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Antioxidants and the bioactivity of endothelium-derived nitric oxide

Douglas Tomasian, John F. Keaney, Joseph A. Vita
DOI: http://dx.doi.org/10.1016/S0008-6363(00)00103-6 426-435 First published online: 18 August 2000
Keywords
  • Endothelial factors
  • Endothelial function
  • Free radicals
  • Nitric oxide

Time for primary view 21 days.

1 Introduction

Over the past two decades, investigators have increasingly recognized the importance of the endothelium as a central regulator of vascular homeostasis. In 1980, Furchgott and Zawadzki [1] reported that acetylcholine-mediated relaxation of isolated aortic tissue is dependent on release of an ‘endothelium-derived relaxing factor’. Subsequent studies by Ignarro and colleagues identified this factor as nitric oxide (NO) [2] or a closely related NO product [3]. In addition to regulating vascular tone, it is now appreciated that endothelium-derived NO also acts to inhibit platelet activity, vascular smooth muscle cell growth, and adhesion of inflammatory cells to the endothelial surface [4].

Loss of the bioactivity of endothelium-derived NO plays a critical role in the pathogenesis of a number of disease states including atherosclerosis and its risk factors [5]. A large body of work suggests that the impaired NO action in atherosclerosis is related to increased oxidative stress in the vascular wall [6]. Since oxidative stress may be defined as an excess of oxidants relative to antioxidant defenses [7], investigators were prompted to examine the possibility that increasing antioxidant availability would restore NO-dependent responses. This article will review the effects of antioxidants on NO bioactivity and the clinical implications of these observations.

2 Endothelium-derived NO in health and disease

NO is synthesized in endothelial cells from l-arginine by the endothelial isoform of NO synthase (eNOS), which is the product of the NOS3 gene [8]. eNOS is constitutively expressed and located to caveolae in the plasma membrane. When bound to caveolin, eNOS is catalytically inactive. In the presence of calcium, calmodulin displaces caveolin and binds to eNOS, and thereby activates the enzyme [8]. Thus, NO production by endothelial cells is stimulated by factors that increase intracellular calcium concentration including receptor-dependent agonists like acetylcholine, bradykinin, substance P, and thrombin [9], and by physical stimuli including shear stress [10]. NO produces vasodilation primarily by activating guanylyl cyclase in vascular smooth muscle cells, which increases intracellular concentrations of cyclic-3′,5′-guanosine monophosphate (cGMP) [11]. cGMP acts in turn as a second messenger, activating cGMP-dependent protein kinase, which decreases cytosolic calcium concentration and modulates ion channel function leading to relaxation of vascular smooth muscle cells [12]. NO may also directly activate calcium-dependent potassium channels in vascular smooth muscle cells, producing hyperpolarization and relaxation [13]. NO inhibits platelet aggregation and adhesion by a guanylyl-cyclase mechanism [14]. Endothelium-derived NO is an inhibitor of leukocyte adhesion at the vessel wall [15], and has anti-mitogenic effects on vascular smooth muscle cells [16]. Thus, it is clear that NO plays a critical role in the regulation of vascular homeostasis (Fig. 1).

Fig. 1

Endothelium-derived NO as a central regulator of vascular homeostasis.

The importance of NO for vascular regulation has been extensively demonstrated in humans. An intra-arterial infusion of acetylcholine or increased blood flow produces vasodilation of coronary arteries [17,18] and peripheral arteries [19,20], and specific inhibitors of NOS may block these responses [21–23]. Normal NO release opposes the vasoconstrictor responses to clinically relevant stimuli including catecholamines [24] and serotonin [25]. NOS inhibition is associated with increased systemic blood pressure [26], decreased blood flow responses to exercise and local ischemia [27,28], and shortened bleeding time [29]. Inhibition of NO production is also associated with increased adhesion of leukocytes [30] or platelets [31] to cultured human endothelial cells. In general, endothelial release of NO acts to limit changes in shear stress and opposes vasoconstrictor, pro-thrombotic, and pro-inflammatory stimuli in the vasculature.

