁷³⁾.

Many fruits and vegetables contain polyphenolic flavonoid compounds⁽Reference Tomas-Barberen and Clifford^74, Reference Hollman and Arts⁷⁵⁾, some of which have been shown to increase the activity of the eNOS enzyme⁽Reference Schroeter, Heiss and Balzer⁷⁶⁾. eNOS is responsible for the generation of the vasodilator NO from l-arginine in the vascular endothelium⁽Reference Moncada and Higgs⁷⁷⁾. A common single-nucleotide polymorphism (G298T) occurs in the eNOS gene that modifies its coding sequence, replacing a glutamate residue at position 298 with an aspartate residue, and this polymorphism has been linked to increased risk of cardiovascular events⁽Reference Hingorani, Liang and Fatibene^78–Reference Hibi, Ishigami and Tamura⁸²⁾.

Two studies have been conducted to investigate the effects of chronic and acute consumption of commercially-available fruit and vegetable puree-based drinks (FVPD) on vasodilation, phytochemical bioavailability and antioxidant status in healthy individuals aged between 30 and 70 years (TW George, C Niwat, S Waroonphan, MH Gordon and JA Lovegrove, unpublished results; TW George, E Paterson, S Waroonphan, MH Gordon and JA Lovegrove, unpublished results). FVPD were chosen to provide a convenient source of fruit and vegetable nutrients and phytochemicals such as vitamin C and flavonoids. FVPD enabled all the participants to receive a standardised product throughout the duration of the studies, which would have been difficult to achieve with fresh produce. The participants of both studies were retrospectively genotyped for the G298T polymorphism and their endothelium-dependent vasodilation responses re-analysed in relation to genotype.