Atherosclerosis: from lesion formation to plaque activation and endothelial dysfunction

Molecular Aspects of Medicine 21 (2000) 99±166 www.elsevier.com/locate/mam

Atherosclerosis: from lesion formation to plaque activation and endothelial dysfunction John F. Keaney Jr.

1

Department of Medicine and Pharmacology, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, Room W507, Boston, MA 02118, USA

Abstract Atherosclerosis is an important source of morbidity and mortality in the developed world. Despite the fact that the association between LDL cholesterol and atherosclerosis has been evident for at least three decades, our understanding of exactly how LDL precipitates atherosclerosis is still in its infancy. At least three working hypotheses of atherosclerosis are now nearing the stage where their critical evaluation is possible through a combination of basic science investigation and murine models of atherosclerosis. As we move forward in our understanding of this disease, e€orts will be increasingly focused on the molecular mechanisms of disease activation that precipitate the clinical manifestations of atherosclerosis such as heart attack and stroke. Two candidates for such investigation involve the events surrounding plaque activation and endothelial dysfunction. Further investigation in these ®elds should provide the necessary insight to develop the next generation of interventions that will reduce the clinical manifestations of this devastating disease. The purpose of this work is to review the major theories of atherogenesis, examine the aspects of atherosclerosis that lead to disease activation and discuss aspects of disease activation that are amenable to treatment. Ó 2000 Published by Elsevier Science Ltd. Keywords: Atherosclerosis; In¯ammation; Endothelium; Nitric oxide; Oxidation

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Atherogenesis . . . . . . . . . . . . . . . . . . . . . . 2.1. Epidemiology and risk factors . . . . . . 2.2. Morphologic features of atherosclerosis 2.2.1. The normal artery . . . . . . . . . . . 2.2.2. Gross morphology . . . . . . . . . . .

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2.3. Response-to-injury hypothesis . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Oxidative modi®cation hypothesis of atherosclerosis . . . . . . . . 2.4.1. LDL does not support foam cell formation . . . . . . . . . . 2.4.2. Mechanisms of LDL oxidation . . . . . . . . . . . . . . . . . . . 2.4.2.1. Structure of LDL . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2.2. Stages of LDL oxidation . . . . . . . . . . . . . . . . . . . 2.4.2.3. Oxidation of LDL in cell-free systems . . . . . . . . . . . 2.4.2.4. Metal ion-induced LDL oxidation . . . . . . . . . . . . . 2.4.2.5. Superoxide and LDL oxidation . . . . . . . . . . . . . . . 2.4.2.6. Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2.7. Glycoxidation reactions . . . . . . . . . . . . . . . . . . . . . 2.4.2.8. Peroxynitrite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2.9. Myeloperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Proatherogenic actions of oxidized LDL . . . . . . . . . . . . . 2.4.4. Evidence to support the oxidative modi®cation hypothesis 2.4.5. Antioxidant studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.6. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.6.1. Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.6.2. Probucol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.7. Problems with the oxidative modi®cation hypothesis . . . . 2.4.8. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.9. Evidence from descriptive studies . . . . . . . . . . . . . . . . . . 2.4.9.1. Evidence from case control studies . . . . . . . . . . . . 2.4.9.2. Evidence from prospective cohort studies . . . . . . . . 2.4.9.3. Randomized trials . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. The response-to-retention hypothesis of early atherogenesis . . . 2.5.1. Evidence to support the response-to-retention hypothesis .

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The concept of disease activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 3.1. The ®brous cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 3.2. Matrix degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

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Vascular homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Oxidative stress and impaired NO bioactivity . . . . . . 4.1.1. Superoxide and NO bioactivity . . . . . . . . . . . . 4.1.2. Lipid peroxidation and NO bioactivity . . . . . . . 4.1.3. Lipid-soluble antioxidants and NO bioactivity . 4.1.3.1. a-Tocopherol and b-Carotene . . . . . . . . . . 4.1.3.2. Probucol . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Water-soluble antioxidants and NO bioactivity . 4.1.4.1. Glutathione . . . . . . . . . . . . . . . . . . . . . . 4.1.4.2. Ascorbic acid . . . . . . . . . . . . . . . . . . . . . 4.1.5. Other means of treating endothelial dysfunction 4.1.5.1. L -arginine . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5.2. Tetrahydrobiopterin . . . . . . . . . . . . . . . . 4.1.5.3. LDL lowering therapy . . . . . . . . . . . . . . . 4.1.5.4. Other treatments . . . . . . . . . . . . . . . . . . .

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Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

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1. Introduction Cardiovascular disease is the major source of morbidity and mortality in the Western World. In 1997, the latest year for which statistics are available, cardiovascular disease claimed 953,110 lives in the United States where presenting 41% of all deaths (American Heart Association, 2000). The magnitude of this problem is simply staggering, cardiovascular disease claims more lives each year than the next seven leading causes of death combined. Approximately 1/6 all of people killed by cardiovascular disease are under the age of 65, and the estimated age-adjusted prevalence of cardiovascular disease is 30% for men, and 24% for women (American Heart Association, 2000). In the black population, this number climbs to 41% for men, and 40% for women (American Heart Association, 2000). Using the United States economic costs, cardiovascular disease consumes in excess of 20 billion dollars in direct healthcare costs, and if one includes lost productivity, the cost climbs to over 250 billion dollars annually (American Heart Association, 2000). Although one might be tempted to believe this is principally a problem of the developed world, the World Health Organization predicts that continued global economic prosperity will likely lead to an epidemic of cardiovascular disease as developing countries continue acquire Western habits. Clearly then, cardiovascular disease is a major public health problem that will only have wider implications over the ensuing decades. The principal manifestations of cardiovascular disease are heart attack and stroke; these represent the clinical sequelae of a systemic vascular process known as atherosclerosis. Data developed from epidemiological studies over the last 40 yrs has shed light on the predilection of cardiovascular disease for the Western world. These ®ndings relate to the fact that atherosclerosis is a disease of aging, and that premature atherosclerosis can be precipitated by a number of clinical conditions, the most prominent of which include excess LDL cholesterol, diabetes mellitus, hypertension, and cigarette smoking (Dawber et al., 1957; Kannel and McGee, 1979). Another major risk factor for premature atherosclerosis is a family history of premature atherosclerosis in a ®rst degree relative. The de®nition of premature atherosclerosis includes a ®rst-degree male relative with clinical evidence of cardiovascular at an age <55, or a ®rst-degree female relative with clinical manifestations of cardiovascular disease prior to age 60. Of these risk factors for cardiovascular disease, excess LDL cholesterol has received the most attention of late. This is perhaps ®tting as it was among the ®rst of the risk factors identi®ed for cardiovascular disease and remains the principal target today for modifying future risk of cardiovascular disease with cholesterol lowering agents. Atherosclerosis is an insidious process that may persist for many years before clinical manifestations become evident. This observation is due to the fact that the processes involved in atherogenesis (i.e., the process of early lesion development) require prolonged exposure to predisposing factors. It is only the latter stages of disease that progress rather rapidly and lead to clinical manifestations. The processes of atherosclerosis lesion development and the clinical events of atherosclerosis are distinct. Due to this distinction, therapies that are e€ective at preventing lesion development may not prove to be e€ective in preventing clinical manifestations of the

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disease. The principal reason is that many therapies are instituted after individuals have established lesions. Indeed, since most individuals initiate atherosclerosis before they are teenagers (Stary, 1983), it would be very dicult to devise a strategy to prevent lesion development. For these reasons, this review will discuss the processes of lesion development, and the clinical manifestations of atherosclerosis separately. Where possible, overlapping mechanisms will be identi®ed and discussed. 2. Atherogenesis 2.1. Epidemiology and risk factors At the outset, one should understand that atherogenesis is a multifactorial process the complexity of which makes it dicult to clearly de®ne the risk attributable to any one risk factor. To be de®ned as a risk factor, a trait must be causally linked to a particular disease in a manner that is strongly and consistently associated with that disease. Because most risk factors do not occur in a vacuum, the apparent strength of the association will depend on whether or not analyses have control for other concurrent conditions that might contribute to the disease. One good example illustrating the need to control for associated variables is obesity. Obesity is clearly associated with an increased risk of atherosclerosis, however, this association seems to be mediated in large part by abnormalities in diabetes, hypertension, and lipoproteins; all known risk factors for atherosclerosis. The purpose of this discussion is not to provide a primer in epidemiology. Rather, this section will focus on established risk factors and their contribution to the development of atherosclerosis. Cigarette smoking. The link between cigarette smoking and heart disease dates back to the 1940s and 1950s when a series of studies unequivocally linked smoking and heart disease (English et al., 1940; Doll and Hill, 1956; Hammond and Horn, 1958). More recently, the Surgeon General's report estimates that smoking increases atherosclerotic disease by more than 50% and doubles the incidence of coronary heart disease (US Department of Health and Human Services, 1989). This excess risk of smoking on coronary disease is readily reduced through smoking cessation. In fact, the risk of heart attack in ex-smokers declines to almost that of non-smokers over two years (Gaziano, 1996). Hypertension. Hypertension is de®ned as a systolic blood pressure in excess of 140 mm Hg or a diastolic blood pressure above 90 mm Hg (Joint National Committee on Detection, 1993). Current estimates indicate that hypertension is more prevalent among blacks than whites, and among men than women, with approximately 30% of the American population qualifying as hypertensive. The elderly also appear to have an increased predilection to hypertension with up to 75% of people over 75 yrs of age qualifying for the risk factor (Joint National Committee on Detection, 1993). There appears to be an approximately linear relation blood pressure elevation and the increased incidence of atherosclerotic vascular disease with an increase of 7 mm Hg and diastolic blood pressure corresponding to a 27% increase in myocardial

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infarction, and 42% increase in stroke (MacMahon et al., 1990). Antihypertensive therapy has proven most e€ective in reducing stroke, with a 5±6 mm mercury reduction in blood pressure corresponding to a 42% reduction in the risk of stroke, but only a 14% reduction in the risk of myocardial infarction (Collins et al., 2000). Diabetes mellitus. Atherosclerotic coronary disease is a major complication of diabetes mellitus, a diagnosis that encompasses approximately 14,000,000 people in the United States. In patients with diabetes the risk of coronary atherosclerosis is 3±5 fold greater than non-diabetics despite controlling for other risk factors (Bierman, 1992; Pyorala et al., 1987). A number of other known risk factors for coronary disease such as hypertension and abnormal lipids are also more common in diabetics than the general population (Bierman, 1992), but despite this association, no more than 25% of the excess coronary atherosclerosis risk from diabetes can be attributed to these known risk factors (Nishigaki et al., 1981). Thus, diabetes represents a major contributing factor to atherosclerosis in the developed world and much of this excess risk has escaped quanti®cation using traditional risk factor analysis. Although improved glucose control in diabetes is associated with a reduction in a number of diabetic complications, coronary atherosclerosis is not reproducibly among these complications reduced by improved diabetic control. Serum cholesterol. The association between cholesterol and atherosclerosis is unequivocal. Among the strongest evidence for this association is an experiment of nature. Familial hypercholesterolemia is an autosomal dominant disorder that affects approximately one in 500 persons from the general population. Heterozygotes for this disease manifest a 2±3 fold elevation in plasma cholesterol that is solely due to an elevation in LDL. The molecular basis for this disease is now well understood and relates to functional defects in the LDL receptor that interfere with LDL clearance. Homozygotes for this disorder demonstrate a 4±6 fold elevation in plasma cholesterol. One of the major features of FH disease is precocious atherosclerosis. In heterozygotes, 85% of individuals have experienced a myocardial infarction by the age of 60, and this age is reduced to 15 in patients who are homozygous for the disease. A similar defect has been reported in rabbits that is also associated with precocious atherosclerosis. With respect to patients in general, approximately 50% of all Americans between the ages of 20 and 74 have cholesterol levels that exceed 200 mg/dl (Lehr et al., 1999). The excess risk of increased LDL cholesterol appears to be linear with a 1% increase in serum cholesterol corresponding to a 2% increase in the risk of coronary heart disease. This risk can be lowered greatly by cholesterol lowering therapy, and with the advent in the last 10 yrs of HMG CoA reductase inhibitors. There has been a dramatic improvement in the morbidity and mortality from cardiovascular disease in patients with hypercholesterolemia. Since most models of atherosclerosis employ LDL cholesterol as a central feature, the remainder of this review will be written from the standpoint that LDL cholesterol is a necessary feature of atherosclerosis. While this may not be true in all respects, it serves as a reasonable working hypothesis to use as a framework for discussion of the known features of both atherosclerosis and the clinical manifestations of coronary vascular disease.

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2.2. Morphologic features of atherosclerosis 2.2.1. The normal artery The arterial wall (Fig. 1) normally consists of three well-de®ned concentric layers: the innermost layer is called the intima, the middle layer is called the media, and the outermost layer is known as the adventitia. These three layers are demarcated by concentric layers of elastin known as the internal elastic lamina (which separates the intima from the media), and the external elastic lamina (which separates the media from the adventitia). The luminal surface of arteries is lined by a single contiguous layer of endothelial cells that sits upon a basement membrane of extracellular matrix, and is bordered by the internal elastic lamina. Endothelial cells are attached to one another by a series of interconnections known as junctional complexes. The amount of extracellular matrix and elastin in the internal elastic lamina is most prominent in medium and largesized arteries. The endothelial cells form a dynamic barrier between the luminal surface of the artery and the stroma of the arterial wall. At one time it was thought that endothelial cells were quite passive in their function, but it is now known that endothelial cells regulate a wide array of functions in the arterial wall including thrombosis, vascular tone, and leukocyte tracking within the arterial wall. Progressing outwards from the internal elastic lamina, the media consists also of a single cell type, in this case, smooth muscle cells. There can be one or many layers of smooth muscle cells depending on the size of the artery. Cells are held together by an extracellular matrix comprised largely of elastic ®bers and collagen, and cells may also be attached together by junctional complexes. The extracellular matrix within the arterial wall is produced predominantly by the smooth muscle cell. This includes collagen, proteoglycans, and elastic ®bers.

Fig. 1. Cross-section of a normal artery.