Atherosclerosis is associated with a loss of the bioactivity of endothelium-derived NO. Intra-coronary infusion of acetylcholine produces vasoconstriction in patients with angiographic evidence of atherosclerosis, while the vasodilator responses to nitroglycerin are maintained [17]. These observations are consistent with a loss of endothelial release of NO and unopposed direct constrictor effects of acetylcholine on vascular smooth muscle, and this mechanism was subsequently confirmed in isolated coronary arterial tissue from patients with coronary artery disease [32]. Vasodilator responses to other stimuli for NO release are also impaired in atherosclerosis including shear stress [18] and serotonin [25].

Loss of endothelium-derived NO occurs early in the atherosclerotic disease process. Flow-mediated dilation in the brachial or femoral artery is impaired in children with familial hypercholesterolemia [33]. Acetylcholine-mediated vasodilation is impaired in the coronary circulation of patients with hypercholesterolemia prior to the development of angiographically apparent atherosclerosis [34] or even ultrasound detectable intimal thickening [35]. Impaired endothelium-dependent dilation is associated with other coronary risk factors including hypertension [36], diabetes mellitus [37], cigarette smoking [38], hyperhomocysteinemia [39], and family history of coronary disease [40]. These findings are consistent with the strong experimental evidence indicating that loss of NO action contributes to the pathogenesis of atherosclerosis [41].

Coronary atherosclerosis is a chronic disease that begins as fatty streaks that progress to advanced atherosclerotic plaques over a period of decades. Eventually, these plaques may sufficiently obstruct the lumen so that coronary flow is limited during periods of increased myocardial oxygen demand, and thus, produce effort angina. Furthermore, it is now recognized that acute coronary syndromes, such as acute myocardial infarction or unstable angina, occur when relatively mild plaques rupture or erode, exposing the sub-endothelium and producing an obstructive or near obstructive thrombus. The precise mechanisms leading to plaque rupture remain undefined, but clearly reflect a fundamental loss of arterial homeostasis. Emerging evidence suggests that plaque inflammation and thinning of the fibrous cap are associated with increased vulnerability to rupture [42].

Impaired endothelial function may contribute to the pathogenesis of both acute and chronic coronary syndromes. Loss of NO-dependent vasodilation in response to flow or circulating catecholamines may increase coronary constriction and limit blood flow during exercise or other stimuli for chronic angina [43,44]. In regard to acute events, impaired NO bioactivity may lead to increased shear stress [45] and plaque inflammation [42], thus promoting plaque rupture or erosion. Loss of NO bioactivity may also exacerbate platelet aggregation at the site of plaque rupture, further worsening the extent of thrombosis and myocardial ischemia [46]. There is emerging evidence that loss of NO bioactivity in the coronary circulation is associated with an increased risk for cardiovascular disease events, supporting its clinical relevance [47].

Thus, impaired NO bioactivity may contribute both to the pathogenesis of atherosclerotic lesions and to their clinical expression during the later stages of the disease process. Other manifestations of endothelial dysfunction or activation that may contribute to inflammation and thrombosis are also believed to play a role in the pathogenesis of coronary artery disease. For example, expression of adhesion molecules, plasminogen activator inhibitor-1, endothelin, and angiotensin II, and decreased production of prostacyclin, all may be relevant to the clinical expression of atherosclerosis [5,46].

3 Oxidative stress and impaired NO bioactivity

In light of the importance of endothelium-derived NO for maintaining normal arterial homeostasis, it is not surprising that there has been intense interest in defining the causes of impaired NO bioactivity in atherosclerosis and related disease states. A large body of work has made it clear that a common feature of many risk factors for atherosclerosis is increased oxidative stress in the vasculature. Indeed, the well-accepted oxidative hypothesis of atherosclerosis [48] emphasizes that oxidative modification of low-density lipoprotein is a key event in atherogenesis that contributes to foam cell formation and initiates the chronic inflammatory process that eventually produces advanced atherosclerotic plaques [49]. Central to this process is an alteration of endothelial cell phenotype with loss of NO bioactivity, as well as increased expression of adhesion molecules and chemotactic, vasoconstrictor, prothrombotic, and growth promoting factors [46,49]. In regard to impaired NO and oxidative stress, investigators have focused on the importance of increased production of reactive oxygen species and the accumulation of products of lipid peroxidation as causative mechanisms.

