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Centre for Vascular Research, University of New South Wales, and Department of Haematology, Prince of Wales Hospital, Sydney, New South Wales, Australia; and Whitaker Cardiovascular Institute, Evans Memorial Department of Medicine, Boston University Medical Center, Boston, Massachusetts
ABSTRACT I. INTRODUCTION A. Atherosclerosis and Its Relationship to Coronary Artery Disease 1. Epidemiology and risk factors A) AGE. B) GENDER. C) OBESITY. D) CIGARETTE SMOKING. E) HYPERTENSION. F) DIABETES MELLITUS. G) SERUM CHOLESTEROL. 2. Morphological features of atherosclerosis A) THE NORMAL ARTERY. B) GROSS MORPHOLOGY OF ATHEROSCLEROTIC LESIONS. C) PLAQUE MORPHOLOGY. D) PLAQUE RUPTURE. B. Hypotheses of Atherogenesis 1. The response-to-injury hypothesis 2. The response-to-retention hypothesis 3. The oxidative modification hypothesis II. REDOX REACTIONS IN THE VASCULATURE A. Oxidative Stress: A Definition B. Oxidants and Markers of Oxidant Events 1. Free radicals or 1e-oxidants 2. 2e-Oxidants C. Sources of Oxidants and Markers of Oxidative Events 1. NAD(P)H oxidases 2. Xanthine oxidase 3. NOS 4. Myeloperoxidase 5. Lipoxygenases 6. Mitochondrial respiration 7. Transition metals 8. Other oxidants D. Antioxidant Defenses 1. Enzymatic antioxidants A) SODS. B) CATALASE AND PEROXIDASES. C) GLUTATHIONE-DEPENDENT ANTIOXIDANT DEFENSES. D) THIOL-DISULFIDE OXIDOREDUCTASES. E) PEROXIREDOXINS. F) NONENZYMATIC REDUCTION OF LIPID HYDROPEROXIDES. 2. Metal sequestration A) BINDING PROTEINS FOR BIOLOGICAL IRON AND COPPER. B) HEME OXYGENASES. 3. Nonproteinaceous antioxidants A) WATER-SOLUBLE ANTIOXIDANTS. B) LIPID-SOLUBLE ANTIOXIDANTS. E. Redox Reactions in Cell Signaling 1. Targets of ROS in signal transduction A) THIOREDOXIN. B) ACONITASE. C) SOLUBLE GUANYLYL CYCLASE. D) RAS. E) TYROSINE KINASES. F) PROTEIN TYROSINE PHOSPHATASES. G) KELCH DOMAIN-CONTAINING PARTNER (KEAP1). H) TRANSCRIPTION FACTORS. 2. Targets for RNS in cellular signaling A) LOW-MOLECULAR-WEIGHT THIOLS. B) PROTEIN THIOLS. C) GLUTATHIOLATION. 3. Summary F. ROS and Cell Proliferation G. Redox Reactions in Cell Death H. Redox Reactions in Platelet Function 1. Platelets and cardiovascular disease 2. ROS and platelet aggregation 3. RNS and platelet aggregation III. OXIDATIVE MODIFICATION HYPOTHESIS OF ATHEROSCLEROSIS A. Original Hypothesis B. Evidence in Support of the LDL Oxidation Hypothesis 1. LDL does not support foam cell formation 2. Oxidative modifications in atherosclerotic lesions A) LIPID OXIDATION. B) PROTEIN OXIDATION. 3. Oxidized LDL is present in atherosclerotic lesions 4. Circulating oxidized LDL 5. Autoantibodies to oxidized LDL 6. Potential proatherogenic activities of oxidized LDL 7. Scavenger receptors and atherosclerosis 8. Antioxidant studies C. LDL Oxidation 1. Putative oxidants involved A) SUPEROXIDE ANION RADICAL. B) RNS. C) MYELOPEROXIDASE. D) LIPOXYGENASES. E) TRANSITION METALS. F) OTHER OXIDANTS. 2. High-uptake or oxidized LDL 3. Minimally modified LDL 4. Role of vitamin E in LDL oxidation A) TOCOPHEROL-MEDIATED PEROXIDATION. D. Antioxidant Status in Atherosclerotic Lesions 1. Proteinaceous antioxidants A) SOD AND CATALASE. B) GLUTATHIONE-RELATED ANTIOXIDANTS. C) HEME OXYGENASE. D) OTHER PROTEINS. 