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The media can vary in size considerably based on the function of a given artery. For example, in very small arteries the media may only be one cell thick, or not present at all, whereas in large arteries like the aorta the media may be many cell layers thick and consist of considerable elastin as part of the extracellular matrix. The principal reason for this di€erence relates to the requirement for elastic recoil during diastole, the period when the heart is not beating. Beyond the external elastic lamina, the adventitia is the outermost layer of the artery. The adventitia typically consists of a loose matrix of elastin, smooth muscle cells, ®broblasts, and collagen. Most of the neural input into blood vessels also traverses the adventitia. At one time, the adventitia was considered quite passive in nature, but contemporary thinking now includes adventitial ®broblasts in the arterial response to insult. 2.2.2. Gross morphology Atherosclerosis typically is manifested in three stages known as early, developing, and mature lesions. Early lesions are characterized by nodular areas of lipid deposition that have been termed ``fatty streaks'' morphologically. These represent lipid®lled macrophages and smooth muscle cells in focal areas of the intima. Fat-soluble dyes readily demonstrate these areas for gross morphologic examination and the principal lipid stained by these fat-soluble dyes is cholesteryl oleate. These early lesions typically develop by age 10, and increase to occupy as much as 1/3 of the aortic surface in the third decade. Developing lesions are sometimes called pearly plaques and represent the next stage beyond fatty streaks. They can be found initially in areas of the coronary arteries, abdominal aorta, and some aspects of the carotid arteries in the third to fourth decade of life. These ®brous plaques are dome-shaped and ®rm, and are covered by a ®bromuscular layer known as a ``cap''. Ultimately, lesions may progress to become complicated and advanced, and these are characterized by calci®ed ®brous areas of the artery with visible ulceration. These are the types of lesions that are often associated with symptoms or arterial embolization. It was once thought that end-organ damage and infarction was due to gradual advancement of these lesions, but we now know a number of functional components in the arterial wall are also important determinants of end-organ damage. 2.3. Response-to-injury hypothesis Based on autopsy studies, it had been known for some time that thrombosis and lipids were involved in atherosclerosis. On of the earliest theories involving thrombosis was the ``incrustation'' theory of Rokitansky (1852) that suggested intimal thickening results from ®brin deposition in the arterial wall. The lipid component of atherosclerosis was encompassed by a competing contemporary hypothesis o€ered by Virchow in 1858 (Virchow, 1989) who proposed that lipid transudation into the arterial wall and complexation with mucopolysaccharides was the initial event in atherosclerosis. Principally as a function of his work on smooth muscle cell

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proliferation, Ross eventually proposed the ``response to injury'' hypothesis of atherosclerosis (Ross et al., 1977). The conceptualization of atherosclerosis as an injurious event to the arterial wall really brought to bear the idea that the development of lesions and the progression of atherosclerotic disease were a dynamic event rather than one characterized by senescence. In 1973, Ross and Glomset (1973) attempted to reconcile a number of observations into a unifying hypothesis they termed the ``response-to-injury''. In this hypothesis, they proposed that the initial step in atherogenesis was endothelial denudation leading to a number of compensatory responses that alter the normal vascular homeostatic properties. For example, this injury increases the adhesiveness of the endothelium for leukocytes and platelets, and tips the local environment from an anticoagulant milieu to a procoagulant one. The recruited leukocytes and platelets release a number of cytokines, vasoactive agents, and growth factors setting the stage for an in¯ammatory response. This in¯ammatory response is characterized by migration and proliferation of smooth muscle cells from the media that become admixed with the in¯ammatory area to form an intermediate lesion. The macrophages recruited to the arterial wall take up deposited LDL lipid to form lipid-laden macrophages also known as foam cells. This is the hallmark of an early atherosclerotic lesion. The accumulation of lipid-laden foam cells perpetuates an in¯ammatory response leading to a localized collection in the arterial wall not unlike an abscess that would be observed in other tissues. This space-occupying lesion may encroach upon the arterial lumen, but the artery can compensate up to a point such that the arterial lumen remains unaltered, the so-called Glagov e€ect (Glagov et al., 1987). The principal mediators of this early in¯ammatory response are macrophages and tea lymphocytes (Jonasson et al., 1986; van der Wal et al., 1989). Within this localized lesion continued in¯ammation can lead to cellular necrosis and further recruitment of monocytes and lymphocytes with a concomitant release of cytokines, growth factors, and proteolytic enzymes. This can set the stage for focal necrosis within the lesion, and can cause autocatalytic expansion of the lesion. As the lesion enlarges the artery can no longer compensate for the encroachment in the lumen, and at some point ¯ow is impaired. The response-to-injury hypothesis was originally based on the notion of endothelium desquamation as a principal event initiating atherosclerosis (Ross and Glomset, 1976). More recently however, it has become clear that developing atherosclerotic lesions are covered by an intact endothelial cell layer, and that endothelial desquamation is the exception rather than the rule. In a recent re®nement to his initial hypothesis, Ross proposed that endothelial dysfunction is sucient to initiate atherogenesis through increased endothelial permeability to atherogenic lipoproteins (Ross, 1999). This hypothesis is not without its problems. It is clear that even in normal artery segments, the rate of LDL entry into the arterial wall exceeds the rate of accumulation (Carew et al., 1984), suggesting that endothelial dysfunction is not strictly necessary for atherogenic lipoprotein entry into the arterial wall. In fact, the accumulation of atherogenic lipoproteins in the arterial wall appears to be concentrated in areas that are predisposed to future lesion development even though

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the rate of entry is similar to normal sites (Schwenke and Zilversmit, 1989a,b). These studies and several others (Schwenke and Carew, 1989a,b; Falcone et al., 1984) indicate that lesion-prone arterial sites show an enhanced retention of atherogenic lipoproteins containing apo B. Such shortcomings in the response-to-injury hypothesis have prompted alternative hypothesis for the initiation of atherosclerosis that will be discussed below. 2.4. Oxidative modi®cation hypothesis of atherosclerosis 2.4.1. LDL does not support foam cell formation Since macrophages were identi®ed as the predominant cell type given rise to foam cells, the hallmark of early lesions, it is not surprising that an intensive e€ort was undertaken to investigate the mechanisms of foam cell formation. Despite the undeniable association of elevated LDL cholesterol levels in atherosclerosis, it is interesting that LDL particles in and of themselves do not appear to be atherogenic in vitro. Initially investigators anticipated that foam cell formation was mediated by the LDL receptor. However, this assumption turned out to be incorrect. One clue to the problem with this assumption is derived from patients with FH disease. These patients lack functional LDL receptors; nevertheless, they manifest precocious atherosclerosis as early as the ®rst decade of life. Therefore, it seems unlikely that a functional LDL receptor in and of itself is required for foam cell formation. Moreover, incubation of LDL with normal macrophages possessing normal LDL receptors does not support foam cell formation (Goldstein et al., 1979a). In fact, high concentrations of LDL generally lead to down-regulation of the LDL receptor as receptor-mediated endocytosis is tightly regulated (Goldstein et al., 1979a). In searching for alternative LDL receptors, Brown and Goldstein observed that chemical modi®cation of LDL in the form of acetylation leads to foam cell formation when incubated with macrophages. The uptake of this chemically modi®ed LDL was shown to take place by way of a saturatable, speci®c receptor later termed the ``acetyl-LDL receptor'' (Goldstein et al., 1979a). This receptor is now one of many so-called ``scavenger receptors'' that are present on macrophages and other cell types (Krieger et al., 1993). In their original paper, Brown and Goldstein (Goldstein et al., 1979a) could not identify any known endogenous means of LDL acetylation in vivo. They did, however, speculate that other modi®cations of LDL as yet undiscovered may facilitate recognition of LDL by the acetyl-LDL receptor. This prediction proved prescient as Henriksen and colleagues (Henriksen et al., 1981) found that incubation of LDL with endothelial cells modi®ed LDL in such a way that it served as a ligand for foam cell formation and macrophages. It is now well established that there is a large family of so-called ``scavenger'' receptors present on macrophages and other cells (Krieger, 1997). The original acetyl-LDL receptor has since been cloned and is now known to exist in two forms termed scavenger receptor A1 and A2 (Kodama et al., 1990). Many other scavenger receptors have also been identi®ed including CD68, CD36, SR-B1, LOX-1, among others (Krieger, 1997). Considerable evidence does suggest that these receptors play a role in atherosclerosis. Mice lacking the SRA gene

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demonstrate a defect in the binding and degradation of modi®ed LDL (Krieger, 1997). In order to investigate the role of SRA receptors in atherosclerosis, these mice were crossed into a well-established mouse model of atherosclerosis, namely mice lacking the apolipoprotein E gene. Mice lacking the apo E gene typically demonstrate atherosclerosis when placed on a high fat diet (Suzuki et al., 1997). However, when these mice also lack the SRA receptor, the extent of atherosclerosis is significantly inhibited (Suzuki et al., 1997). Similarly, macrophages derived from mice lacking the CD36 scavenger receptor demonstrate reduced uptake of modi®ed LDL (Nozaki et al., 1995) and reduced atherosclerosis when crossed with the apo E null mouse (Febbraio et al., 2000). Thus, scavenger receptors appear to play an important role in the development of atherosclerosis. It remains to be seen what the precise contribution of each class of scavenger receptors and whether genetic variations in these receptors have any implications for the development of atherosclerosis and its clinical events in a patient population. One obvious question with the discovery of the family of scavenger receptors is their precise role that favored their persistence throughout evolution. This appears to involve the recognition and disposal of a variety of modi®ed biomolecules. The current theory of the oxidative modi®cation hypothesis states that LDL becomes oxidized in the arterial wall where it then lends itself to cellular uptake and foam cell formation. Of critical importance to this hypothesis is the mechanism of LDL oxidation both in vitro and in vivo. We know considerably more about the former than the latter, and in the ensuing pages we will discuss what is known about the mechanisms of LDL oxidation. 2.4.2. Mechanisms of LDL oxidation 2.4.2.1. Structure of LDL. The precise characterization of LDL oxidation has been problematic. One, this is largely a function of both the complexity and heterogeneity of human LDL both amongst individuals and in response to dietary variation. Human LDL is a particle containing both lipid and protein that is typically isolated by ultracentrifugation between the densities of 1.019 and 1.063 g/ml (Gotto and Farmer, 1988). Physically, LDL is spherical with a diameter that ranges between 19 and 25 nm, and a molecular weight between 1.8 and 2.8 million (Keaney and Frei, 1994). The lipid composition of LDL is outlined in Table 1. Given an assumed molecular weight of approximately 2.5 million, an LDL particle consists of a lipophilic core containing approximately 1600 molecules of cholesteryl ester, and 170 molecules of triglyceride (Esterbauer et al., 1990). The LDL particle surface is also embraced by a single apolipoprotein B-100 (apo B). Apo B is a glycosylated with approximately 4500 amino acids corresponding to a molecular weight of 550,000 kDa (Gotto and Farmer, 1988). With respect to the lipid classes within an LDL particle, a prototypical particle contains 2700 fatty acid molecules (Esterbauer et al., 1990; Yla-Herttuala et al., 1998), about half of these fatty acids are polyunsaturated fatty acids (PUFAs) with the predominant PUFAs being linoleic acid (18:2) and arachidonic acid (20:4), and a relatively minor quantity of docosahexaenoic acid (22:6) Table 1 (Esterbauer et al.,

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Table 1 Lipid composition of human LDLa nmol/mg LDL protein Mean  S.D. Total phospholipids Phosphatidylcholine Lysophosphatidylcholine Sphingomyelin

1300  227 818  143 145  25 336  59

Fatty acids Linoleic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Arachidonic acid Docosahexaenoic acid Free fatty acids Triglycerides Free cholesterol Total cholesterol Conjugated dienes

2000  541 1260  375 80  44 260  118 825  298 278  100 53  31 48 304  140 1130  82 4090 Not detectable

a

mol/mol LDL Mean 700  122 450  78 80  14 185  32 2700 1100  298 693  206 44  24 143  65 454  164 153  55 29  17 26 170  78 600  44 2200

Taken from Keaney and Frei (1994).

1987). The content of PUFAs can vary considerably amongst LDL populations on an individual basis with some reports demonstrating that the content of linoleic acid may vary as much as 100% (Esterbauer et al., 1990). The importance of this fact is emphasized by the knowledge that LDL lipid peroxidation is generally restricted to PUFAs and therefore, LDL samples may vary considerably with respect to their susceptibility for oxidative modi®cation. Another important characteristic of LDL-associated antioxidants (Table 2) is that, by nature, they are lipid soluble. The most abundant lipid soluble antioxidant in LDL by far is a-tocopherol with approximately 6±8 molecules per particle. All of the other lipid soluble antioxidants are presents at amounts <1 molecule per LDL particle. These include c-tocopherol, uniquinol-10, b-carotene, lycopene, cryptoxanthine, and a-carotene (Table 2). Of these lipid soluble compounds, it appears Table 2 Antioxidant content of human LDLa Antioxidant

nmol/mg LDL protein Mean  S.D.

mol/mol LDL Mean

Vitamin E (a ‡ c-tocopherol) Ubiquinol-10 b-carotene Lycopene Cryptoxanthine a-carotene

15.5  2.9 0.65  0.28 0.53  0.47 0.41  0.20 0.25  0.23 0.22  0.25

7.95 0.33 0.27 0.21 0.13 0.11

a

Values are derived from Esterbauer et al. (1992), Frei and Gaziano (1993).

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only that a-tocopherol and ubiquinol-10 possess important antioxidant activity within the LDL particle. Since most of these compounds are contained in the diet, one might expect the content of lipid soluble antioxidants will vary considerably among individuals as a function of several factors including diet and the rate of fat absorption. 2.4.2.2. Stages of LDL oxidation. The oxidative modi®cation of LDL has been arbitrarily divided into three stages. The ®rst is known as the initiation of lipid peroxidation and involves the initial formation of radical species within the particle. The second stage is known as the propagation stage of oxidation and represents the portion of LDL oxidation involving a chain reaction (i.e., each radical produced in the particle yields more that one subsequent radical). The ®nal stage of LDL oxidation is known as decomposition predominantly because lipid hydroperoxides formed within the LDL particle decompose into reactive aldehydes and ketones. This leads the modi®cation of the apo B moiety of LDL and changes the net charge of LDL. This division of LDL oxidation into stages is quite arbitrary and used for conceptual purposes only. The actual oxidation process is a continuum that is not readily divided into such distinct stages. A stylized scheme of lipid peroxidation is contained in Fig. 2. The earliest event in the modi®cation is the initiation of lipid peroxidation shown in the ®gure as abstraction of hydrogen from a PUFA. In this case the oxidant is hydroxyl radical, an extremely potent free radical with a very high reactivity and short half-life (10ÿ9 s). The predilection for an oxidant attacking a hydrogen bound to a carbon that is ¯anked by two double bonds is based on the carbon hydrogen bond energy. The hydrogen shown in Fig. 1 is termed a bis-allylic methylene group and the ¯anking double bonds reduce the carbon hydrogen bond energy to between 75 and 70 kcal/ mol (Wagner et al., 1994). Despite all our knowledge about lipid peroxidation, the precise entity responsible for the initiation of lipid peroxidation in biologic systems is not known. A number of species have been proposed that include hydroxyl radical, Fe2‡ =Fe3‡ =O2 (Minotti and Aust, 1987), peroxynitrite, tyrosyl radical, and even enzyme systems such as lipoxygenase. This question of initiation is extremely important since LDL isolated from plasma is generally free of pre-formed of lipid hydroperoxides based on the most sensitive techniques (Sattler et al., 1994). Once lipid hydroperoxides have been established in the LDL particle, it is relatively easy to generate radical species especially in the presence of metal ions as shown in Eqs. (1) and (2). LOOH ‡ Me…n‡1† ‡ ! LOOá ‡ Men‡ ‡ H‡

…1†

Men‡ ‡ LOOH ! LOá ‡ OH ‡ Me…n‡1†

…2†

In lipid systems that contain trace amount or preformed lipid hydroperoxides (LOOH), metal ions (Me) such as copper or iron can catalyze the decomposition of lipid hydroperoxides into peroxyl …LOOá † and alkoxyl …LOá † radicals as shown above. These peroxyl and alkoxyl radicals readily react with other adjacent bis-allylic

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Fig. 2. Scheme for lipid peroxidation. In this scheme, hydroxyl radical initiates lipid peroxidation through the abstraction of hydrogen (A) from a bis-allylic methylene group in a polyunsaturated fatty acid (PUFA). The carbon-centered radical so formed undergoes molecular rearrangement to form a conjugated diene compound exhibiting UV absorbance at 234 nm (B). The carbon-centered radical then reacts readily with molecular oxygen to form a lipid peroxyl radical (C) that may then propagate lipid peroxidation through the abstraction of hydrogen from an adjacent PUFA forming both a lipid hydroperoxide and another carbon-centered radical (D), the lipid peroxyl radical.

methylene groups (LH) by extracting hydrogen atoms and forming lipid hydroperoxides and lipid hydroxides, as well as carbon-centered radicals by the scheme outlined below LOOá ‡ LH ! LOOH ‡ Lá

…3†

LOá ‡ LH ! LOH ‡ Lá

…4†

Lá ‡ O2 ! LOOá

…5†

This decomposition of lipid hydroperoxides and the secondary generation of alkoxyl and peroxyl radicals has been termed re-initiation of LDL lipid peroxidation (Gokce and Frei, 1996) since the existence of pre-formed hydroperoxides within the LDL particle indicates prior oxidative events (van der Wal et al., 1989). Whatever the mechanism, the introduction of a free radical into a lipoprotein particle by nature must generate a chain reaction. The reason for this is quite straight forward, free radicals contain unpaired electrons whereas non-radical species (such as those in an LDL particle) are spin-paired. The product of such a reaction must, invariably, contain an uneven number of electrons and result in the production of a free radical. This radical product must then react with another non-radical species, and by this