3.1 Superoxide anion

There is clear evidence that superoxide anion production in the vasculature is closely linked to the bioactivity of endothelium-derived nitric oxide. Superoxide anion is produced in all cells as a consequence of normal oxidative metabolism. Superoxide anion reacts with NO to form peroxynitrite in a diffusion-limited process, and since peroxynitrite is much less effective as an activator of guanylyl cyclase, this reaction results in a marked reduction in the bioactivity of NO [50]. Formation of peroxynitrite has further consequences, since this potent oxidant can initiate lipid peroxidation and oxidize critical thiols or tyrosine residues [51]. In experimental systems, there is evidence that ambient levels of superoxide modulate the bioactivity of endothelium-derived NO [52]. For example, inhibiting endogenous superoxide dismutase (SOD) decreases endothelium-dependent vasodilation, without decreasing net production of NO as determined by measuring oxidation products of NO [53]. This finding is consistent with ‘inactivation’ of NO by superoxide.

Superoxide production is increased in a number of disease states including hypercholesterolemia [54], hypertension [55], and diabetes mellitus [56]. The precise mechanisms leading to this increase remain uncertain. Several enzymatic sources of superoxide anion in atherosclerosis have been identified including xanthine oxidase [54], NADH/NADPH oxidase [57], and NOS itself [58]. Enhancing vascular superoxide dismutase activity or inhibiting the enzymatic source of superoxide anion production is associated with improved endothelium-dependent vasodilator function in atherosclerosis and related disease states [55,59]. Although experimental studies strongly support a role for increased superoxide anion production in impaired endothelium-derived NO bioactivity in atherosclerosis, specific evidence for this mechanism in human subjects is not available, in part due to a lack of specific superoxide scavengers that can gain access to the vascular wall.

3.2 Lipid peroxidation

Another consequence of increased production of reactive oxygen species in the vasculature is lipid peroxidation, and there is strong experimental evidence that this process adversely influences endothelial function and the bioactivity of NO. For example, oxidized low-density lipoprotein (ox-LDL) is cytotoxic to endothelial cells [60]. Ox-LDL promotes the recruitment of inflammatory cells to the vessel wall, which may increase local production of reactive oxygen species leading to ‘inactivation’ of NO as described above [61]. There is evidence that ox-LDL [62] and products of lipid peroxidation [63] can react with NO directly and eliminate its biological activity. Ox-LDL also decreases eNOS protein levels in endothelial cells [64], thus potentially decreasing production of NO. Finally, products of lipid peroxidation, including lysophosphatidylcholine, may interfere with signal transduction and receptor-dependent stimulation of NOS activity [65,66] and with activation of guanylyl cyclase [67].

In human subjects, there is indirect evidence that lipid peroxidation is relevant to endothelial function. For example, there is an inverse correlation between endothelium-derived NO activity in forearm resistance vessels and plasma antibodies against ox-LDL [68] or epitopes of ox-LDL in plasma [69]. Endothelium-dependent vasodilation in the coronary circulation has also been reported to correlate with the susceptibility of LDL to ex vivo oxidation in patients treated with the lipid soluble antioxidant probucol [70]. A direct link between lipid peroxidation and endothelial dysfunction remains to be established in humans.