2. Nonproteinaceous antioxidants A) AQUEOUS ANTIOXIDANTS. B) LIPID-SOLUBLE ANTIOXIDANTS. E. Inhibition of LDL Oxidation F. Problems With the Oxidative Modification Hypothesis 1. LDL oxidation versus atherosclerosis A) IN VITRO LDL OXIDATION VERSUS ANTIOXIDANT AND LIPID OXIDATION IN THE VESSEL WALL. B) LDL OXIDATION: CAUSE OR CONSEQUENCE OF ATHEROSCLEROSIS. C) OXIDIZED LDL VERSUS OTHER OXIDIZED TARGETS. 2. Cardiovascular disease epidemiology and antioxidant intake A) PROSPECTIVE COHORT STUDIES. B) RANDOMIZED CONTROLLED STUDIES: PRIMARY PREVENTIONS. C) RANDOMIZED CONTROLLED STUDIES: SECONDARY PREVENTIONS. D) VITAMIN E IN COMBINATION WITH OTHER ANTIOXIDANTS. IV. ROLE OF OXIDATIVE MODIFICATIONS OTHER THAN LOW-DENSITY LIPOPROTEIN OXIDATION AND CLINICAL MANIFESTATIONS OF CORONARY ARTERY DISEASE A. Concept of Disease Activity B. Plaque Disruption 1. The fibrous cap A) MATRIX DEGRADATION. B) MATRIX PRODUCTION. C. Vasomotor Function 1. Endothelial dysfunction 2. Oxidative events and endothelial dysfunction A) SUPEROXIDE ANION RADICAL. B) LIPID PEROXYL RADICALS. C) PEROXYNITRITE AND N2O3. D) MYELOPEROXIDASE AND HYPOCHLOROUS ACID. E) HYDROGEN PEROXIDE. 3. Antioxidants and endothelial function A) ASCORBIC ACID, TETRAHYDROBIOPTERIN, AND GLUTATHIONE. B) LIPID-SOLUBLE ANTIOXIDANTS. D. Adhesion Molecules V. RECONCILING AVAILABLE DATA ON OXIDATIVE EVENTS AND ATHEROSCLEROSIS A. Oxidative Events and Atherosclerosis Are Not Causally Linked B. Incomplete Knowledge of Oxidants Involved in Atherosclerosis C. The Oxidative Response to Inflammation Hypothesis of Atherosclerosis D. Conclusions ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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Atherosclerosis is the major source of morbidity and mortality in the developed world. The magnitude of this problem is profound, as atherosclerosis claims more lives than all types of cancer combined and the economic costs are considerable. Although currently a problem of the developed world, the World Health Organization predicts that global economic prosperity could lead to an epidemic of atherosclerosis as developing countries acquire Western habits.
Atherosclerosis is characterized by the accumulation of cholesterol deposits in macrophages in large- and medium-sized arteries. This deposition leads to a proliferation of certain cell types within the arterial wall that gradually impinge on the vessel lumen and impede blood flow. This process may be quite insidious lasting for decades until an atherosclerotic lesion, through physical forces from blood flow, becomes disrupted and deep arterial wall components are exposed to flowing blood, leading to thrombosis and compromised oxygen supply to target organs such as the heart and brain. The loss of heart and brain function as a result of reduced blood flow is termed heart attack and stroke, respectively, and these two clinical manifestations of atherosclerosis are often referred to as coronary artery disease and cerebrovascular disease. To simplify any discussion of the underlying pathology in these clinical syndromes, coronary artery disease and cerebrovascular disease are commonly referred to by the collective term cardiovascular disease. With respect to the underlying pathology of atherosclerosis, there are a number of environmental and genetic "cardiovascular risk" factors that warrant consideration.