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mechanism, a single radical will produce damage to a number of subsequent PUFAs and hence the term ``chain reaction''. If one traces the sequence outlined in Fig. 2, one can see that the carbon-centered radical rapidly takes up oxygen to produce a lipid peroxyl radical (C) that may abstract a hydrogen from an adjacent PUFA regenerating a lipid hydroperoxide and another carbon-centered radical to begin the cycle of events again (Fig. 1). This chain reaction portion of LDL oxidation takes place during the propagation phase, which is characterized by the consumption of polyunsaturated fatty acids (PUFAs) and the formation of lipid hydroperoxides within the LDL particle. Lipid hydroperoxides, in particular phospholipid hydroperoxides, formed within the LDL particle can be hydrolyzed to lysophospholipids, and fatty acid hydroperoxides by a phospholipase A2 activity that has is intrinsic to apo B (Parthasarathy and Barnett, 1990), or a platelet activating factor (PAF) acetylhydrolaselike activity that also appears to be associated with LDL (Steinbrecher and Pritchard, 1989). Such decomposed hydroperoxides can undergo beta-scission reactions that form aldehyde compounds, and those measured during this process include 4-hydroxynonenal, malondialdehyde, and 2,4-heptadienal (Esterbauer et al., 1987). Aldehydes have a predilection for amino groups and readily form Schi€ bases with the lysine groups in apo B (Haberland et al., 1984; Steinbrecher, 1987). The net result of these reactions is to change the charge of apo B, as lysine residues are positively charged at physiologic PH and their charge is lost during the formation of a Schi€ base. This is manifested as an increased electrophoretic mobility of LDL upon electrophoresis, and this change in charge is important with respect to LDL receptor recognition. In particular, modi®ed apo B with its increased net negative charge is no longer recognized by the apo B/E LDL receptor (Brown and Goldstein, 1986) and instead recognized by any number of these ``scavenger receptors'' is in macrophages (Goldstein et al., 1979a; Sparrow et al., 1989; Freeman et al., 1991). Recognition by the scavenger family of receptor is essential for the formation of foam cells. As discussed above, receptor-mediated endocytosis of native LDL is tightly regulated whereas scavenger receptor-mediated LDL uptake is not. Uptake of modi®ed LDL by the scavenger receptor pathway is at least 3±10 fold more ecient than native LDL uptake (Henriksen et al., 1983). 2.4.2.3. Oxidation of LDL in cell-free systems. With its relatively high content of PUFAs, LDL oxidation does occur spontaneously (Gurd, 1960), but also requires many months of low temperatures (Lee, 1980). Early investigations into the oxidation of LDL involved the relative ecacy of a number of metal ions in producing LDL oxidation (Ray et al., 1954) with the most e€ective agent identi®ed being copper (Ray et al., 1954; Nichols et al., 1961), and the demonstration that metal ion chelators such as ethylenediaminetetraacetic acid (EDTA) e€ectively inhibited LDL oxidation (Ray et al., 1954; Schuh et al., 1978). Subsequent investigation demonstrated that the protein moiety of LDL also underwent modi®cation initially described as degradation (Schuh et al., 1978).

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2.4.2.4. Metal ion-induced LDL oxidation. The precise mechanisms involved in metal ion-induced LDL oxidation have been dicult to elucidate. As shown in Fig. 2, lipid peroxidation is initiated by the abstraction of a bis-allylic hydrogen atom from LDL PUFAs. With respect to metals, there is no chemical evidence that copper or iron are capable of abstracting hydrogen atoms directly, and initiated lipid peroxidation (Halliwell and Gutteridge, 1990). One proposed mechanism to get around this problem has been the participation of copper or iron in the generation of hydroxyl radical from hydrogen peroxide via Fenton chemistry, as shown below. ‡ 2Oáÿ 2 ‡ 2H ! H2 O2 ‡ O2

…6†

H2 O2 ‡ Me…nÿ1†‡ !á OH ‡ OHÿ ‡ Men‡

…7†

n‡ ! O2 ‡ Me…nÿ1†‡ Oáÿ 2 ‡ Me

…8†

However, there are several problems with this model. The equations outlined above demonstrate that superoxide is a critical component of this model and consistent with this assumption, SOD has been shown to inhibit copper-mediated LDL oxidation in vitro (Lynch and Frei, 1993; Parthasarathy et al., 1989). However, careful examination reveals that this e€ect may be due to non-speci®c binding of copper ions to SOD in a redox-inactive form rather than dismutation of superoxide (Jessup et al., 1993). Another problem with the model relates to observations that catalase does not appear to inhibit iron- or copper-induced LDL oxidation (Lynch and Frei, 1993), yet hydrogen peroxide is required for the Fenton reaction (Hammond and Horn, 1958) depicted above. Finally, ecient scavengers of aqueous hydroxyl radicals appear ine€ective in inhibiting metal ion-induced LDL oxidation (Lynch and Frei, 1993). One alternative to the initiation of lipid peroxidation by hydroxyl has been the metal catalyzed breakdown of pre-formed lipid hydroperoxides as described in Eqs. (1) and (2). However, the existence of pre-formed lipid hydroperoxides in LDL is controversial. While some investigators claim hydroperoxides are present in vivo (Esterbauer et al., 1992), many of these ®ndings could be due to the method of LDL isolation. In fact, rapid isolation of LDL from plasma produces lipoproteins that contain no detectable hydroperoxides (Sattler et al., 1994), whereas traditional prolonged methods of LDL isolation produce abundant quantities of lipid hydroperoxides in LDL (Shwaery et al., 1998). Furthermore, it appears that pre-formed hydroperoxides are not necessary for lipid peroxidation as removal of all hydroperoxides in LDL with the synthetic peroxidase ebselen generates hydroperoxide-free LDL that is still susceptible to copper- and iron-mediated oxidation (Lynch and Frei, 1993). Once the initiation of lipid peroxidation is established, propagation of lipid peroxidation is readily accomplished by copper or iron as outlined above in Eqs. (1) and (2). This process appears to be quite dependent upon the relative distribution of oxidized versus reduced transition metal ions (Minotti and Aust, 1987). For example, incubation of LDL with Cu2‡ is associated with its rapid reduction to Cu1‡ while co-incubation of LDL with Fe3‡ does not yield as ecient a reduction of iron (Lynch

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and Frei, 1995). In fact, LDL appears to have considerable capacity for the reduction of copper, and one might wonder where all the reducing activity comes from. This quandary was addressed recently when it was found that a-tocopherol serves as a reductant for copper in human lipoproteins and is responsible for triggering the initiation of LDL oxidation (Kontush et al., 1996). One potential problem with metal ion-induced LDL oxidation is the fact that the existence of extracellular free metal ions or their low-molecular-weight chelates has not been de®nitively established. Redox-active metal ions have been found in homogenates of atherosclerotic tissue (Smith et al., 1992; Swain and Gutteridge, 1995; Lamb et al., 1995), but comparable treatment of normal tissue has not been examined. Low concentrations of albumin also inhibit metal ion-dependent LDL oxidation (Thomas, 1992), and albumin is the most abundant protein in plasma. Thus, one cannot be con®dent that the requirements for Fenton chemistry can be met in the extracellular space of the arterial wall. 2.4.2.5. Superoxide and LDL oxidation. Superoxide is the one electron reduction product of molecular oxygen and its production has been implicated in LDL lipid peroxidation in vivo (Lynch et al., 1997). In¯ammatory cells produce superoxide as a means of aiding the host defense mechanism (Klebano€, 1980) and the biochemical basis for superoxide production by these cells is well understood. Activation of in¯ammatory cells is associated with NADPH oxidase activity that catalyzes the direct reduction of molecular oxygen to superoxide. Monocyte activation is known to promote LDL oxidation (Hiramatsu et al., 1987; Cathcart et al., 1989) and this reaction is prevented in the presence of superoxide scavengers. Most importantly, LDL oxidation by in¯ammatory cells appears to require superoxide production as cells defective in this feature are not able to promote LDL oxidation (Hiramatsu et al., 1987). Superoxide is implicated in LDL oxidation by other cells as well. In fact, cellmediated oxidation of LDL has been demonstrated by all the major cell types in the vascular wall including endothelial cells (Henriksen et al., 1981) and smooth muscle cells (Morel et al., 1984; Heinecke et al., 1984). Evidence supporting superoxide and cell-mediate LDL oxidation is derived from observations that it is inhibited by superoxide dismutase (Heinecke et al., 1986; Stenbrecher, 1988) and that superoxide generated enzymatically (Lynch and Frei, 1993), or by radiolysis (Bedwell et al., 1989) also promotes LDL oxidation in the presence of metal ions. However, these studies su€er from many of the same drawbacks as metal ion-mediated LDL oxidation since cell-mediated oxidation is almost uniformly inhibited in the presence of metal ion chelators. Thus, there is considerable doubt as to whether superoxide plays a role in oxidizing LDL in vivo consistent with the notion that superoxide at neutral PH has an absolute requirement for metal ions in the promotion of LDL oxidation (Lynch and Frei, 1993). 2.4.2.6. Lipoxygenase. The lipoxygenases are intracellular enzymes that add oxygen to polyunsaturated fatty acids (Yamamoto, 1992). These enzymes are present in all the major cell types of the arterial wall and have been observed in association with

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atherosclerotic lesions (Yamamoto, 1992; Witztum and Steinberg, 1991; Berliner and Heinecke, 1996). These ®ndings have fueled speculation that lipoxygenases may play an important role in the oxidation of LDL that leads to atherosclerosis. Such speculation has been bolstered by ®ndings that a combination of soy bean lipoxygenase and phospholipases oxidize LDL in vitro (Sparrow et al., 1988). Consistent with this speculation, lipoxygenase inhibitors prevented LDL oxidation by cultured cells (Parthasarathy et al., 1989) although these ®ndings are not without problems. For example, a number of lipoxygenase inhibitors are not speci®c, and they also block metal ion-mediated LDL oxidation suggesting they have signi®cant antioxidant properties (Jessup et al., 1991; Sparrow and Olszewski, 1991). The hypothesis that lipoxygenases contribute to LDL oxidation has recently been bolstered by observations that disruption of the lipoxygenase gene in mice diminishes atherosclerosis (Cyrus et al., 1999). Not withstanding this enthusiasm, one must question how an intracellular enzyme such as lipoxygenase facilitates LDL oxidation which is predominantly an extracellular process. Among the proposed mechanisms for this phenomenon include the idea that lipoxygenases produce lipid hydroperoxides that are then transferred into LDL thereby ``seeding'' LDL and facilitating re-initiation of lipid peroxidation as described above (Parthasarathy et al., 1989). If this hypothesis were correct, one would expect to ®nd lipoxygenase products in early atherosclerotic lesions, and this indeed is the case. The oxidation of LDL in vitro with 15-lipoxygenase exhibits a ratio of S/R stereoisomers for 13-hydroxyoctadecanoic acid (13-HODE) that is approximately 2.5/1. As expected, free radical-mediated LDL oxidation produces a ratio that approaches 1. In two studies examining lipids from atherosclerotic plaques, the S/R ratio of 13-HODE was 1.12 in advanced atherosclerotic lesions (Folcik et al., 1995), and 1.08 in early atherosclerotic lesions (Kuhn et al., 1997). These small, but statistically apparent increases in stereospeci®c 15-lipoxygenase products are consist with the participation of 15-lipoxygenase in LDL oxidation. 2.4.2.7. Glycoxidation reactions. As discussed above, diabetes is a potent risk factor for premature atherosclerosis. The increased risk of atherosclerosis associated with diabetes has not been well identi®ed, but one proposed mechanism includes excess glucose since hyperglycemia is a hallmark of diabetes. This so-called ``glucose hypothesis'' suggests that oxidation reactions induced by glucose help modify LDL to forms that support the initiation of atherosclerosis and lesion development. This hypothesis draws some support from studies showing that intensive glycemic control in type I diabetics produces reduced vascular complications (Kuhn et al., 1990). The molecular mechanism of glucose-mediated biological oxidation was ®rst identi®ed in 1912 by Maillard (1919). The Maillard reaction accounts for the glucose-dependent non-enzymatic modi®cation of proteins that accompanies hyperglycemia. Initially, this reaction involves the combination of the aldehyde group of glucose (present in the open-chain form) with amine groups in proteins to form a Schi€ Base followed by Amadori rearrangement to form the product fructoselysine. This process is reversible and underlies the formation of hemoglobin A1C , a wellrecognized marker of chronic glycemic control. The persistence of the fructoselysine

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produce may yield irreversible oxidation, or glycoxidation of fructoselysine to yield a host of products which have been termed in aggregate, advanced glycation endproducts (AGEs). These irreversible oxidation products have been found to correlate with the vascular and renal complications of diabetes, and thus represent an attractive source of oxidation to explain precocious atherosclerosis in diabetes (McCance et al., 1993). Although this may explain one mechanism for enhanced atherosclerosis in diabetes, it does not provide an adequate explanation for LDL modi®cation in atherogenesis in non-diabetic subjects. For this reason, other sources of oxidative stress capable of producing LDL oxidation are necessary. 2.4.2.8. Peroxynitrite. Peroxynitrite is a well-characterized product of the radical± radical combination reaction of superoxide and nitric oxide (Beckman et al., 1990). Peroxynitrite is a two-electron oxidant that oxidizes LDL lipids and converts LDL into a form that is recognized by macrophage scavenger receptors (Graham et al., 1993). The possibility that peroxynitrite or other reactive nitrogen species might support LDL oxidation during atherosclerosis has been examined in some detail. Atherosclerotic lesions contain detectable levels of 3-nitrotyrosine (a marker of peroxynitrite-mediated oxidation) that are 80-fold higher that in circulating LDL (Leeuwenburgh et al., 1997). This observation is consistent with the notion that peroxynitrite participates in the formation of oxidized LDL in the arterial wall. 2.4.2.9. Myeloperoxidase. In the host-defense response, phagocytes produce superoxide in such high concentrations that spontaneous dismutation into hydrogen peroxide is signi®cant (Fridovich, 1983). One target of this hydrogen peroxide is the phagocyte-secreted heme protein myeloperoxidase, which interacts with hydrogen peroxide to generate a host of antimicrobial species (Klebano€, 1980; Hurst and Barette, 1989). Active myeloperoxidase has been observed in human atherosclerotic lesions (Daugherty et al., 1994), and colocalizes with in¯ammatory cells. In vitro, myeloperoxidase readily oxidizes LDL, perhaps through the production of tyrosyl radical (Savenkova et al., 1994). Among the markers for tyrosyl radical formation is di-tyrosine, and it appears that tyrosyl radical may play a role in LDL oxidation in vivo since dityrosine in detected in LDL isolated from atherosclerotic lesions (Leuwenburgh et al., 1997). Other important products of myeloperoxidase are hypochlorous acid (HOCl) and chlorine gas. These oxidants produces chlorinated biomolecules, and therefore create relatively speci®c markers for their oxidative damage. 3-chlorotyrosine is formed in vitro in LDL incubated with myeloperoxidase-peroxide-chloride system (Hazen et al., 1996). If one isolates LDL from human atherosclerotic tissue, 3-chlorotyrosine is also readily detected suggesting a role for myeloperoxidase and these oxidants in the modi®cation of LDL (Hazen and Heinecke, 1997). 2.4.3. Proatherogenic actions of oxidized LDL In addition to its activity to support foam cell formation, oxidized LDL (oxLDL) also has a number of other proatherogenic properties that are outlined in Table 3. Among the early events of atherosclerosis is endothelial cell activation, and products

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Table 3 Potential proatherogenic activities of oxidized LDL (OxLDL)a             a

OxLDL has supports macrophage foam cell formation OxLDL-derived products are chemotactic for monocytes and T-cells and chemostatic for tissue macrophages OxLDL-derived products are cytotoxic and can induce apoptosis OxLDL is mitogenic for smooth muscle cells and macrophages OxLDL can alter in¯ammatory gene expression of vascular cells OxLDL can increase expression of macrophage scavenger receptors OxLDL can induce expression and activate PPARc , thereby in¯uencing many gene functions OxLDL is immunogenic and elicits autoantibody formation and activated T-cells Oxidation renders LDL more susceptible to aggregation, which independently leads to enhanced uptake. OxLDL is a substrate for sphingomyelinase, which aggregates LDL OxLDL may enhance procoagulant pathways by induction of tissue factor and platelet aggregation Products of OxLDL can aversely impact arterial vasomotor properties

