Heart and Metabolism

Clinical effects of free radical scavengers

Thomas Heitzer
Department of Cardiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany
Correspondence: Dr Thomas Heitzer
Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Innere Medizin,
Kardiologie, Martinistr 53, 20246 Hamburg, Germany,
tel: +49-4042803-3972, fax: +49-4042803-5766, e-mail: heitzer@uke.uni-hamburg.de

Abstract

Increased production of free radicals has been implicated in key processes of atherosclerosis, including oxidative modification of low-density lipoprotein and endothelial dysfunction, thereby promoting a vascular inflammatory response. Recent negative results of large clinical trials using “antioxidant” supplementation have raised questions about the effect of vitamin E and the source of free radical production in vivo. Free radicals, such as superoxide, rapidly react with nitric oxide, leading to endothelial dysfunction, a well-known cardiovascular risk factor and feature of atherosclerosis. Recent studies have shown that endothelial dysfunction can serve as a prognostic marker of future adverse events. Since angiotensin-converting enzyme inhibitors and statins are known to improve endothelial function and to reduce vascular oxidative stress, these mechanisms may produce beneficial effects on morbidity and prognosis in high-risk patients. We need sensitive and specific methods to assess the oxidative burden in vivo and to identify patients at risk.
n Heart Metab. 2003;19:11–17.

Keywords: Atherosclerosis, free radicals, antioxidants, vitamin C, vitamin E, endothelium,
prognosis, oxidative stress.

Introduction
In the 20 years since Brown and Goldsteins’s observation that oxidative modification of low-density lipoprotein (LDL) leads to foam cell formation, there has been intense interest in the relationship between oxygen-derived free radical formation and atherosclerosis. In vitro and animal model studies provided a growing body of evidence that reactive oxygen species not only promote lipid peroxidation but also stimulate smooth muscle cell growth and initiate expression of proinflammatory responses, all of which contribute to the development and progression of atherosclerosis [1]. The most persuasive data came
from animal models of atherosclerosis in which studies with antioxidants, such as vitamin C, vitamin E, probucol, and coenzyme Q, showed a significant decrease in the degree of LDL oxidation and the extent of atherosclerotic lesions [2]. However, clinical studies investigating the effect of antioxidant supplementation on cardiovascular risk have produced mixed results.

Antioxidants and cardiovascular risk
Epidemiological studies have suggested that increased intake of dietary antioxidants lowers the risk of atherosclerosis. High-dose vitamin E intake has been associated with a significant reduction in cardiovascular diseases (Table I) [3–5].

Table I. Observational and randomized trials of antioxidant vitamins.

Similarly, an inverse relation between vitamin C intake and major coronary events was found when subjects reporting an intake of 50 mg or more of vitamin C per day were shown to have a lower death rate from all cardiovascular diseases [12]. However, a major limitation of such nonrandomized studies is the possibility that confounding factors may account for the decreased risk, because the effects of other aspects of diet or lifestyle on disease rates cannot be ruled out.
Several randomized, double-blind, placebo-controlled trials have been conducted in recent years (Table I). The Finnish Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study failed to show any effect on coronary heart disease in male smokers [6]. The results of the Cambridge Heart Antioxidant Study (CHAOS) were encouraging: cardiovascular death and nonfatal myocardial infarction declined by 47% when patients with established coronary artery disease were treated with 400 to 800 IU vitamin E per day [8]. Three large, randomized, double-blind intervention trials using antioxidant vitamin supplementation have been reported and all three were negative [9–11]. The Heart Outcomes Prevention Evaluation (HOPE) trial in patients at high risk for cardiovascular events used 400 IU vitamin E daily together with ramipril in a 2 ´ 2 factorial design. Vitamin E had no effect, but ramipril conferred significant protection [9]. In patients after myocardial infarction, the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI) trial compared synthetic vitamin E (300 mg daily) and omega-3 polyunsaturated fatty acids (1 g daily) in a 2 ´ 2 factorial design [10]. Over a 3.5-year follow-up, vitamin E had no effect on the composite end point of death, nonfatal myocardial infarction, and stroke. The Heart Protection Study in 20,536 patients with atherosclerotic disease found no significant benefits on major coronary events using antioxidant vitamin supplementation [11].
The negative results of these trials raise important questions about the role of reactive oxygen species in atherosclerotic vascular disease.