4 Antioxidants and the bioactivity of endothelium-derived NO

Given the relationship between increased oxidative stress in the vasculature and impaired endothelial vasodilator function, it was natural for investigators to consider the possibility that augmenting antioxidant defenses would have a beneficial effect. In the past 6 years, a large number of studies have examined the effects of antioxidant supplementation on endothelial function [71,72]. The majority of studies on this subject used α-tocopherol (the principal component of vitamin E) or ascorbic acid (vitamin C), the principal lipid-soluble and water-soluble natural antioxidants, respectively. In the remainder of this review, we will focus on the experimental and human evidence that these two natural low-molecular weight antioxidants may improve the bioactivity of endothelium-derived NO and review the clinical implications of these findings.

4.1 α-Tocopherol

In 1993, Keaney et al. [73] reported that feeding rabbits a diet containing α-tocopherol or β-carotene prevented the development the endothelial vasomotor dysfunction associated with an atherogenic diet (Fig. 2). This beneficial effect of α-tocopherol in the setting of hypercholesterolemia was confirmed by other investigators [74]. The protective effects of α-tocopherol could not be attributed to a change in lipoprotein profile or reduction in the extent of atherosclerotic lesion formation [73]. Furthermore, these effects did not necessarily relate to the resistance of LDL to ex vivo oxidation [75]. Instead, the bioactivity of endothelium-derived NO correlated with the accumulation of these lipid soluble-antioxidants in vascular tissue [73]. These observations suggested a tissue-specific effect, rather than an effect dependent only on the prevention of LDL oxidation [76]. For example, augmenting tissue levels of α-tocopherol is associated with an increased resistance to the adverse effects of ox-LDL on NO bioactivity, an effect that is mediated in part by preventing the activation of protein kinase C [66]. α-Tocopherol supplementation also provides protection against ox-LDL-mediated cytotoxicity [77] and limits cell-mediated LDL oxidation and consequent production of monocyte chemotactic protein-1 (MCP-1) in vascular cell co-culture systems [78]. In addition to the beneficial effects in hypercholesterolemia, α-tocopherol supplementation also has been shown to improve the bioactivity of endothelium-derived NO in other disease states, including diabetes mellitus [79].

Fig. 2

Lipid soluble antioxidants preserve the bioactivity of endothelium-derived NO in hypercholesterolemia. Aortic rings from rabbits fed chow (▴), 1% cholesterol (○), 1% cholesterol with β-carotene (□), and 1% cholesterol with α-tocopherol (♢) were exposed to increasing concentrations of acetylcholine (ACH), calcium ionophore A23187, or sodium nitroprusside. NO-mediated relaxation to ACH and A23187 were impaired in cholesterol fed rabbits, but were normal when the cholesterol diet was supplemented with either α-tocopherol or β-carotene. Modified with permission from Ref. [73].

The effects of α-tocopherol supplementation on vascular function has been extensively examined in human subjects. However, in contrast to the consistent beneficial effects observed in experimental models, the results in humans have been mixed. Gillian et al. [80] demonstrated no beneficial effect on endothelium-dependent vasodilation of forearm microvessels following treatment with α-tocopherol in combination with β-carotene and ascorbic acid in patients with hypercholesterolemia. Other studies also failed to demonstrate a beneficial effect of α-tocopherol on microvascular function in patients with hypercholesterolemia [81], diabetes mellitus [82] or proven coronary artery disease [83,84]. In contrast, several small studies have shown a beneficial effect of α-tocopherol supplementation on endothelium-dependent flow-mediated dilation of the conduit brachial artery [85,86] and in patients with multiple coronary risk factors [87].

Apart from the obvious species differences, the reasons for the consistently positive effects of α-tocopherol in experimental models and the mixed results in human subjects are unclear. One potential difference is the phase of the disease. The experimental studies described above examined the bioactivity of endothelium-derived NO early in the disease process, shortly after initiation of a hypercholesterolemic diet. In contrast, the currently available human studies have focused on patients with long standing risk factors or proven coronary artery disease. Another difference relates to the studied vascular bed. Most of the negative human studies examined microvessels in the forearm [80,81,83]. In contrast, the positive studies involving whole animals generally examined conduit vessels (aorta, coronary or carotid artery) [73,74,88], and many of the studies that examined conduit arteries in humans have shown a beneficial effect [85,86]. A large scale, well-controlled study examining the effect of α-tocopherol on conduit vessel function, particularly in epicardial coronary artery function would help clarify this issue. It is notable that the lipid-soluble synthetic antioxidant probucol, in combination with lipid-lowering therapy, has been reported to improve endothelial vasomotor function in the coronary circulation of patients with coronary atherosclerosis [89].