Modi®ed from Tsimikas and Witztum (2000).

of oxLDL have been shown to facilitate this activation (Kume and Gimbrone, 1994; Khan et al., 1995). Hypercholesterolemia is a cardinal feature for experimental models of atherosclerosis, and it is associated with increased LDL entry into the arterial wall and reduced egress (Schwenke and Carew, 1989a,b). This is thought to be due to increased LDL retention within the arterial wall (see the response-to-retention hypothesis), and it is thought LDL undergoes oxidation while retained within the arterial wall. Once oxidized, LDL increases its substrate suitability for sphingomyelinase, an enzyme that is known to aggregate LDL and thereby enhance its uptake by macrophages (Williams and Tabas, 1998). Initial oxidation of LDL within the arterial wall forms a minimally modi®ed form of LDL (MM-LDL) that has a number of properties in and of itself. For example, MM-LDL stimulates adjacent endothelial cells and smooth muscle cells to synthesize and secrete monocyte chemotactic protein-1 (MCP-1) (Cushing et al., 1990). This local production of MCP-1 (Navab et al., 1991) as well as the expression of endothelial leukocyte adhesion molecules (Cybulsky and Gimbrone, 1991), are responsible for the recruitment of monocytes that undergo activation-di€erentiation in the subendothelial space to become macrophages. A role for MCP-1 in the recruitment of in¯ammatory cells is supported by the isolation of MCP-1 protein and mRNA from macrophage rich regions of human and rabbit atherosclerotic aorta (Yla-Herttuala et al., 1989). Moreover, evidence consistent with a requirement for MCP-1 in atherogenesis is derived from studies indicating that mice lacking the MCP-1-receptor demonstrate decreased lesion formation in two murine models of atherosclerosis (Boring et al., 1998; Gosling et al., 1999). This chemokine-mediated co-localization of monocytes and LDL in the subendothelial space provides a facile environment for more extensive LDL modi®cation. The formation of oxidized LDL in the subendothelial space also facilitates the progression of atherosclerosis through other mechanisms. OxLDL is chemotactic for monocytes (Quinn et al., 1987) and T lymphocytes (McMurray et al., 1993), perhaps

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through LDL accumulation of lysophosphatidylcholine during oxidation (Steinbrecher et al., 1984). Macrophages exposed to oxLDL demonstrate impaired mobility (Quinn et al., 1987) suggesting that oxLDL could prevent their egress from the arterial wall. Furthermore, the accumulation of oxidized LDL and its by-products within the arterial wall would be expected to produce impaired bioactivity of endotheliumderived nitric oxide (Kugiyama et al., 1990) that could, conceivably enhance endothelial permeability (Kubes and Granger, 1992) and stimulate local accumulation of LDL and monocytes (Kubes et al., 1991). OxLDL has also been shown to be immunogenic by eliciting the production of auto-antibodies (Parums et al., 1990; Salonen et al., 1992) and the formation of immune complexes that can also facilitate macrophage internalization of LDL (Klimov et al., 1985; Grith et al., 1988). The gene expression pattern in the arterial wall is also subject to in¯uence by modi®ed forms of LDL. For example, MM-LDL as well as oxLDL can induce macrophage scavenger receptor expression thereby enhancing foam cell formation and LDL uptake (Mietus-Snyder et al., 1997). A number of genes associated with in¯ammation are also upregulated by oxLDL such as SAA, ceruloplasmin, and heme oxygenase (Liao et al., 1993). In addition, a recent discovery is the e€ect of oxLDL on macrophage expression of peroxisome proliferator …PPARc † expression. This has been shown to alter scavenger receptor (CD36) expression and alter the expression of pro-in¯ammatory genes (Nicholson et al., 2000). 2.4.4. Evidence to support the oxidative modi®cation hypothesis Initially, there was considerable skepticism about the idea that LDL oxidation could occur in vivo. This skepticism stems from the knowledge that numerous antioxidants are present in plasma and extracellular ¯uid that prevent oxidation of LDL (Keaney and Frei, 1994; Dabbagh and Frei, 1995). There now are numerous lines of evidence to indicate that LDL oxidation does occur in vivo. For example, one can generate antibodies against the protein component of oxLDL. In particular, aldehyde-modi®ed epsilon amino groups of lysine such as malondialdehyde-lysine (MDA-lysine), or 4-hydroxynonenal-lysine (4-HNE-lysine) have been generated and used as a tool to determine if such epitopes exist in atherosclerotic lesions. Using this approach, a number of investigators have demonstrated that antibodies raised against such epitopes react with components of atherosclerotic lesions, and do not react with normal arterial segments (Palinski et al., 1989; Haberland et al., 1988; Palinski et al., 1990; Palinski et al., 1994; Hulten et al., 1996). With respect to human studies, compared to normal age-match controls, patients with coronary atherosclerosis have elevated levels of endogenous antibodies to oxLDL (Salonen et al., 1992). Furthermore, levels of plasma oxLDL are increased in patients who have su€ered an acute myocardial infarction compared with age-matched controls (Holvoet et al., 1995). The oxidation-speci®c epitopes discussed above are speci®c for the adduct (i.e., MDA-lysine or 4-HNE-lysine), but not for their existence in LDL per se. One could argue that the immunological evidence presented above actually corresponds to these epitopes on proteins other than apo B. To address this issue, Yla-Herttuala and colleagues extracted LDL from atherosclerotic lesions of humans and experimental

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119

animals (Yla-Herttuala et al., 1989). The compositional analysis of this LDL suggested that it closely resembled LDL produced ex vivo with copper or other oxidants. Most importantly, LDL isolated from these lesions demonstrated rapid uptake by the macrophage scavenger receptors in a manner that competed e€ectively for LDL that had been oxidized ex vivo (Yl a-Herttuala et al., 1989). Finally, if one examines atherosclerotic lesions directly for the presence of oxidized lipids, one can ®nd evidence of both lipid peroxidation (Suarna et al., 1995) and protein modi®cation (Leeuwenburgh et al., 1997) that are similar to those observed during ex vivo oxidation of LDL. Thus, there is clear evidence that LDL oxidation occurs in vivo. A more pressing question, however, is whether this oxidation of LDL is a prerequisite for atherosclerotic lesion formation and the progression of atherosclerosis. 2.4.5. Antioxidant studies Principally as a consequence of the oxidative modi®cation hypothesis, a number of studies have been performed to examine the antioxidant protection of LDL and ultimately, to apply this knowledge to animal studies in an attempt to inhibit atherosclerosis. Table 4 contains selected small-scale clinical trials of antioxidant supplementation in humans and its e€ect on ex vivo LDL oxidation. As one can appreciate, the bulk of the trials have been done with vitamin E as it is the major lipid-soluble antioxidant in human plasma and lipoproteins. In general, all of the trials have demonstrated that oral consumption of vitamin E results in an increased resistance of LDL particles to ex vivo oxidation. This e€ect appears to be observed with a number of doses (Dieber-Rotherneder et al., 1991). Although not shown in the table, most of the studies involving b-carotene alone did not demonstrate any particular e€ect of b-carotene to increase the resistance of LDL particles to oxidation (Reaven et al., 1993). Other lipid soluble antioxidants such as ubiquinol 10 (Mohr et al., 1992) and probucol (Reaven et al., 1992; Cristol et al., 1992) have demonstrated increased LDL resistance to oxidation in patients treated with these compounds. Thus, there is considerable precedent that oral supplementation with lipid-soluble antioxidants increases LDL particle resistance to oxidation. With this in mind, let us examine animal studies undertaken to examine the e€ect of antioxidant supplementation on atherosclerosis. 2.4.6. Animal studies 2.4.6.1. Vitamin E. Among the most obvious lipid soluble antioxidants to be studied for the prevention of atherosclerosis is vitamin E. Table 5 contains selected studies of vitamin E on the inhibition of atherosclerosis in experimental animals. It is interesting to note that many earlier studies of vitamin E supplementation and its e€ect on atherosclerosis predate awareness of the oxidative modi®cation hypothesis (Morgulis et al., 1938; Stamler et al., 1954; Beeler et al., 1962). Another feature of these earlier studies is a cholesterol-lowering e€ect of vitamin E in these experimental animals (Brattsand, 1975; Wilson et al., 1978; Westrope et al., 1982; Prasad and Kalra, 1993). This is particularly important as cholesterol reduction with vitamin E has produced a reduction in atherosclerosis, however, one cannot be convinced that

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Table 4 Selected human studies of antioxidant supplementation and LDL oxidationa Antioxidant(s) Dose

Weeks

N

LDL levels

Oxidative stress

E€ect on LDL oxidation

References

Smoking

+

Harats et al. (1990)

Cu2‡

+

Cu2‡

+ + + +

Dieber-Rotherneder et al. (1991)

Vitamin C

1.5 g/d

4

4

Vitamin E a-tocopherol

600 mg/d 150 IU/d

3

4 2

a-tocopherol

225 IU/d 800 IU/d 1200 IU/d 800 IU/d

12

2 2 2 12

a-tocopherol

1600 mg/d

20

7

" 2.5-fold

Cu2‡ , EC

+

" 54% " 106% " 53% " 141%

Jialal and Grundy (1992) Reaven et al. (1993)

(AT) b-carotene (BC) a-tocopherol+ b-carotene Vitamin C+ a-tocopherol+ b-carotene Vitamin C+

60 mg/d

12

8

" 19-fold

Cu2‡ , EC

)

1600 mg/d +60 mg/d 2 g/d+ 1600 mg/d +60 mg/d 900 mg/d+

12

8

+

8

8

24

22

AT " 1.3-fold Cu2‡ , EC BC " 22-fold Vitamin C n.d. Cu2‡ , EC AT " 1.3-fold BC " 37-fold Cu2‡

+

a-tocopherol+ b-carotene Ubiquinol-10

200 mg/d+ 18 mg/d 300 mg/d 11d

Abbey et al. (1993)

3

" 4-fold

AAPH

+

Probucol

250 mg/d

6 lg/mg protein 12 lg/mg protein

Cu2‡ , EC

+

Mohr et al. (1992) Reaven et al. (1992)

Cu2‡

+

24

1 g/d Probucol

250 mg/d

11 7

16

26

+

Cristol et al. (1992)

a

Cu2‡ refers to copper ions in solution; AAPH refers to 2,2-azobis (2-amidinopropane) hydrochloride, an aqueous peroxyl radical generator; EC refers to endothelial cells in culture. N refers to number of subjects.

this is due to any of the antioxidant activity of vitamin E. This can be seen in Table 5, with the exception of the mouse studies, a large number of the latter studies did not demonstrate any material e€ect of vitamin E to reduce atherosclerosis. Studies in the transgenic mice have generally been positive for an e€ect of vitamin E to inhibit atherosclerosis although a few more studies will be required to see if this is a universal feature. The precise reasons behind such discrepant observations are not clear. Possible explanations included material di€erences in atherosclerosis between models, as well as altered antioxidant metabolism in these two species. Further study will be required to sort out such di€erences.

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Table 5 Selected studies of vitamin E and atherosclerosisa Study

Model

Vitamin E dose

% Change compared to no supplement Serum/plasma Lesion cholesterol area

Williams et al. (1992b) Prasad and Kalra (1993) Willingham et al. (1993) Kleinveld et al. (1994) Fruebis et al. (1995) Shaish et al. (1995) Kleinveld et al. (1995) Fruebis et al. (1997) Schwenke and Behr (1998) Crawford et al. (1998) Pratico et al. (1998) a

Modi®ed WHHL rabbits Cholesterol-fed rabbits WHHL rabbits WHHL rabbits WHHL rabbits Cholesterol-fed rabbits Oleate-fed WHHL rabbits Linoleate-fed WHHL rabbits WHHL rabbits Cholesterol-fed rabbits Cholesterol-fed LDLR-de®cient micej Apo E ()/)) mice

5000 mg/kg diet 40 mg/kg/d 2000 mg/kg diet 250 mg/kg diet 1000 mg/kg diet 100 mg/kg diet 250 mg/kg diet 250 mg/kg diet 1000 mg/kg diet 1375 mg/kg diet 1000 mg/kg diet 2000 mg/kg chow

)38 )23 )19 0 0 0 0 0 0 )17 0

)32 )73 0 0 0 0 0 0 0 0 )60

0

)66

All values for changes in cholesterol and lesion area are at study termination unless otherwise indicated.

The interpretation of animal antioxidant supplementation studies is not entirely straightforward. Many of these studies su€er from a lack of standardization as control diets amongst studies are not comparable. In particular, the tocopherol content of ``standard'' laboratory chows can vary quite considerably. This may have major implications on experimental ®ndings as reported in the literature (Lehr et al., 1999; Willy et al., 1995). 2.4.6.2. Probucol. Probucol is a cholesterol-lowering drug that is known to possess considerable antioxidant activity (Marshall, 1982; Parthasarathy et al., 1986). Early studies performed with probucol demonstrated that it inhibited the development of atherosclerosis in rabbits (Kritchevsky et al., 1971; Tawara et al., 1986), as well as monkeys (Wissler et al., 1983). Subsequent to these studies, probucol became the test compound for the oxidative modi®cation hypothesis (Kita et al., 1987; Carew et al., 1987). Kita and colleagues (Kita et al., 1987) treated Watanabe Heritable Hyperlipidemic (WHHL) rabbits with probucol and observed an 87% reduction in lesion area compared to those animals not treated with probucol. In addition, lipoproteins derived from probucol-treated rabbits were much more resistant to copper-mediated oxidation than LDL particles derived from the control animals (Kita et al., 1987). The authors concluded the reduction of atherosclerosis was due to an antioxidant e€ect, however, the treated group did demonstrate a 17% reduction in total cholesterol compared to the control group, complicating the analysis. This issue was dealt with in a subsequent study by Carew et al. (1987) in which the cholesterollowering e€ect of probucol was o€set by another treatment group with lovastatin, an HMG-CoA reductase inhibitor that lowers cholesterol without antioxidant protection. Both lovastatin and probucol lowered the total cholesterol in WHHL rabbits

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similarly, however, probucol treatment provided an additional 48% reduction in the extent of atherosclerosis compared to lovastatin (Carew et al., 1987). These data were taken a strong evidence that probucol inhibits atherosclerosis independent of cholesterol-lowering with the idea that this additional e€ect was directly related to its antioxidant activity. A number of other studies have been conducted which also support the notion that probucol reduces atherosclerosis, perhaps, through its antioxidant activity. In one such study, WHHL rabbits were treated with a structural analog of probucol that had no cholesterol-lowering properties (Mao et al., 1991). Compared to probucol, both compounds inhibited LDL oxidation and both reduced atherosclerosis consistent with an antioxidant-mediated reduction in lesion formation. Subsequent studies in primates (Sasahara et al., 1994), cholesterol-fed rabbits (Shaish et al., 1995), and hamsters (Parker et al., 1995) have also demonstrated a reduction in atherosclerosis with probucol. Other antioxidants have also been tested for their ability to inhibit atherosclerosis. Once such compound is N ; N 0 -diphenyl-phenylenediamine (DPPD), an aniline compound that inhibits LDL oxidation ex vivo. Inclusion of DPPD in the diet of cholesterol-fed rabbits signi®cantly reduces atherosclerosis and cholesterol accumulation in the aorta (Sparrow et al., 1992). This ®nding is also associated with enhanced LDL resistance to oxidation ex vivo (Sparrow et al., 1992). Similar ®ndings have been extended to a murine model of atherosclerosis with a signi®cant inhibition of atherosclerosis and LDL oxidation in apo E (±/±) mice fed a high-fat diet (Tangirala et al., 1995). Similarly, combined supplementation of butylated hydroxytoluene and butylated hydroxyanisole (two lipid-soluble antioxidant compounds) prevents atherosclerosis in butter-fed rabbits (Wilson et al., 1978), whereas butylated hydroxytoluene alone reduces atherosclerotic lesions in cholesterol-fed rabbits (Byorkhem et al., 1991). Finally, Cynshi and co-workers (Cynshi et al., 1998) have tested a synthetic compound (BO-653) that contains structural components of both vitamin E and probucol for its e€ect on atherosclerosis. In both rabbit and murine models of atherosclerosis, BO-653 inhibits atherosclerosis signi®cantly (Cynshi et al., 1998). Thus, a number of lipid-soluble antioxidant compounds have been used to demonstrate an association between ex vivo, inhibition of LDL oxidation, and a reduction in atherosclerosis. Not all studies have been positive, and a number of inferences between antioxidant protection and inhibition of atherosclerosis have been indirect. This lack of direct evidence has presented a problem for the oxidative modi®cation hypothesis (for review, see Stocker, 1999). 2.4.7. Problems with the oxidative modi®cation hypothesis Despite the evidence outlined above supporting the role of LDL oxidation in atherosclerosis, some skepticism has arisen concerning an absolute requirement for LDL oxidation to initiate atherosclerosis. In particular, there has been diculty consistently linking the resistance of LDL to oxidation with the antiatherogenic action of certain antioxidants. For example, although probucol inhibits both LDL oxidation and atherosclerosis in cholesterol-fed (Shaish et al., 1995; Daugherty