1. Epidemiology and risk factors
Over the last 40 years, a number of clinical and laboratory variables have proven predictive of the incidence of cardiovascular disease and thus qualify as cardiovascular disease risk factors. In the following, we will restrict our discussion to those traits that are strongly and consistently associated with cardiovascular disease in a manner that is independent of other traits.
A) AGE.
Although it is not subject to modification, age is among the most important risk factors for predicting incident cardiovascular disease. This concept is, perhaps, best illustrated if one considers the risk of developing cardiovascular disease over a 10-year period. Based on experience in the United States, the average risk of developing cardiovascular disease for a 30- to 34-year-old male is 
3% (1040). This number rises some sevenfold to 21% for a comparable individual aged 60–64 yr. Prediction of coronary heart disease uses risk factor categories (1040). The exact magnitude of age-related risk compared with other cardiovascular disease risk factors is illustrated by work from the Framingham Heart Study that has resulted in a 14-point scoring system to predict incident 10-yr cardiovascular disease. In this system, increasing risk is characterized by a higher score, and up to 7 points can be attributed to age alone. Thus age is an overriding risk factor for incident cardiovascular disease.
B) GENDER. Numerous observational studies have indicated that males exhibit excess risk for cardiovascular disease compared with age-matched women (40). There has been considerable speculation that estrogens offer a "protective" effect to women, as cardiovascular disease accelerates in women after menopause. However, this speculation has been difficult to substantiate, as the treatment with estrogen has not reduced the incidence of cardiovascular disease of postmenopausal women (419). Alternatively, some of this apparent protection could be due to the fact that women exhibit relatively higher concentrations of high-density lipoprotein (HDL) cholesterol than do age-matched men. Nevertheless, incident cardiovascular disease is less common in premenopausal women than their age-matched male counterparts.
C) OBESITY. There is now a growing appreciation that obesity, defined as an excess body weight with an abnormal high preponderance of body fat, is a condition that increases the incident risk of cardiovascular disease. The exact mechanism(s) to explain this phenomenon, however, are controversial. A number of other risk factors for cardiovascular disease, such as hypertension, low HDL cholesterol, and diabetes mellitus, often coexist with obesity (1041). This relation between obesity and cardiovascular disease has become of considerable concern as the prevalence of obesity in the developed world is increasing at an alarming rate.
D) CIGARETTE SMOKING.
The notion that cigarette smoking is linked to heart disease dates back to a series of studies that unequivocally linked smoking and the incidence of myocardial infarction (208, 231, 355). More recently, the Surgeon General's report estimates that smoking increases atherosclerotic disease by 50% and doubles the incidence of coronary artery disease (962a). There is now considerable confidence that smoking is causally related to coronary artery disease, as smoking cessation is quite effective in lowering the future risk of the disease. In fact, the risk of heart attack in ex-smokers approaches that of nonsmokers in only 2 years (296).
E) HYPERTENSION. Hypertension is defined as a systolic blood pressure in excess of 140 mmHg or a diastolic blood pressure above 90 mmHg (734). The current estimates indicate that the elderly are particularly predisposed to hypertension, with up to 75% of people over 75 years of age qualifying for this diagnosis (734). There appears to be an approximately linear relation between blood pressure elevation and the increased incidence of atherosclerotic vascular disease (573). The causal nature of this association is supported by numerous studies demonstrating that both heart attack and stroke are significantly reduced in hypertensive patients with the institution of antihypertensive therapy (380).
F) DIABETES MELLITUS. Approximately 17 million people in the United States, or 6.2% of the population, carry the diagnosis of diabetes mellitus (165). In patients with diabetes, the risk of coronary atherosclerosis is three- to fivefold greater than in nondiabetics despite controlling for other risk factors (60, 739). 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 (60), but despite this association, no more than 25% of the excess coronary atherosclerosis risk from diabetes can be attributed to these known risk factors (670). Thus diabetes represents a major contributing factor to atherosclerosis.
G) SERUM CHOLESTEROL.