4.2 Ascorbic acid

The effects of ascorbic acid on the bioactivity of endothelium-derived NO have also been extensively studied in human subjects and to a lesser extent in experimental animals. In contrast to α-tocopherol, a consistent beneficial effect of ascorbic acid has been demonstrated. The initial studies of this question were actually performed in human subjects, and subsequent experimental studies have clarified the mechanisms of benefit.

Ting et al. [90] demonstrated that intra-arterial infusion of supraphysiological concentrations of ascorbic acid improved endothelium-dependent vasodilation in forearm resistance vessels of patients with Type 2 diabetes mellitus. Levine et al. [91] demonstrated an acute improvement in brachial artery flow-mediated dilation in patients with coronary artery disease following a single oral dose of ascorbic acid that increased plasma levels within the physiological range. Chronic ascorbic acid in coronary disease patients was subsequently shown to also have a sustained beneficial effect (Fig. 3) [92].

Fig. 3

Effect of acute and chronic ascorbic acid treatment on brachial artery flow-mediated dilation. NO-dependent flow-mediated dilation (upper panel) and nitroglycerin-mediated dilation (lower panel) of the brachial artery were assessed using high resolution vascular ultrasound at baseline, 2 h after a 2-g dose of ascorbic acid or placebo, and after 30 days of ascorbic acid 500 mg/day or placebo in 46 patients with proven coronary artery disease. Ascorbic acid significantly improve flow-mediated dilation after acute and chronic treatment (P=0.005 by repeated measures ANOVA), but had no effect on nitroglycerin-mediated dilation. Reproduced by permission from Ref. [92].

These initial observations have been extensively confirmed. Physiological concentrations of ascorbic acid reverse endothelial dysfunction in conduit vessels of patients with congestive heart failure [93], cigarette smoking [94], hyperhomocysteinemia [95], and vasospastic angina [96]. Intra-arterial infusion producing supraphysiological concentrations of ascorbic acid improve microvascular function in patients with hypercholesterolemia [97], hypertension [98–100], and smoking [101].

These remarkably consistent findings prompted studies to elucidate the mechanism of this beneficial effect of ascorbic acid on the bioactivity of endothelium-derived NO. Ascorbic acid is a potent antioxidant with a number of effects including the ability to inhibit lipid peroxidation [102] and scavenge superoxide anion [103]. Together with glutathione, ascorbic acid is one of the two primary water-soluble antioxidants inside cells and, as such, is an important determinant of cellular redox state [104]. Any or all of these effects might influence the bioactivity of endothelium-derived NO.

Given the importance of superoxide anion as a mechanism of endothelial dysfunction in atherosclerosis [54], many investigators assumed that ascorbic acid exerts its beneficial effect by scavenging superoxide anion [90,91]. However, consideration of the kinetics of the relevant reactions suggests that this assumption may be overly simplistic. The reaction between superoxide anion and NO is extremely rapid with a rate constant (1.9×1010 M/s) near the limit of diffusion [105]. The rate constant for the reaction between ascorbic acid and superoxide anion is 105 lower, and based on measured concentrations of NO adjacent to the endothelial surface (0.1–1 μM) [106], one would predict that supraphysiological levels of ascorbic acid (>10 mM) would be required to protect NO against ‘inactivation’ by superoxide. Jackson et al. [103] recently confirmed this prediction using an ex vivo model of superoxide-mediated vascular dysfunction. Although scavenging superoxide anion might explain improved NO action in studies that involved intra-arterial infusion of supraphysiological concentrations of ascorbic acid (1–10 mM) [90,97–101], this mechanism is unlikely to explain the benefits observed following acute or chronic administration of physiological doses of ascorbic acid [91,92,94,95,107].