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et al., 1989), and WHHL (Kita et al., 1987; Carew et al., 1987; Nagano et al., 1992) rabbits, vitamin E provides signi®cant protection against LDL resistance to oxidation without any e€ect of atherogenesis in the very same models (Shaish et al., 1995; Fruebis et al., 1995; Kleinveld et al., 1995). These ®ndings imply that manipulating ex vivo LDL resistance to oxidation does not necessarily translate into reduced atherosclerosis. Another complicating matter is the observation in one study demonstrated a reduction of atherosclerosis with b-carotene in cholesterol-fed rabbits without any change in LDL resistance to oxidation (Shaish et al., 1995). Thus, there is no secure evidence that measurements of ex vivo LDL resistance to oxidation predict the antiatherogenic activity of lipid-soluble antioxidants. One major problem with this analyses is linking an ex vivo assay (LDL resistance to oxidation) to events that occur within the vascular wall. To address this question, Witting and colleagues examined lipid peroxidation within atherosclerotic aorta and tested the e€ect of both probucol and its metabolite, bisphenol on atherosclerosis and lipid peroxidation in WHHL rabbits (Witting et al., 1999). In this study, both bisphenol and probucol enhanced the resistance of circulating LDL to oxidation. Both compounds strongly inhibited aortic accumulation of lipid oxidation products, however, only probucol had an inhibitory e€ect on atherosclerosis whereas bisphenol did not. More importantly, the extent of atherosclerosis in this study did not correlate with the aortic content of oxidized lipids. This study suggests that aortic accumulation of oxidized lipids is not a prerequisite for the initiation and progression of atherosclerosis in WHHL rabbits. These data question the absolute requirement for LDL oxidation in the initiation of atherosclerosis, and suggest a further study is needed to precisely de®ne of oxLDL in atherosclerosis. 2.4.8. Clinical studies A number of clinical trials have been conducted to explore the relation between antioxidant status and atherosclerosis and selected examples are contained in Table 6. There is currently a wealth of epidemiological data examining the interaction between antioxidant intake, or antioxidant status in the development of vascular disease. The data consists of a collection of descriptive studies, case-controlled studies, prospective cohort studies, and a limited, but growing number of randomized trials. 2.4.9. Evidence from descriptive studies Descriptive studies examine the characteristics of a population and its associated disease rates, and compare the data from one time period, or one country to the next. In general these studies su€er from the inability to control for potentially confounding factors such as di€erences in diet, genetics, and environmental factors. However, these studies may be quite valuable as they can generate hypotheses to be tested by more rigorous epidemiological means. A total of ®ve descriptive studies published since 1975 have shown in inverse association between fresh fruit and vegetable consumption in cardiovascular disease

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Table 6 Selected studies and randomized trials of antioxidants and vascular disease Study

Study population

Findings

Descriptive studies Verlangieri et al. (1985)

United States

Gey and Puska (1989)

Sixteen European regions

Inverse association of fruit and vegetable consumption and cardiovascular mortality rate Inverse association of mean plasma vitamin E concentrations and cardiovascular mortality

Case-control studies Riemersma et al. (1989) Ramirez and Flowers (1980) Prospective studies Nurses' Health Study (Stampfer et al., 1993) Health Professionals' Follow-up Study (Rimm et al., 1993) NHANES Study (Enstrom et al., 1992) Losonczy et al. (1996)

110 cases of angina and 394 normal subjects 101 cases of CAD on angiogram and 49 normal subjects

Lower plasma vitamin E concentrations in cases vs. controls Lower leukocyte ascorbic acid levels in cases vs. controls

87,000 US female nurses

Inverse association between CAD events and intake of vitamin E Inverse association between CAD events and vitamin E or b-carotene intake* Inverse association between cardiovascular mortality and vitamin C intake Reduced CAD events in subjects taking vitamin E vs. those not taking vitamin E

39,000 US male Health professionals 11,349 US men and women 11,178 elderly US citizens

Randomized, double-blind, Placebo-controlled trials ATBC (The 2900 Finnish male smokers ATBCILCPSG, 1994) Physicians' Health Study (Hennekens et al., 1996) CHAOS (Stephens et al., 1996) GISSI (GISSI Investigators, 1999) HOPE (Yusuf et al., 2000)

22,071 US male physicians 2002 British men and women with angiographic CAD 11,324 Patients with MI 9541 Patients at high risk for MI

No e€ect of either b-carotene or vitamin E on coronary artery disease events No e€ect of b-carotene on CAD events 77% reduction in CAD events with vitamin E as compared with placebo No e€ect of vitamin E, positive e€ect for PUFA No e€ect of vitamin E, positive e€ect for angiotensin converting enzyme inhibition with ramipril

rates (Acheson and Williams, 1983; Armstrong et al., 1975; Ginter, 1979; Gey and Puska, 1989). In some cases these studies examined the intake of certain vitamins (i.e., vitamin C, vitamin E) in cardiovascular disease mortality rates. The strongest theme from these studies is an observed trend across populations that fresh fruit and vegetable consumption tends to protect against cardiovascular disease. Whether this is due to the dietary intake of antioxidants or the replacement by fresh fruits and vegetables of potentially harmful dietary components (e.g., animal fats) cannot be determined of (Gaziano, 1999).

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2.4.9.1. Evidence from case control studies. Case control studies consist of data gathered retrospectively on dietary or lifestyle exposures of interest as well as data on a variety of potentially confounding variables. Individual cases are compared with appropriate controls in an attempt to isolate the e€ect of a variable of interest from potential confounders. Three such studies, one study each to support an association between vitamin E (Riemersma et al., 1991), vitamin C (Ramirez and Flowers, 1980), and tissue b-carotene levels (Kardinaal et al., 1993) in cases with cardiovascular disease compared to controls, suggest that natural antioxidants reduce the risk of cardiovascular disease. The strength of these conclusions is still limited, for example, the selection of cases and controls may introduce bias, and the chosen controls may also not adequately represent the intended population. Furthermore, uncharacterized or unknown variables may have a signi®cant impact on results that go unexamined. 2.4.9.2. Evidence from prospective cohort studies. Prospective studies o€er an advantage of measuring exposure of variables prior to the development of disease, which minimizes the impact of selection and recall bias. This study design also minimizes the e€ects that the disease may have on exposure related variables such as dietary habits. Data from two such studies, the Nurses Health Study (Stampfer et al., 1993), and the Health Professionals Follow-Up Study (Rimm et al., 1993) support an inverse association between the development of coronary artery disease events, and the intake of vitamin E. A third study conducted in elderly US citizens suggests an inverse relation between coronary artery disease events such as heart attack and stroke, and the dietary intake of vitamin E (Losonczy et al., 1996). Only one of these studies, the Health Professionals Follow-Up Study (Rimm et al., 1993) supported such an association for b-carotene. An association for other vitamins was not supported by these studies. 2.4.9.3. Randomized trials. A number of randomized trials have now been completed which examine the e€ect of antioxidant supplementation on cardiovascular events. Although not designed to look at cardiovascular events, the a-tocopherol, b-carotene (ATBC) trial examined the e€ect of b-carotene in vitamin E on 29,000 ®nish male smokers (The ATBCILCPSG, 1994). They found no e€ect of either vitamin on coronary artery disease events although there was a pro-carcinogenic e€ect of bcarotene in these smokers. Among the more celebrated antioxidant trials, the Cambridge Heart Antioxidant Study (CHAOS) trial (Stephens et al., 1996) examined the e€ect of vitamin E as secondary prevention for cardiovascular disease in 2002 British men and women with established coronary artery disease. In this trial treatment with vitamin E was associated with a 77% reduction in non-fatal heart attacks, but no change in mortality. Perhaps the most complete trial is the Heart Outcomes Prevention Evaluation (HOPE) trial (Yusuf et al., 2000). In this trial over 9000 men and women were randomized to vitamin E or placebo and followed for a mean of 412 years. In this study, there was no signi®cant e€ect of vitamin E to evaluate the e€ects of cardiovascular disease at a vitamin E dose of 400 IU per day.

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In summary then, there is no clear consensus that antioxidant supplementation appears to ameliorate the e€ects of atherosclerosis. The precise reasons for these observations are not yet clear, although several possibilities exist. First, it may be true that there is absolutely no relation between lipid peroxidation and the process of atherosclerosis. This seems highly unlikely given the available scienti®c evidence, however, it may be true that the relative importance of lipid peroxidation is modest. Another possibility relates to the dose response between antioxidants and any e€ect on cardiovascular disease. It could be that in well-nourished populations most individuals are already receiving the maximum bene®t of antioxidant treatment through a healthy diet. A ®nal possibility is that only certain individuals are in a state of heightened oxidative stress and would bene®t from antioxidant therapy. Since we have no good assays for identifying those patients su€ering from excessive oxidative stress, it stands to reason that indiscriminately treating patients with antioxidant therapy may not facilitate the detection of an e€ect. For whatever the reason, it is clear that general, routine, supplementation of individuals with lipid-soluble antioxidants in the hopes that cardiovascular disease will be prevented does not appear to be a viable strategy. 2.5. The response-to-retention hypothesis of early atherogenesis We have already discussed the problems associated with both the ``response-toinjury'' and ``oxidative modi®cation'' hypotheses of atherosclerosis. A number of studies suggest that retention of lipoproteins within the arterial wall has important implications for atherogenesis. In general, the rate of LDL entry into normal segments of the arterial wall exceeds the rate of accumulation (Carew et al., 1984) suggesting that LDL egress is the limiting factor for the accumulation of LDL in the arterial wall. Moreover, the accumulation of atherogenic lipoproteins in the arterial wall appears to be concentrated in areas predisposed to future lesion development even though the rate of entry is similar to normal sites (Schwenke and Zilversmit, 1989a,b). These studies and several others (Schwenke and Carew, 1989a,b; Falcone et al., 1984) indicate that lesion-prone arterial sites show an enhanced retention of atherogenic lipoproteins containing apo B. These data suggest that it is the retention of lipoproteins within the arterial wall that is the inciting event for atherosclerosis as opposed to any oxidative modi®cation of such lipoproteins. 2.5.1. Evidence to support the response-to-retention hypothesis The central tenant of the response-to-retention hypothesis is that high plasma concentrations of atherogenic lipoproteins are required and that persistent retention within the arterial wall sets the stage for lesion development. Within two hours of injecting LDL into rabbits, arterial retention of LDL and its micro aggregates can be observed (Nievelstein et al., 1991). The precise mechanisms involved in the retention of atherogenic lipoproteins within lesion-prone arterial sites is not clear. There is, however, considerable evidence that components of the extracellular matrix participate in this process. Apo B retained within the arterial wall is closely associated with arterial proteoglycans (Yl a-Herttuala et al., 1987; Camejo et al., 1993). Lipolytic enzymes within the extracellular matrix also appear to play a role. For example,

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lipoprotein lipase (LpL) enhances the adherence of LDL in vitro to proteoglycans (Williams et al., 1991). This phenomenon appears to occur in vessels enriched with LpL in vitro (Rutledge and Goldberg, 1994), and is independent of LpL enzymatic activity (Williams et al., 1992a). Once retained within the arterial wall, LDL can form micro aggregates perhaps through the action of secretory sphingomyelinase, a normal component of the arterial wall (Xu and Tabas, 1991). There are a number of important issues that arise from LDL that is entrapped within the arterial wall. LDL bound proteoglycans appears to be more susceptible to oxidation (Hurt-Camejo et al., 1992), and once oxidized, the LDL can obviously take on all the proatherogenic activities discussed above for both ox-LDL. The retained LDL is also subject to action by arterial wall sphingomyelinase (Xu and Tabas, 1991) that can generate ceramides with a number of well documented e€ects such as induction of apoptosis and mitogenesis (Hannun, 1994; Joseph et al., 1994). Most importantly, aggregated LDL is avidly taken up by macrophages and smooth muscle cells (Ismail et al., 1994) and thus, can support foam cell formation (Vijayagopal et al., 1992). The precise nature of this aggregated LDL uptake by macrophages and smooth muscle cells appears to involve receptors distinct from the LDL receptor (Vijayagopal et al., 1993). Interestingly, macrophage conversion to foam cells induces further release of LpL (O'Brien et al., 1992) providing for enhanced retention of LDL and further expansion of LDL aggregates within the arterial wall. A recent study lends considerable credence to the response-to-retention hypothesis. Boren and colleagues constructed a transgenic mouse strain overexpressing human apo B-100 and demonstrated that dietary induction of hypercholesterolemia caused severe atherosclerosis. In contrast, mice expressing a mutated form of apo B that does not bind proteoglycans (but does bind the LDL receptor) develop almost no lesions despite a similar degree of hypercholesterolemia (Boren et al., 1998). These results tend to suggest that LDL association with proteoglycans is an important initial step in the formation of atherosclerotic lesions. 3. The concept of disease activity Thus far we have reviewed a number of theories involved in the initiation events of atherosclerosis. Although di€erent in some respects, all of these theories involve the eventual recruitment of in¯ammatory cells to the arterial wall and a response to foam cell formation within the arterial intima. We have also reviewed brie¯y the events leading to lesion formation and the establishment of mature atherosclerotic lesion. The ``classical'' mature atherosclerotic lesion (Fig. 3A) involves a central core of foam cells with extracellular cholesterol arranged in so-called cholesterol ``clefts''. There is typically also a considerable amount of necrotic debris, and overlying the central core is a ®brous cap comprised of extracellular matrix, smooth muscle cells, and collagen. Lesions may remain quiescent in this state for many years. Such lesions may never produce any symptoms during the course of a patient's lifetime. At some point, in order to e€ect atherosclerotic disease activity (i.e., heart attack or stroke), the lesion must become activated. Lesion activation is initiated by rupture of the

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Fig. 3. Morphological features of atherosclerotic lesions. (A) The ``classical'' atherosclerotic lesions consists of a lipid core, with a ®brous plaque. The core often contains cholesterol crystals and necrotic debris. Stable atherosclerotic plaques contain cells and extracellular matrix in a thick ®brous cap. In contrast, (B) unstable plaques contain fewer cells and matrix in a thinner cap with an abundance of macrophages in the ``shoulder'' regions of the cap.

atherosclerotic plaque such that the plaque contents are exposed to the luminal surface of the artery (Fig. 3B). Contact with blood components in the plaque then leads to a thrombotic response that may precipitate a vascular event. In the following section we will review what is known about lesion activation and the events that contribute to this process. 3.1. The ®brous cap For many years, end-organ ischemia was thought to result as a consequence of progressive narrowing of the arterial lumen and loss of blood ¯ow. Although rupture