Why has vitamin E supplementation failed?
The above studies seem to cast doubt on the oxidative modification theory of atherosclerosis, but there are several reasons to believe that this is not justified. (1) Although vitamin E effectively scavenges lipid peroxyl radicals, it has limited activity against other oxidants, such as superoxide, peroxynitrite, and hypochlorous acid, which have been implicated in atherosclerosis. The rate constant for the reaction of vitamin E with O2- is five orders of magnitude slower than the rate of reaction of O2- with endogenous antioxidant enzymes and molecules such as superoxide dismutase and nitric oxide [13]. (2) Oral intake of vitamin E only modestly increases its plasma and tissue levels. Given the slow rate constants for vitamin E’s reaction with O2- and other radicals, these modest increases in its concentration are unlikely to affect biological processes. (3) Many of the oxidative reactions that contribute to atherosclerosis occur in the cytoplasm, nucleus, and interstitial space. Vitamin E is concentrated in lipid layers and in the LDL particle and is therefore unlikely to affect these events. (4) Vitamin E may have adverse, pro-oxidant effects. The tocopheroxyl radical generated when vitamin E reacts with a radical can promote lipid peroxidation by attacking polyunsaturated fatty acids [14].
Given these considerations, it is quite possible that the use of vitamin E or other antioxidant vitamins will not be the best approach to limit vascular oxidative stress.

What are the sources of pathogenic oxygen radicals in atherosclerosis?
Preventing free radical production may represent a much more efficient way to inhibit the detrimental effects of vascular oxidant stress than trying to scavenge free radicals using vitamins. This requires a better understanding of the enzyme systems involved in their production. There are several potential enzymatic sources of reactive oxygen species including xanthine oxidase, (reduced) nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidases, nitric oxide synthase, the mitochondrial electron transport chain, arachidonic acid pathway enzymes lipoxygenase and cyclo-oxygenase, cytochrome P-450, peroxidase, and other hemoproteins [15]. In addition, infiltrating phagocytic cells, which contain the high-capacity O2--generating flavoenzyme NADPH oxidase, may be another important source of reactive oxygen species in atherosclerotic blood vessels. The nonphagocytic NAD(P)H oxidase seems to be a major source of reactive oxygen species in endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts. Its activity is regulated by cytokines (tumor necrosis factor-a) and hormones (angiotensin II), and has been shown to be upregulated in experimental models of atherosclerosis, hyperlipidemia, diabetes, hypertension, and balloon denudation [16]. In addition, segments of human vessels obtained from patients undergoing routine coronary artery bypass surgery have shown increased NAD(P)H oxidase-dependent vascular O2- production [17].
Increased free radical production not only leads to LDL peroxidation but also to endothelial dysfunction by inactivating nitric oxide, a well-known feature of atherosclerosis.

Oxidative stress induces endothelial dysfunction
The endothelium plays an integral role in maintaining vascular tone and function, in part by synthesis and release of vasoactive substances such as nitric oxide. Nitric oxide not only produces vasodilatation but also has potent antiatherogenic properties, including inhibiting adhesion molecule expression, preventing smooth muscle cell proliferation, reducing lipid peroxidation, and inhibiting platelet aggregation. Over the past decade, numerous experimental and clinical studies have demonstrated that oxidant stress is a major cause of endothelial dysfunction [18]. In particular, nitric oxide reacts rapidly with O2-, resulting in the formation of the peroxynitrite anion and loss of nitric oxide bioactivity (Figure 1).

Figure 1. Mechanisms for oxidative stress-induced endothelial dysfunction leading to acceleration of atherosclerotic processes. O2–, superoxide; NO, nitric oxide.

Accordingly, treatment with strong reducing agents such as vitamin C has consistently demonstrated beneficial effects on endothelial function in the coronary and peripheral circulation of patients [19]. Of note, because of the low rate constant of the reaction between vitamin C and superoxide, vitamin C must be given intra-arterially in sufficiently high concentrations (plasma concentration »10 mmol/L) to compete effectively with nitric oxide for superoxide [20]. Oral vitamin C has been shown to be rather unsuccessful. These findings are consistent with the notion that excessive production of superoxide contributes to endothelial dysfunction and decreased nitric oxide bioavailability.