Given the potential role of lipid peroxidation as a mechanism of impaired bioactivity of endothelium-derived NO in atherosclerosis, the ability to inhibit lipid peroxidation could contribute to the beneficial effects of ascorbic acid. However, this mechanism is unlikely to be important in studies demonstrating a beneficial effect within a few hours of treatment [91]. Furthermore, initial studies have shown no relationship between the beneficial effects of ascorbic acid treatment on endothelial function and plasma markers of lipid peroxidation (F2-isoprostanes) and protein oxidation (o,o′-dityrosine), but these markers may not accurately reflect changes at the level of the vessel wall [92].

Finally, several recent studies suggest that ascorbic acid may improve the bioactivity of endothelium-derived nitric oxide by influencing cellular redox state. Ascorbic acid is capable of sparing intracellular glutathione from oxidation. Although controversial, there is evidence that glutathione availability may be important for NO in experimental models [108–110]. and in human subjects [111–113]. Furthermore, there is evidence that ascorbic acid may have direct effects on the activity of NOS, possibly through an effect on essential cofactors for the enzyme, including tetrahydrobiopterin [114–116]. This effect to increase NO production by eNOS is evident at physiologically relevant extracellular and intracellular concentrations of ascorbic acid [115,116]. The importance of tetrahydrobiopterin in this regard is further supported by the observation that tetrahydrobiopterin treatment improves endothelium-dependent vasodilation in patients with hypercholesterolemia [117]. In summary, while the exact mechanisms remain uncertain, there is clear and consistent evidence that ascorbic acid supplementation increases the bioactivity of endothelium-derived NO.

4.3 Other antioxidants

A number of other natural and synthetic antioxidants may influence the bioactivity of endothelium-derived NO, and the effects of supplementation of many of these antioxidants has been examined in experimental systems and in human subjects. For example, the synthetic antioxidant probucol potently inhibits oxidation of LDL and improves endothelium-dependent vasodilation in experimental hypercholesterolemia [118]. This effect may relate both to reduced lipid peroxidation and to a decrease in vascular production of superoxide anion [118]. As discussed above, probucol in combination with lipid-lowering therapy has been shown to improve endothelium-dependent vasodilation in the coronary circulation of patients with coronary artery disease [89].

Flavonoids are a class of polyphenolic compounds found in plants, including grapes and tea, and they have the ability to scavenge reactive oxygen species and inhibit lipid peroxidation [119,120]. Recently, there has been considerable interest in the effects of flavonoids in cardiovascular disease and on vascular function [121]. An experimental study demonstrated a beneficial effect of red wine on endothelial function in isolated vascular tissue [122]. In an uncontrolled human study, Stein et al. [123] reported improved brachial artery flow-mediated dilation and increased resistance of LDL to ex vivo oxidation following ingestion of purple grape juice for 14 days, an effect likely attributable to increased flavonoid intake.

Finally, a considerable portion of cellular antioxidant defense is provided by enzymatic antioxidants including superoxide dismutase, catalase, and glutathione peroxidase, and there has been considerable interest in examining the importance of these compounds for the bioactivity of endothelium-derived NO. As discussed above, SOD activity is closely linked to NO bioactivity in experimental systems. The benefit of administration of these large proteins would be predicted to be limited, because of inability to access the vascular wall and intracellular space. In fact, studies examining the effects of enzymatic SOD on endothelium-dependent vasodilation in hypertension [124] and hypercholesterolemia [125] have failed to demonstrate a beneficial effect. However, low-molecular weight mimics of these compounds might have utility. Furthermore, a molecular strategy that increases cellular expression of these enzymes might prove more successful.