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of the atherosclerotic plaque was proposed in the ®rst half of this century (Leary, 1934), it is only in the past 15 yrs that this phenomenon has been appreciated in the genesis of clinical events in atherosclerosis. Autopsy studies have consistently identi®ed a number of morphologic features that are associated with recently ruptured atherosclerotic plaque. These include a large necrotic core of lipid and cellular debris, a thin ®brous cap that is often eccentric, and often a relatively modest protrusion into the arterial lumen (Davies and Thomas, 1985; Davies et al., 1989). One theory for these morphological characteristics concerns mechanical stresses on the ®brous cap. In particular, the presence of a large, soft lipid core compromises the structural integrity of the cap as the core is unable to bear mechanical forces and focuses available forces on the ®brous cap (Richardson et al., 1989). A large portion of these forces are concentrated at the junction of the ®brous cap with a normal vessel, the so-called ``shoulder'' region (Fig. 3A and B). This region of the plaque, coincidentally, is the region most likely to rupture based on autopsy studies (Davies and Thomas, 1985). Although these are common features of atherosclerotic plaques that have ruptured, not all plaques with this morphology will indeed fracture. In fact, only small proportion of atherosclerotic plaques are vulnerable to rupture, and this fact underlies the diculty in developing an adequate animal model for this phenomenon. Nevertheless, the vascular biology of the ®brous cap has come into focus in recent years, and has provided insights into modulating atherosclerotic lesions, thus, making them more stable and less prone to rupture. Among the major components of the atherosclerotic plaque is extracellular matrix. Normally, extracellular matrix production and turnover is remarkably slow (Seyer and Kang, 1992). In atherosclerosis, however, the environment of injury is associated with enhanced synthetic activity of major matrix components such as elastin, collagen, and proteoglycans (Libby, 1995). The population of in¯ammatory cells such as foam cells, and monocyte-derived macrophages within the plaque produces an environment that is replete with the cytokines and growth factors. A number of these agents have important implications for matrix production as some cytokines, such as transforming growth factor-b (TGF-b) may stimulate collagen synthesis whereas others (interferon-c) suppress it (Folcik et al., 1995). One proposed mechanism of cap weakening is an insucient production of matrix within the cap. 3.2. Matrix degradation Once a ®brous cap is formed over an atherosclerotic lesion, it is subject to a number of stresses due to both blood pressure and shear on the luminal surface of the artery. In the best of circumstances this cap can withstand such forces based upon its structural integrity. A wealth information now indicates that the structural components of the ®brous cap be degraded by a variety of matrix-degrading enzymes. These enzymes include serine proteases tissue-type and urokinase-type plasminogen activators, plasmin, the matrix metalloproteninases (MMPs), and cysteine proteases. Tables 7 and 8 contains a number of known matrix-degrading enzymes, their locations and typical substrates. Since the matrix metalloproteninases are capable of degrading all of the major components of the extracellular matrix,

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Table 7 Matrix-degrading enzymes relevant to atherosclerosisa Type

Examples

Examples of substrates

Serine protease

Plasmin, urokinase, cathepsin G, TPAb Cathepsins, B, D, H, L, N, and S Interstitial collagenase (MMP-1) Gelatinase A (MMP-2) Stromelysin-1 (MMP-3)

Fibrin, ®bronectin, laminin, some proteoglycans Wide range, including collagen, proteglycans, and elastin Collagens I, II, III, VII, and X

Cysteine protease Matrix metalloproteinases

PUMP-1 (MMP-7) Neutrophil collagenase (MMP-8) Gelatinase B (MMP-9) Stromelysin-2 (MMP-10) Stromelysin-3 (MMP-11) Metalloelastase (MMP-12) MT-MMP (MMP-14) a b

Collagens IV, V, VII, and X Collagens III, IV, V, and IX; laminin, ®bronectin, elastin, proteoglycans Gelatin, ®bronectin, laminin, collagen type IV, procollagenase, and proteoglycan core protein Collagens I, II, and III and proteoglycans Collagens IV, V, VII, and X Collagens III, IV, V, and IX; laminin, ®bronectin, elastin, proteoglycans Gelatin, ®bronectin, and proteoglycans Elastin Collagen IV, gelatin, and progelatinase A

Adapted from Libby and Lee (1997). Tissue-type plasminogen activator.

Table 8 Human studies of cholesterol lowering therapy and endothelial vasomotor functiona Study ®rst author

Baseline total cholesterol (mg/dl)

Intervention

Treatment duration (mo)

Improvement demonstrated

Coronary circulation Leung et al. (1994) Egashira et al. (1994) Treasure et al. (1995) Anderson et al. (1995)

275  31 272  8 230  33 209  33

6 6 5.5 12

+ + + 

Yeung et al. (1996)

204  32

Cholestyramine Pravastatin Lovastatin Lovastatin plus cholestyramine Simvastatin

6

)

Forearm circulation O'Driscoll et al. (1997) Drury et al. (1996) Vogel et al. (1995) Tamai et al. (1997)

255  33 209  17 200  12 195  34

Simvastatin Pravastatin Simvastatin LDL apheresis

1 5 yrs 3 Immediate

+ + + +

a

Adapted from Dillon and Vita (2000).

there under a multiple levels of regulation. All are under transcriptional regulation that has not been particularly well characterized. After synthesis, however, the MMPs typically must be activated through cleavage of the N-terminus by the activity of serine proteases such as plasmin or urokinase (Werb et al., 1977). A ®nal level of regulation involves a class of proteins known as the tissue inhibitors of

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metalloproteninases (TIMPs) (DeClerk et al., 1994). These proteins bind to matrix metalloproteninase active sites and inhibit the enzymes. With respect to the integrity of the ®brous cap, a number of matrix metalloproteninases have been observed in atherosclerotic plaques. For example, human lesions contain MMP-3 and MMP-1 (Henney et al., 1991), with the former demonstrating intense staining in the shoulder regions of atherosclerotic plaques (Nikkari et al., 1995). The synthesis of MMPs within the vascular wall is now an area of intense investigation. Smooth muscle cells and culture predominantly express MMP-2, a gelatinase that participates in cellular migration and only degrades non-®brillar collagen (Galis et al., 1994). Under resting conditions, cultured smooth muscle cells express little of the enzymatic activity required to cleave ®brillar collagen (MMP-1). One must keep in mind, however, that the atherosclerotic plaque is typically bathed in a milieu of cytokines and growth factors. Under these conditions, smooth muscle cell production of MMPs is quite di€erent. In the presence of tumor necrosis factor-a or interleukin-1b, vascular smooth muscle cells readily produce MMP-1, and MMP-3, two enzymes capable of degrading structural collagen (Table 7). Other cell types within the atheroma have also been implicated in MMP production. Macrophages from arterial lesions in rabbits demonstrate production of MMP-1 and MMP-3, consistent with cell culture ®ndings (Galis et al., 1995). This activity of macrophages may important implications for plaque strength as there appears to be an inverse correlation between macrophage density and the mechanical strength of human aortic plaques (Lendon et al., 1991). Consistent with this notion, macrophage in®ltration of the ®brous cap and its shoulder regions is a common ®nding in morphologically unstable lesions (van der Wal et al., 1994; Moreno et al., 1992). Rupture of the atherosclerotic plaque leads to the exposure of the lipid core to the blood vessel lumen. This has implications for promoting disease activity in atherosclerosis as the lipid core has a number of important features. For example, the matrix derived from the core of atheromatous lesions is considerably more thrombogenic in vitro than matrix derived from other parts of the arterial wall (Fernandez-Ortiz et al., 1994). One possible reason for this observation is the abundant content of tissue factor within the core of atherosclerotic lesions (Libby, 1995). Tissue factor activates factors IX and X after binding to the coagulation factor VIIa (Nemerson, 1995), thereby activating the clotting cascade. In addition to matrix degradation, depopulation of the ®brous cap may also contribute to plaque rupture (Falk, 1992). In particular, ®brous caps that demonstrate rupture tend to have an imbalance between the number of macrophages and smooth muscle cells with many more macrophages than stable plaques and many fewer smooth muscle cells (Falk, 1992). One explanation for this observation is that activated T lymphocytes are also found within atherosclerotic plaques (Hansson et al., 1989). A major cytokine derived from T cells is interferon-c, and vascular smooth muscle cells exposed to cytokines such as interferon-c demonstrate apoptosis (Bennett et al., 1995; Geng et al., 1996). One could certainly envision a situation where in¯ammation in speci®c areas of the atherosclerotic plaque may promote smooth muscle cell apoptosis, and confer some defect in the synthetic capacity of the

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atherosclerotic plaque. This combined with shear forces on the plaque cap could promote rupture in the shoulder regions. If one assumes that plaque vulnerability is determined, in part, by the activity of matrix-degrading enzymes, one would predict manipulations known to reduce to cardiovascular events should also reduce matrix-degrading enzyme activity. This prediction has proven to be true in experimental animals. Aikawa and colleagues (Aikawa et al., 1998) produced experimental atherosclerosis in rabbits by balloon injury and an atherogenic diet. Rabbits were then continued on diets with or without high cholesterol. Animals continuing on the high cholesterol diet demonstrated lesions consisting of many macrophages, and considerable MMP-1 expression. In the group that did not continue on high cholesterol diet, proteolytic activity decreased substantially and the aortic content of interstitial collagen increased. These results suggest that cholesterol-lowering prompts a change in the arterial wall characterized by lower matrix metalloproteinases activity and increased collagen retention. These features would be expected to provide plaque stabilization. In summary, development of the atherosclerotic lesion involves the generation of a ®brous cap that overlies a lipid core. In the best of circumstances, this ®brous cap remains thick and replete with smooth muscle cells that produce collagen adding to the stability of the plaque. Under these circumstances, shear forces and mechanical stress from arterial pressure on the plaque are not met with any structural failure, and the lipid core remains isolated from the circulation. However, under certain circumstances often associated with in¯ammation and cytokines, components of the plaque may change such that the shoulder regions of the atherosclerotic plaque become replete with macrophages and matrix metalloproteinases activity. In addition, T cells populating the plaque may produce cytokines that promote apoptosis of vascular smooth muscle cells. Under this scenario, the plaque becomes more acellular and contains less interstitial collagen. This sets the stage for structural failure of the plaque during the periods of high mechanical stress leading to plaque rupture and exposure of the lipid core to the circulation. Since the lipid core contains a number of prothrombotic components, this prompts thrombosis that may then propagate to include the entire arterial lumen and produce vascular insuciency with catastrophic consequences such as heart attack or stroke. Further work will be needed to precisely identify the events of plaque instability and allow us to devise strategies to promote integrity of the ®brous cap and prevent plaque rupture. 4. Vascular homeostasis Although the section above makes a compelling case that we do not understand all of the events associated with plaque rupture, it is clear that lesion activation also involves a fundamental lapse in the homeostatic mechanism of the blood vessel (Keaney and Vita, 1995; Levine et al., 1995). Normal vascular homeostasis provides for adequate end-organ perfusion through the continuous control of vascular tone, blood ¯ow, and constitutive inhibition of thrombosis. The endothelium, as the interface between blood ¯ow and vessel wall, is an important and critical component

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of vascular homeostasis. Among the more important features of the endothelium is its role as a local site for the integration of paracrine and autocrine signals. For example, the endothelium can respond to cytokine production within the vascular wall by up regulating adhesion molecules on its surface that help promote leukocyte recruitment into the blood vessel (Luscinskas and Gimbrone, 1996). The endothelium also constitutively produces substances such as prostaglandin I2 …PGI2 †, a potent inhibitor of platelet activation (Radomski et al., 1987). Endothelial cell synthesis of heparins and thrombomodulin also promote blood ¯uidity through the reduction of thrombin activity at the endothelial surface (Ursini et al., 1995; Avissar et al., 1989). Finally, the endothelium exerts control over ®brinolysis through the synthesis of both tissue-type plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) (Loskuto€ and Edgington, 1977). Thus, the normal activity of the endothelium maintains a state favoring ®brinolysis over thrombosis. Another product of the vascular endothelium that maintains blood ¯ow and vascular tone is nitric oxide (NO), this species is a free radical synthesized constitutively in the endothelium by an oxido-reductase enzyme known as endothelial nitric oxide synthase (eNOS). A number of important homeostatic functions appear to depend on endothelium-derived NO. For example, vascular tone (Quyyumi et al., 1995), smooth muscle cell phenotype (Garg and Hassid, 1989), platelet adhesion (Azuma et al., 1986), and leukocyte recruitment (Kubes et al., 1994) all appear to be controlled, at least in part, by nitric oxide from the endothelium. Experimental animals lacking the eNOS exhibit spontaneous hypertension (Olesen et al., 1988), an enhanced thrombotic response (Freedman et al., 1996), and defective vascular remodeling (Rudic et al., 1998). In patients, impaired nitric oxide action has been associated with spontaneous arterial thrombosis (Freedman et al., 1996). Thus, a predominance of available evidence indicates that the production of NO from the endothelium is an important component of vascular homeostasis. Nitric oxide elicits its bioactivity through two principal mechanisms. The ®rst involves the binding of NO to the heme moiety of soluble guanylyl cyclase resulting in enzymatic activation and the synthesis of 30 ,50 -cyclic guanosine monophosphate (cGMP) (Arnold et al., 1977). There is an association between vascular cyclic GMP and vasodilation due to nitric oxide (Ignarro et al., 1984) as well as NO-mediated inhibition of platelet activity (Moro et al., 1995; Radomski et al., 1997). Another mechanism of NO bioactivity relates to the formation of adducts of nitric oxide. Under aerobic conditions NO forms oxides of nitrogen that combine with thiol groups to form S-nitrosothiols (Wink et al., 1993), and this reaction has been implicated in NO-mediated activation of potassium channels (Bolotina et al., 1994), calcium channels (Xu et al., 1998), and the modulation of hemoglobin oxygen anity (Stamler et al., 1997). The component of vascular homeostasis contributed by nitric oxide has been the subject of intense investigation with respect to atherosclerosis. The major reason being that endothelium-derived NO bioactivity is impaired in patients who demonstrate frank atherosclerosis, or known risk factors for premature atherosclerosis. The vasodilatory responses to agents that stimulate endothelium-derived NO are abnormal in the coronary arteries of patients with atherosclerosis (Ludmer et al.,

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1986), diabetes (Nitenberg et al., 1993), and hypertension (Panza et al., 1993). This appears to be a systemic process as NO-mediated responses are also impaired in forearm resistance arterials (Panza et al., 1993; Creager et al., 1990; Johnstone et al., 1992), as well as other conduit arteries such as the brachial artery (Anderson et al., 1989; Celermajer et al., 1993). This abnormality in vascular homeostasis typically precedes the clinical manifestations of vascular disease. Individuals with hypercholesterolemia (Vita et al., 1990), advanced age (Celermajer et al., 1994), and cigarette smoking (Kugiyama et al., 1996) typically demonstrate impaired vasoactive responses to agents that stimulate endothelial NO production. Genetic factors may also play a role as patients with a family history have atherosclerosis also demonstrate impaired NO-mediated arterial vasodilation (Clarkson et al., 1997). Thus, atherosclerosis is characterized by a broad defect in vascular homeostasis that also includes abnormal NO-mediated vascular homeostasis. This defect is thought to play an important role in the clinical manifestations of vascular disease as patients with abnormal vascular function are more likely to develop vascular events than those whose vascular function is relatively well-preserved (Suwaidi et al., 2000; Schachinger et al., 2000). 4.1. Oxidative stress and impaired NO bioactivity The exact mechanism producing abnormal bioactivity in the setting of vascular disease and atherosclerosis remains a subject of intense investigation. However, considerable evidence now indicates that NO bioactivity is particularly sensitive to oxidative stress. Although oxidative stress is a poor term, it has come to gain acceptance as a means to describe an imbalance between oxidants and antioxidants in favor of the former. With respect to vascular disease and atherosclerosis, there is considerable evidence that there is an increase in the production of oxidants, and this has important implications for NO bioactivity. The principal oxidants involved in impairment of NO bioactivity will be discussed in detail below focusing on the mechanistic aspects by which NO bioactivity is impaired. 4.1.1. Superoxide and NO bioactivity It has been known for some time that atherosclerotic blood vessels demonstrate abnormal functional responses (Henry and Yokoyama, 1980; Heistad et al., 1984) and the knowledge that endothelium-dependent vasorelaxation is due, in part, to nitric oxide (Ignarro et al., 1987) has fueled considerable investigation to determine the mechanisms responsible for impaired endothelial function in atherosclerosis. To gain insight into the mechanism of vascular dysfunction and atherosclerosis, Minor and colleagues (Minor et al., 1990) measured the elaboration of nitrogen oxides from normal, hypercholesterolemic, and atherosclerotic blood vessels. They then correlated this with the amount of relaxation produced by those blood vessels and the result is shown is Fig. 4. Diseased blood vessels exhibit considerably less relaxation per unit of nitrogen oxide released suggesting that NO may be inactivated in pathologic states. This source of NO inactivation has since been identi®ed as superoxide (Ohara et al., 1993; Keaney et al., 1995), and a number of disease states