Endothelial dysfunction, oxidative stress, and cardiovascular risk
Several groups have studied the prognostic value of endothelial dysfunction by obtaining long-term follow-up of patients with and without significant coronary artery disease [21–23]. These studies demonstrated that patients with endothelial dysfunction are at increased risk for cardiovascular events, including death from cardiovascular causes, myocardial infarction, ischemic stroke, coronary angioplasty, and coronary bypass graft. Although the precise mechanisms underlying the association between endothelial dysfunction and cardiovascular risk are unknown, vascular oxidative stress may not only contribute to endothelial dysfunction but also to coronary artery disease activity. This suggestion is supported by a recent 5-year follow-up study, finding that the beneficial effects of intra-arterial administration of vitamin C on endothelial function are much more pronounced in patients with subsequent cardiovascular events compared with patients without adverse events (Figure 2) [23]. The degree of improvement by vitamin C may indicate the amount of vascular oxidative stress in these patients. This strongly supports the concept that increased oxidative stress contributes to the progression of atherosclerotic disease and may therefore be an important determinant of clinical events.

Figure 2. (A) Endothelial function assessed by acetylcholine-induced blood flow in patients with (squares) and without (circles) cardiovascular events during saline and vitamin C infusion. Vitamin C improved endothelial function in both groups. However, the effect of vitamin C was significantly larger in patients with events than in those without events. *P < 0.05 vs saline; **P < 0.001 vs saline. (B) Kaplan-Meier analysis demonstrating cumulative proportion of patients without cardiovascular events during follow-up. Effect of vitamin C on endothelial function is divided into values below and above the median. (Adapted from [23].)

“Antioxidant” effects of angiotensin- converting enzyme (ACE) inhibitors and statins
Angiotensin II potently stimulates vascular free radical production from sources such as NAD(P)H oxidase, leading to endothelial dysfunction and promoting a vascular inflammatory response. Angiotensin II type 1 (AT1) receptor antagonists or angiotensin-converting enzyme (ACE) inhibitors have been shown to normalize vascular oxidative stress and to reduce the progression of atherosclerosis [24, 25]. ACE inhibitors are known to improve endothelial dysfunction and reduce the rate of death from cardiovascular causes. Thus, ACE inhibitors and AT1 receptor antagonists are believed to be potent antiatherosclerotic drugs likely due to their antioxidative properties.
The beneficial effects of HMG-CoA reductase inhibitors appear to extend beyond their effects on serum cholesterol levels. Indeed, recent experimental and clinical evidence indicates that some of the “pleiotropic” effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, and decreasing oxidative stress and vascular inflammation [26]. Indeed, statins dramatically decrease superoxide production and increase bioavailability of nitric oxide by stimulating and upregulating endothelial nitric oxide synthase.

Search for markers of oxidative stress
Despite convincing evidence that free radicals play a central role in vascular disease, there are few methods of direct measurement of oxidative stress in patients. Assessment of plasma concentrations of antioxidants such as vitamin C, vitamin E, carotenoids, ubiquinol-10, and glutathione can be used [27]. Estimation of in vivo LDL oxidation has been proposed by measuring malondialdehyde, conjugated dienes in circulating LDL, or plasma titers of autoantibodies to oxidized LDL [28]. F2-isoprostanes have recently begun to emerge as a new class of free radical catalyzed products of arachidonic acid metabolism supposed to be far more specific than the conventional methods of malondialdehyde [29]. However, none of these markers has been accepted as a satisfactory candidate to identify patients at high risk due to oxidative stress. Biomarkers are urgently needed to identify such high-risk populations and to assess whether therapy effectively lowers the oxidative burden.

Conclusion
The recent disappointing trials of antioxidant therapy raise doubts about the ability of vitamins to augment antioxidant defence mechanisms in vivo. Despite ample experimental evidence that free radicals play a pivotal role in atherosclerotic disease, the methods currently available to assess the degree of oxidative stress and the efficacy of antioxidant therapy in vivo are quite limited. Quantitatively accurate indices of the oxidative burden of patients at risk would substantially support clinical research in this area.

REFERENCES

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