5 Clinical implications of the effects of antioxidants on endothelium-derived NO

An important corollary to the oxidative hypothesis of atherosclerosis is the possibility that antioxidant treatment will reduce atherosclerosis. Indeed, there is good evidence that probucol reduces the extent of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit model of atherosclerosis [126]. In studies involving other models and other antioxidants, the effects on atherosclerosis severity are mixed, but the weight of evidence suggests that antioxidants can limit atherogenesis by multiple mechanisms that are not simply limited to the inhibition of LDL oxidation [127].

In humans, there is very strong epidemiological and observational study evidence that individuals with greater dietary intake of antioxidants vitamins or increased levels of antioxidants in plasma or tissue are at lower risk for cardiovascular disease events [128]. Depending on the nutritional status of the patient population, this effect has been demonstrated for both vitamin E [129,130] and ascorbic acid [131,132]. However, randomized trials have produced less certain results.

Vitamin E has been studied most extensively. One randomized study, the Cambridge Heart Antioxidant Study (CHAOS), demonstrated a marked reduction in non-fatal myocardial infarction in patients randomized to treatment with 400–800 IU of vitamin E/day compared to patients receiving placebo [133]. However, three subsequent randomized studies have failed to confirm these findings and demonstrated no beneficial effect of vitamin E for the secondary [134,135] or primary prevention [136] of cardiovascular disease. Several studies using β-carotene, another lipid soluble antioxidant, have also been convincingly negative [136–138]. To date, there have been no large-scale, randomized studies that examined the effect of ascorbic acid treatment on cardiovascular disease.

Thus, despite the strong evidence that increased dietary intake of antioxidants has benefit against cardiovascular disease events, the results of randomized trials with vitamin E have been disappointing. The reasons for these apparently discrepant results are unclear. It is possible that long-term antioxidant intake plays a role in primary prevention while relatively short-term treatment is ineffective in patients with advanced disease. Alternatively, increased dietary intake of antioxidants could be a marker for some other unrelated health-promoting behavior.

Despite the mixed results of recent randomized trials with vitamin E, there has been and continues to be great interest in how increased intake of antioxidant vitamins reduces cardiovascular disease risk. Investigators have postulated that antioxidants might slow the development or progression of atherosclerotic lesion formation and several angiographic trials have suggested modest effects of vitamin E [139]. and ascorbic acid [140] in selected populations. However, a randomized trial using probucol failed to demonstrate reduction or slowed progression of the angiographic severity of femoral artery atherosclerosis [141].

The studies reviewed in this paper strongly suggest that antioxidant vitamin supplementation has a beneficial effect on the bioavailability of endothelium-derived NO. As discussed above, this effect has the potential to reduce the risk of acute and chronic coronary disease events by promoting vasodilation and inhibiting platelet activity and inflammation. As for other interventions that reduce cardiovascular risk, including lipid-lowering therapy [46], these mechanisms are likely more important that any effect on the anatomic severity of atherosclerotic lesions. These findings fit well with the concepts that atherosclerosis is a dynamic process and that the focus of therapy should be to restore vascular homeostasis.

6 Conclusions

In summary, there is strong evidence that endothelium-derived NO plays a critical role in regulating vascular homeostasis and that the bioactivity of endothelium-derived NO is impaired in atherosclerosis and related disease states. Loss of NO bioactivity in this setting is attributable to increased oxidative stress, particularly increased production of superoxide anion and accumulation of products of lipid peroxidation. As would be expected from these observations, increasing antioxidant oxidant defense by antioxidant supplementation has the capacity to restore endothelial vasomotor function. These effects may explain, in part, the association between increased intake of vitamin E or ascorbic acid and reduced cardiovascular disease risk. Further study is needed to determine the relative importance of dietary antioxidant intake compared to antioxidant supplements, and specifically whether ascorbic acid supplementation is beneficial for secondary prevention.

Acknowledgements

Drs Vita and Keaney are the recipients of Established Investigator Awards from the American Heart Association. Portions of the work reviewed in this paper were supported by grants from the National Institutes of Health (HL52936, HL53398, and HL55993).

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