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Fig. 4. Arterial relaxation and NO production in rabbit aorta. Rabbits were treated with a 1% cholesterol diet for either 2±5 weeks (hypercholesterolemia) or 4 months (atherosclerotic). Arterial relaxation and NOx (nitrite+nitrate) were examined in response to 1 lM acetylcholine or 10 lM calcium ionophore A23187. Reproduced from The Journal of Clinical Investigation (1990) 86:2109±2116 by copyright permission of the American Society for Clinical Investigation.

such as hypercholesterolemia, diabetes, and hypertension are now noted to be associated with an increase in the steady state ¯ux of superoxide within the vascular wall (Griendling and Alexander, 1997). This change in the ambient superoxide level has important implications for nitric oxide. It is known that superoxide and NO combine rapidly in a radical-radical combination reaction that occurs at the di€usion limit …k ˆ 1:9  1010 Mÿ1 sÿ1 † (Kissner et al., 1997) to form peroxynitrite as depicted in reaction (9) ÿ á Oáÿ 2 ‡ NO ) O±O±N ˆ O

…9†

The relevance of this reaction on NO bioactivity is emphasized by experimental evidence that it occurs in vivo (Leeuwenburgh et al., 1998; Ischiropoulos et al., 1992). The product of this reaction, peroxynitrite, has the capacity to activate guanylyl cyclase (Mayer et al., 1995; Tarpey et al., 1995), although it does so much less potently than nitric oxide itself (Tarpey et al., 1995). Therefore, any interaction of NO and superoxide in the vascular wall will result in a reduction of NO bioactivity. Since endothelial cells produce NO and superoxide, small changes in the relative ¯ux of either species should have important implications for NO bioactivity. This prediction has been proven true in experimental systems proving that SOD improves the vascular relaxation response to endothelium-derived NO (Rubanyi et al., 1986; Gryglewski et al., 1986). Both receptor-stimulated NO release from endothelial cells as well as the basal constitutive production appear to be responsive to ambient levels of superoxide (Rubanyi et al., 1986; Gryglewski et al., 1986). Further evidence that ambient superoxide is important for NO bioactivity comes from experiments in which endogenous SOD activity has been inhibited. For example, inactivation of

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Cu±Zn SOD through chelation with diethyldithiocarbamate results in impaired release of bioactive NO (Omar et al., 1991). Chronic inhibition of Cu±Zn SOD through copper-de®ciency also results in impaired NO-mediated arterial relaxation (Lynch et al., 1997; Schuschke et al., 1992) that is associated with an increased vascular ¯ux of superoxide detected by chemiluminescence (Lynch et al., 1997). Available evidence, therefore, indicates that vascular SOD activity is an important component for intact NO responses. Despite the consensus that ambient superoxide helps determine NO bioactivity, considerable controversy exists as to the source of superoxide especially in the setting of vascular disease. All layers of the vascular wall have demonstrated evidence for superoxide production (Pagano et al., 1995; Pagano et al., 1997; Miller et al., 1998). Immunohistochemical data suggests that at least in the aortic adventitia, there is evidence for NADPH oxidoreductase activity (Pagano et al., 1995) and the presence of known subunits of the neutrophil NADPH oxidase such as p22phox , gp91phox , p47phox , p67phox (Pagano et al., 1997). Other studies in cultured endothelial cell homogenates indicate that NADH oxidoreductase is a principal source of superoxide (Mojazzab et al., 1994). In the setting of vascular disease, other sources of superoxide have been implicated including xanthine oxidase (Ohara et al., 1993) that may actually be bound to the surface of the endothelium (White et al., 1996). This ®nding would be consistent with studies in patients with hypercholesterolemia that demonstrate a partial improvement in NO-mediated arterial relaxation with an inhibitor of xanthine oxidase, oxypurinol (Cardillo et al., 1997). The signaling events involved in stimulating NADPH oxidase activity in atherosclerosis are now being elucidated. Warnholtz and colleagues found that superoxide production in cholesterol-fed rabbits was increased signi®cantly, and this response could be inhibited by an angiotensin-II type I receptor antagonist (Warnholtz et al., 1999). These results suggest that angiotensin-II, at least in part, mediates the increase in superoxide production observed with hypercholesterolemia. In considering the sources of superoxide, one must consider that nitric oxide synthase is capable of reducing molecular oxygen to produce superoxide (Pou et al., 1992; Vasquez-Vivar et al., 1998; Xia et al., 1998a). This may have particular importance for hypercholesterolemia-induced vascular superoxide generation as endothelial cells exposed to LDL exhibit enhanced superoxide generation from eNOS (Pritchard et al., 1995). The precise mechanisms underlying these observations are not yet clear, but may have to do with depletion of intracellular tetrahydrobiopterin …BH4 †, a co-factor for eNOS that appears important in determining relative superoxide production from this enzyme (Vasquez-Vivar et al., 1998). Although considerable data suggests that vascular superoxide is an important determinant of NO bioactivity, it is clear that the entire spectrum of impaired NO bioactivity in hypercholesterolemia and atherosclerosis cannot be attributed solely to superoxide. For example, even in studies reporting improved NO bioactivity by increasing vascular superoxide dismutase (Mugge et al., 1991; White et al., 1994), the e€ect on NO bioactivity was incomplete. Consistent with these observation, chronic SOD inhibition appears to impair receptor-dependent stimuli for NO production to

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a greater extent than non-receptor-dependent stimuli (Lynch et al., 1997). This ®nding suggests that NO elaboration may also be impaired during later stages of atherosclerosis (Oemar et al., 1989). One such candidate for impaired NO elaboration is lipid peroxidation. 4.1.2. Lipid peroxidation and NO bioactivity It is important to realize that other sources of oxidative stress are present in atherosclerosis beyond superoxide. In particular, peroxynitrite is readily formed from superoxide and NO and this product is a potent oxidant (Beckman and Crow, 1993). As an oxidant, peroxynitrite has a number of important activities including the transfer of oxygen atoms (Beckman and Crow, 1993), oxidation of sulfhydryls (Moreno and Pryor, 1992), and the initiation of lipid peroxidation (Graham et al., 1993). Other important sources of oxidative stress beyond peroxynitrite may include redox-active copper from ceruloplasmin (Ehrenwald et al., 1994), tyrosyl radical from myeloperoxidase (Heinecke et al., 1993; Heinecke, 1997), lipoxygenase (Folcik et al., 1995), and hypochlorous acid, another product of myeloperoxidase (Malle et al., 1995). The process of lipid peroxidation (Fig. 2) is of some consequence for NO bioactivity. Lipid peroxyl radicals produced during this process combine readily with peroxynitrite to form lipid peroxynitrite derivatives (Padmaja and Huie, 1993; Rubbo et al., 1994) e€ectively quenching bioactive NO (O'Donnell et al., 1999). Lipid peroxidation also leads to the formation of oxLDL (Yla-Herttuala et al., 1989) that may either directly inactivate NO (Chin et al., 1992) or reduce eNOS in endothelial cells (Liao et al., 1995). Indirect e€ects of lipid peroxidation include interruption of G-protein-coupled signal transduction leading to impaired NO production (Kugiyama et al., 1990). Thus, lipid peroxidation has a number of important consequences for NO bioactivity. 4.1.3. Lipid-soluble antioxidants and NO bioactivity In light of the aforementioned evidence that lipid peroxidation impairs NO bioactivity, it follows that antioxidants that reduce vascular lipid peroxidation should improve NO bioactivity. As shown in Table 1, the main lipid-soluble antioxidants are a-tocopherol (vitamin E), b-carotene, and ubiquinol-10. These three compounds represent the main lipid soluble antioxidants in humans and, with the exception of b-carotene (Jialal et al., 1991; Gaziano et al., 1995), their main action is thought to be the inhibition of lipid peroxidation (Esterbauer et al., 1989). 4.1.3.1. a-Tocopherol and b-Carotene. The activity of a-tocopherol and b-carotene with respect to NO bioactivity has been examined in rabbits consuming a 1% cholesterol diet that typically demonstrate impaired endothelium-derived NO bioactivity as assessed by arterial relaxation (Jayakody et al., 1985; Verbeuren et al., 1986). This defect induced by cholesterol-feeding has been linked to an increase in vascular oxidative stress (Ohara et al., 1993; Keaney et al., 1995). We found that cholesterolfed rabbits treated with either b-carotene or a-tocopherol demonstrate near normal NO-mediated arterial relaxation responses to both acetylcholine and A23187

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(Keaney et al., 1993). At least with a-tocopherol, this dietary treatment was associated with enhanced antioxidant protection as assessed by lipoprotein resistance to copper-mediated oxidation (Keaney et al., 1993). Similar studies in the coronary (Andersson et al., 1994) and carotid (Stewart-Lee et al., 1994) arteries of cholesterolfed rabbits have also demonstrated that a-tocopherol preserves NO-bioactivity. In rats, a species not normally susceptible to atherosclerosis, cholesterol-feeding has been associated with impaired endothelium-dependent arterial relaxation and this e€ect of cholesterol is abrogated by dietary vitamin E (Lutz et al., 1995). The role of a-tocopherol in preserving NO bioactivity has not been limited solely to atherosclerosis and hypercholesterolemia. For example, aortic segments isolated from streptozotocin-induced diabetic rats demonstrate impaired NO-mediated arterial relaxation that improves signi®cantly with a-tocopherol supplementation in the diet (Keegan et al., 1995; Karasu et al., 1997a,b). Similar observations have been reported in isolated perfused rat coronary arteries (Rosen et al., 1996). To the extent that diabetes represents a state of heightened oxidative stress (Gopaul et al., 1995; Davi et al., 1999; Keaney and Loscalzo, 1999), these data provide additional evidence that antioxidant protection in vivo is important in maintaining normal NO bioactivity in the setting of chronic vascular disease. Since LDL oxidation impairs NO bioactivity (Kugiyama et al., 1990), and atocopherol inhibits LDL oxidation (Dieber-Rotherneder et al., 1991), one might speculate that a-tocopherol preserves NO bioactivity by inhibiting LDL oxidation. Available evidence suggests that this contention may be overly simplistic. For example, cholesterol-fed rabbits treated with two di€erent regimens of a-tocopherol demonstrates strikingly di€erent e€ects on NO bioactivity. At an a-tocopherol dose of 110 IU/d, NO-mediated arterial relaxation is preserved in cholesterol-fed rabbits and LDL is protected from ex vivo copper-mediated oxidation (Keaney et al., 1993, 1994). In contrast, a 10-fold higher dose of a-tocopherol produces worse NO bioactivity than cholesterol feeding alone despite excellent antioxidant protection of LDL (Keaney et al., 1994). Thus, antioxidant protection of LDL alone is insucient to preserve NO bioactivity in the cholesterol-fed rabbit model. a-Tocopherol is a very ecient scavenger of lipid peroxyl radicals, nevertheless, it is clear that atherosclerosis exhibits lipid peroxidation in the vascular wall despite the presence of a-tocopherol (Suarna et al., 1995). In light of this knowledge, one favorable action of a-tocopherol may be the protection of vascular cells against the deleterious e€ects of lipid peroxidation by-products. Consistent with this notion, isolated arterial segments derived from a-tocopherol-de®cient rabbits demonstrate impaired NO-mediated arterial relaxation upon exposure to ox-LDL (Keaney et al., 1996). In contrast, arterial segments derived from animals supplemented with atocopherol contain 100-fold more tocopherol and exhibit a marked resistance to the e€ects of ox-LDL (Keaney et al., 1996). Similar e€ects can be demonstrated using cultured endothelial cells (Jay et al., 1997). Recent data has shed light on the mechanism of such observations. Exposure of arterial tissue to oxidized LDL results in protein kinase C stimulation (Kugiyama et al., 1992) and this e€ect can be prevented by incorporation of a-tocopherol into endothelial cells (Fig. 5) (Keaney et al., 1996). Since protein kinase C stimulation impairs NO bioactivity (Sugiyama et al.,

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Fig. 5. a-Tocopherol prevents oxLDL-induced protein kinase C stimulation in endothelial cells. Human aortic endothelial cells were loaded with a-tocopherol (AT) and incubated with oxidized LDL (oxLDL), native LDL (nLDL), or media (control) for 4 h. After washing, protein kinase C activity was determined with an in situ assay (Williams and Schrier, 1992). Reproduced from The Journal of Clinical Investigation (1996) 98:386±394 by copyright permission of the American Society for Clinical Investigation.

1994; Ohgushi et al., 1993; Flavahan et al., 1991) and a-tocopherol prevents protein kinase C stimulation (Keaney et al., 1996; Boscoboinik et al., 1991; Freedman et al., 1996), these data point to the e€ect of a-tocopherol on protein kinase C as the mechanism for improved NO bioactivity. It is worth noting that similar e€ects have been observed in platelets (Freedman et al., 1996) and smooth muscle cells (Boscoboinik et al., 1991). 4.1.3.2. Probucol. The e€ect of probucol to limit atherosclerosis has been discussed in detail above. In the setting of atherosclerosis, supplementation with probucol results in its accumulates within the vascular wall (Shaish et al., 1995; Keaney et al., 1995). Since probucol accumulates in vascular tissue, and it is a potent inhibitor of LDL oxidation in a variety of oxidizing systems (Marshall, 1982; Parthasarathy et al., 1986), it is no surprise that its activity to modulate NO bioactivity has been examined. Probucol has been tested for its e€ect on endothelium-derived NO in cholesterolfed (Keaney et al., 1995; Simon et al., 1993; Inoue et al., 1998) and LDL receptorde®cient (Hoshida et al., 1997) rabbits. With respect to its antioxidant activity, probucol treatment as 1% of the diet did eliminate the increase in plasma (Simon et al., 1993; Inoue et al., 1998) and aortic (Keaney et al., 1995) lipid peroxides associated with cholesterol feeding as assessed by thiobarbituric acid-reactive substances. In all the animal studies conducted thus far, probucol treatment universally preserved NO bioactivity measured as endothelium-dependent arterial relaxation in response to either acetylcholine or A23187 (Inoue et al., 1998; Keaney et al., 1995;

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Simon et al., 1993). In normal animals, probucol does not appear to alter NO bioactivity (Simon et al., 1993), suggesting that its e€ect is limited to vascular disease states. The e€ect of probucol is also not limited to atherosclerosis, since it also improves NO bioactivity in alloxan-induced diabetes (Tesfamariam and Cohen, 1992). Although probucol potently inhibits LDL oxidation in ex vivo assays (Marshall, 1982; Parthasarathy et al., 1986), its mechanism of action in these animal models appears unrelated to this e€ect. Cholesterol-fed rabbits typically demonstrate an increased vascular steady-state superoxide ¯ux that can be detected by chemiluminescence methods (Ohara et al., 1993). In two separate studies, probucol treatment leads to a reduction in the increased vascular ¯ux of superoxide (Keaney et al., 1995; Inoue et al., 1998). This e€ect of probucol is not related to superoxide scavenging by the compound (Keaney et al., 1995). Reducing the vascular ¯ux of superoxide inhibits arterial wall lipid peroxidation (Keaney et al., 1995; Inoue et al., 1998) and accumulation of lysophosphatidylcholine (Keaney et al., 1995), two phenomena that have been linked to reduced NO bioactivity in animal models (Lynch et al., 1997; Flavahan, 1992). Thus, probucol improves NO bioactivity in experimental animals principally through a direct e€ect on arterial tissue. The precise nature of this e€ect is not known, but may involve the signals needed to stimulate superoxide production. Human studies examining the e€ect of lipid soluble antioxidants on NO bioactivity are more mixed than animal studies. Studies examining antioxidant e€ects on NO bioactivity in resistance vessels of patients with vascular disease (McDowell et al., 1994; Elliott et al., 1995; Gilligan et al., 1994). In contrast, the e€ect of lipidsoluble antioxidants on NO responses in most conduit arteries has been somewhat more consistent. Koh and colleagues examined the e€ect of a-tocopherol on postmenopausal women and observed an improvement in NO-mediated endotheliumdependent vasodilation (Koh et al., 1998). Similar ®ndings have been reported in patients with elevated remnant lipoprotein levels (Motoyama et al., 1998) in the coronary circulation in response to vitamin E treatment. The reader is directed to a recent review of human studies examining the e€ect of antioxidants on vascular function (Du€y et al., 1999). 4.1.4. Water-soluble antioxidants and NO bioactivity With the popularity of the ``oxidative modi®cation hypothesis'', there has been considerable investigation into water-soluble antioxidants and vascular function. The principal water-soluble antioxidants in the arterial wall are glutathione (GSH) and ascorbic acid. Glutathione is typically present in plasma at concentrations below 1 lM (Wendel et al., 1980), whereas ascorbic acid is normally considerably more abundant (30±150 lM) in plasma (Keaney and Frei, 1994). In contrast, both compounds demonstrate relatively high concentrations within the cell cytosol reaching concentrations that range from 1 to 5 mM (Bray and Taylor, 1993; Bergsten et al., 1994). Considerable evidence suggests that both intracellular antioxidant compounds are required for normal cellular viability and function. All cells appear to demonstrate the active transport and/or synthesis of GSH and vitamin C and Animals deprived or depleted of either compound become morbid and may die as a consequence (Martensson et al., 1993; Martensson and Meister, 1991).

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4.1.4.1. Glutathione. The potential role of intracellular GSH in the bioactivity and production of NO has been controversial. Early studies involving the manipulation of intracellular GSH in porcine (Murphy et al., 1991) and bovine (Hecker et al., 1992) endothelial cells produced concordant changes between GSH and the release of NO, consistent with the role for GSH in endothelial cell NO production. However, speci®c correlation between the intracellular thiol levels and the release of NO has been problematic. Nitric oxide production from cultured bovine aortic endothelial cells by bradykinin is impaired by thiol alkylation with N-ethylmaleimide or thiol oxidation with 2,20 -dithiodipyridine (DTDP). However, DTDP treatment was not linked to a demonstrable reduction in endothelial cell GSH (Hecker et al., 1992), suggesting some other activity of DTDP may interfere with NO release. In a separate study with bovine cells, glutathione depletion using buthionine sulfoximine also failed to demonstrate any reduction in bioactive NO despite a >90% reduction in GSH (Mugge et al., 1991). Thus, these early studies in animals cells provided no consensus that intracellular GSH is important in endothelium-derived NO production. Studies in human cells, however, have been more consistent with the role of intracellular GSH in NO production. The treatment of human umbilical vein endothelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that covalently modi®es GSH, reduced both intracellular GSH and endothelial NO production (Ghigo et al., 1993). Conversely, increasing endothelial cell GSH with GSH ester increased cellular NO production in a manner that strongly correlated with intracellular GSH (Ghigo et al., 1993). It is dicult to reconcile the inconsistent ®ndings of the in vitro studies outlined above. One possibility involves the non-speci®c action of many thiol modulating agents. For example, although CDNB reduces intracellular glutathione, it also has activity to inhibit thioredoxin reductase, an enzyme important in maintaining protein thiols in a reduced state. Since protein thiols are known to be important for eNOS activity (Patel et al., 1996), this may represent one mechanism for observed ®ndings. In contrast to the data obtained in vitro, emerging data from human studies indicate that glutathione may have a role in modulating endothelium-derived NO. Patients with documented atherosclerosis and coronary artery disease are characterized by impaired NO-mediated arterial relaxation in the coronary (Ludmer et al., 1986) and brachial circulations (Celermajer et al., 1992). Treatment of such patients with L-to-oxo-4-thiazolidine carboxylate, an agent that selectively increases intracellular glutathione (Williamson and Meister, 1981; Boesgaard et al., 1994), produced improved NO bioactivity in the brachial artery (Vita et al., 1998). These ®ndings are consistent with recent evidence from resistance vessels in the femoral circulation. Prasad and colleagues (Prasad et al., 1999) examined the e€ect of glutathione in patients with atherosclerosis or its risk factors. Infusion of glutathione markedly enhanced the response to aceylcholine, a receptor-mediated agonist for NO production (Prasad et al., 1999). This enhancement of NO-mediated vasodilation was associated with an increase in the plasma cGMP content of the femoral vein, consistent with the notion that glutathione enhances NO bioactivity. Similar results have been observed in the coronary circulation. For example, in patients with

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a vasospastic angina, intracoronary glutathione improves NO bioactivity in response to acetylcholine (Kugiyama et al., 1998). Although emerging evidence supports a role for glutathione and NO bioactivity, the mechanisms responsible for this e€ect remain to be elucidated. Early studies with iNOS demonstrated that glutathione was necessary for optimal enzymatic activity (Stuehr et al., 1990). More recent studies have con®rmed this e€ect for neuronal NOS (Komori et al., 1995; Hofmann and Schmidt, 1995). The e€ect of glutathione appears unique as other thiols such as cysteine are considerably less e€ective (Komori et al., 1995; Hofmann and Schmidt, 1995). The precise events of this phenomenon are best understood for neuronal NOS, where thiol-mediated enhancement of enzymatic activity is associated with direct thiol binding to the heme moiety of nNOS cooperatively with tetrahydrobiopterin (Gorren et al., 1997). With respect to antioxidant activity of glutathione, one possible mechanism might include the prevention of NOS inactivation by peroxynitrite that is produced during simultaneously generation of NO and superoxide by the enzyme when substrate conditions are limiting (Vasquez-Vivar et al., 1998; Fitzgerald et al., 1986). Recent studies demonstrating that tissue GSH is reduced in the setting of hypercholesterolemia resulting in enhanced oxidative damage tend to support this notion (Ma et al., 1997). 4.1.4.2. Ascorbic acid. Ascorbic acid is a versatile antioxidant, it scavenges a wide range of biologically relevant oxidizing species including superoxide, aqueous peroxyl radicals, hydrogen peroxide, and hydroxyl radical (Nishikimi, 1975; Bodannes and Chan, 1979; Halliwell et al., 1987; Frei et al., 1989). As a consequence of this potent antioxidant activity, the e€ect of ascorbic acid on NO bioactivity has been examined. For example, coronary artery disease patients treated with oral ascorbic acid demonstrate a signi®cant improvement in NO bioactivity within the brachial artery and plasma concentrations of ascorbic acid remain within the normal range (Levine et al., 1996). In contrast, a number of other studies have demonstrated a salutary e€ect of ascorbic acid on NO-mediated arterial relaxation using pharmacological concentrations. For example, diabetic (Ting et al., 1996) or hypercholesterolemic (Ting et al., 1997) patients treated with high-dose intra-arterial ascorbic acid (about 10 mM) demonstrated enhanced endothelial function in forearm resistance vessels. Similar e€ects have been observed with other clinical conditions associated with atherosclerosis such as smoking (Heitzer et al., 1996), congestive heart failure (Hornig et al., 1998), and hypertension (Koh et al., 1998; Motoyama et al., 1998). There has been considerable speculation concerning the mechanism of ascorbic acid action. At ®rst glance, the fact that ascorbic acid scavenges superoxide suggest that superoxide scavenging may be responsible for the improved NO bioactivity (Koh et al., 1998; Ting et al., 1996; Ting et al., 1997; Heitzer et al., 1996; Hornig et al., 1998). Direct studies in isolated arterial segments suggest a more careful interpretation of the data is warranted. Isolated arteries exposed to superoxide demonstrate impaired NO-mediated arterial relaxation that improves with physiologic concentrations of SOD (about 1±3 lM) (Fig. 6) (Rubanyi et al., 1986; Jackson et al.,

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Fig. 6. Ascorbic acid does not readily substitute for SOD in preserving NO bioactivity. (A) Contracted vessels were assayed for NO bioactivity as relaxation in response to acetylcholine in the presence of no additions (squares), superoxide from pyrogallol (circles), pyrogallol with 300 IU/ml SOD (diamonds), or 300 IU/ml SOD alone (down triangles). (B) NO bioactivity in response to acetylcholine in the presence of no additions (squares), pyrogallol (circles), or pyrogallol with 0.1 (up triangles), 1.0 (diamonds), or 10 (down triangles) mM ascorbic acid.  P < 0:05 vs no additions only by two-way ANOVA. Taken from Jackson et al. (1998) with permission.

1998; Ignarro et al., 1988; O'Keefe et al., 1996). However, ascorbic acid cannot substitute for SOD at physiologically relevant concentrations (Jackson et al., 1998). The need for pharmacological concentrations of ascorbic acid can be predicted based on available kinetic data considering that the interaction between superoxide and ascorbic acid …105 Mÿ1 sÿ1 † occurs approximately 105 -fold less rapidly than the interaction of superoxide with NO …1010 Mÿ1 sÿ1 † (Kissner et al., 1997). Thus, simple superoxide scavenging appears to provide an incomplete explanation for the e€ect of ascorbic acid on NO bioactivity. The requirement for pharmacological concentrations of ascorbic acid in these acute infusion studies has recently been demonstrated. Sherman and colleagues (Sherman et al., 2000) observed that a high-dose of ascorbic acid (24 mg/min) in the forearm improved NO-mediated arterial relaxation in hypertensive patients. In contrast, a ten-fold lower dose designed to produce arterial concentrations of ascorbate between 100 and 300 lM did not improve NO responses (Sherman et al., 2000). These data suggest that for ascorbic acid to act through superoxide scavenging, pharmacological concentrations are needed in keeping with in vitro data (Jackson et al., 1998). The question of how ascorbic acid acts to improve NO bioactivity at normal plasma concentrations (Levine et al., 1996) has recently been addressed. Incubation of endothelial cells culture leads to the intracellular accumulation of ascorbate and

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improved NO bioactivity (Heller et al., 1999; Huang et al., 2000). This e€ect appears to be the result of increased synthesis of NO from endothelial cells as measured by the accumulation of nitrogen oxides or enzymatic production of L -citrulline (Huang et al., 2000). Interestingly, intracellular manipulation of glutathione status does not have the same e€ect in this system. The reason for these observations stems from the fact that increasing intracellular ascorbic acid also increases BH4 within the endothelial cell, whereas glutathione manipulation has no e€ect in this regard (Huang et al., 2000). Thus, intracellular ascorbic acid modulates endothelial cell NO production through a BH4 -dependent mechanism. 4.1.5. Other means of treating endothelial dysfunction 4.1.5.1. L -arginine. After the initial description that endothelial dysfunction was a prominent feature of patients with atherosclerosis (Ludmer et al., 1986), there was considerable interest in identifying treatments that might reverse endothelial dysfunction in patients. Unlike those outlined above, such studies were not speci®cally directed at any particular hypothesis of atherosclerosis. For example, one of the early observations concerning NO production was its dependence on L -arginine as a substrate (Palmer et al., 1988). This observation prompted a series of studies examining the e€ect of L -arginine supplementation on vascular function. The administration of L -arginine has now been shown to enhance endothelium-derived NO bioactivity in a number of disease states including coronary atherosclerosis (Quyyumi et al., 1997), variant angina (Egashira et al., 1996), hypercholesterolemia (Drexler et al., 1991; Creager et al., 1992). These observations suggest that a de®ciency of L -arginine may exist in patients with vascular disease. This does not agree completely with the known enzymology of NOS as the KM for L -arginine is approximately 2:9 lM (Pollock et al., 1991), which is well below established intracellular and plasma L -arginine concentrations (Harrison, 1997). One possible explanation for this ``arginine paradox'' is that newly imported L -arginine may be a better substrate for NO synthesis than the intracellular pool. This idea is supported by observations that the L -arginine transporter is physically associated with NOS and endothelial cells (McDonald et al., 1997). Another possible mechanism for the arginine paradox relates to the presence of endogenous inhibitors of NOS. Once such inhibitor is asymmetric-dimethyl arginine (ADMA) (Vallance et al., 1992). There are observations that vascular disease is associated with increased plasma levels of ADMA (Boger et al., 1998; Goonasekera et al., 1997), suggesting that the observations with L -arginine are due to displacement of ADMA. Thus, it is clear that L -arginine treatment improves NO-mediated arterial relaxation, however the mechanisms for such observations are still the subject of intense investigation. 4.1.5.2. Tetrahydrobiopterin. Another major cofactor for NOS is BH4 . This cofactor is important for coupling the activation of oxygen with nitric oxide production (Vasquez-Vivar et al., 1998; Bec et al., 1998; Xia et al., 1998b) and available evidence indicates this cofactor improves NO responses in patients. For example, administration of BH4 improves endothelial function in hypercholesterolemic patients (Stroes et al., 1997), cigarette smokers (Higman et al., 1996; Heitzer et al., 2000), and

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experimental models of diabetes (Pieper, 1997). These data suggest that intracellular BH4 status may be limited in the setting of vascular disease. 4.1.5.3. LDL lowering therapy. Given that all the major theories of atherogenesis involve LDL cholesterol (see above), it is not surprising that cholesterol lowering has been investigated as a means to improve endothelial function. Moreover, lipid-lowering therapy has been shown to convincingly reduce the incidence of cardiovascular events, and this occurs in the setting of modest reductions in atherosclerotic lesions (Grundy, 1998). Such ®ndings prompt speculation that cholesterol-lowering therapy improves the function of arteries. A number of studies have investigated this and they are outlined in Table 8. Treatment with a variety of cholesterol-lowering therapies has been shown to improve endothelial function over durations as short as one month. In a very interesting study, Tamai et al. (1997) showed that LDL apheresis improved endothelial function immediately in patients with relatively modest levels of LDL cholesterol. These data underlie a speci®c interaction between LDL and endothelial dysfunction that appears readily amenable to treatment (see Table 8). 4.1.5.4. Other treatments. A number of other interventions associated with reduced cardiovascular risk also appear to translate into improved endothelial function. For example, post-menopausal women treated with estrogen appear to have reduced cardiovascular events (Stampfer et al., 1997), and consistent with this observation, treatment of these same patients with estrogens appears to improve endothelial function (Lieberman et al., 1994). Similarly, angiotensin converting enzyme inhibitors are known to reduce coronary artery disease events (Pfe€er et al., 1989), and treatment of patients with quinapril for six months improves coronary artery endothelial function (Mancini et al., 1996). Thus, there appears to be a close association between interventions that improve cardiovascular outcome and the improvement of endothelial function. Further studies will be required to identify the precise mechanism for this association. 5. Summary Atherosclerosis is an important source of morbidity and mortality in the developed world. Despite the fact that the association between LDL cholesterol and atherosclerosis has been evident for at least three decades, our understanding of exactly how LDL precipitates atherosclerosis is still in its infancy. At least three working hypotheses of atherosclerosis are now nearing the stage where critical evaluation is possible through a combination of basic science investigation and murine models of atherosclerosis. As we move forward in our understanding of this disease, e€orts will be increasingly focused on the molecular mechanisms of disease activation that precipitate the clinical manifestations of atherosclerosis such as heart attack and stroke. Two candidates for such investigation involve the events surrounding plaque activation and endothelial dysfunction. Further investigation in these ®elds should provide the necessary insight to develop the next

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