The association between LDL cholesterol and atherosclerosis has been established based, in part, on an experiment of nature. Familial hypercholesterolemia is an autosomal dominant disorder that affects 1 in 500 persons from the general population. Heterozygotes for this disease manifest a two- to fivefold elevation in plasma LDL cholesterol that is due to a functional impairment of the LDL receptor, resulting in a defect in LDL clearance. Homozygotes for this disorder demonstrate a four- to sixfold elevation in plasma cholesterol that produces precocious atherosclerosis. In heterozygotes, 85% of individuals have experienced a myocardial infarction by the age of 60, and this age is reduced to 15 yr in patients homozygous for the disease (325). In the general population, the cardiovascular disease risk from increased LDL cholesterol is supported by observations that cholesterol-lowering therapy greatly diminishes the clinical manifestations of atherosclerosis, particularly since the advent of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (i.e., statins) that profoundly lower LDL cholesterol (326).
In contrast to the situation with LDL cholesterol, the relation between HDL cholesterol and atherosclerosis is an inverse one (320). The causal nature of this association is also supported by an experiment of nature, Tangier disease (682). This autosomal codominant condition is characterized by the essential absence of HDL cholesterol levels due to a defect in the ATP binding cassette transporter-1 (69, 584, 790) that impairs cholesterol efflux from cells (525). Tangier patients demonstrate a tissue cholesterol-loading syndrome, characterized by large tonsils, neuropathy, and premature coronary artery disease in some kindreds (682). Thus considerable evidence supports the inverse relation between coronary artery disease and serum levels of HDL cholesterol.
2. Morphological features of atherosclerosis
A) THE NORMAL ARTERY. The arterial wall normally consists of three well-defined concentric layers that surround the arterial lumen, each of which has a distinctive composition of cells and extracellular matrix. The layer immediately adjacent to the lumen is called the intima, the middle layer is known as the media, and the outermost layer comprises the arterial adventitia. These three layers are demarcated by concentric layers of elastin, known as the internal elastic lamina that separates the intima from the media, and the external elastic lamina that separates the media from the adventitia.
A single contiguous layer of endothelial cells lines the luminal surface of arteries. These cells sit on a basement membrane of extracellular matrix and proteoglycans that is bordered by the internal elastic lamina. Although smooth muscle cells are occasionally found in the intima, endothelial cells are the principal cellular component of this anatomic layer and form a physical and functional barrier between flowing blood and the stroma of the arterial wall. Endothelial cells regulate a wide array of processes including thrombosis, vascular tone, and leukocyte trafficking among others.
Progressing outwards from the internal elastic lamina, the media consists principally of smooth muscle cells arranged in layers, the number of layers depending on the arterial size. An extracellular matrix consisting largely of elastic fibers and collagen with a lesser content of proteoglycan holds the smooth muscle cells together. An increasing content of elastin is typical of larger arteries that need to provide for considerable elastic recoil during diastole, the time period between ejections of blood from the heart.
The adventitia is the outermost layer of the artery and typically consists of a loose matrix of elastin, smooth muscle cells, fibroblasts, and collagen. Most of the neural input into blood vessels also traverses through the adventitia. At one time, the adventitia was considered inactive with respect to vascular homeostasis; however, recently it has become clear that the adventitia, through the production of reactive oxygen species (ROS), may play an important role in controlling vascular remodeling and nitric oxide (NO) bioactivity (766).
B) GROSS MORPHOLOGY OF ATHEROSCLEROTIC LESIONS. Atherosclerosis manifests itself histological as arterial lesions known as plaques that have been extensively characterized (878–880) into six major types of lesions that reflect the early, developing, and mature stages of the disease (Fig. 1). In lesion-prone arterial sites, adaptive thickening of the intima is among the earliest histological changes. As macrophages accumulate lipid, type II lesions form as nodular areas of lipid deposition that are also known as "fatty streaks," and these represent lipid-filled macrophages (i.e., foam cells). Continued foam cell formation and macrophage necrosis can produce type III lesions that contain small extracellular pools of lipid. Types II and III lesions are readily apparent through the use of fat-soluble dyes that stain cholesterol esters accumulated in macrophages and the extracellular space. These early lesions are often evident by age 10 (877) and can occupy as much as one-third of the aortic surface by the third decade. Developing lesions represent the next two types of lesions and, as shown in Figure 1, are characterized by significant areas of extracellular lipid that represents the "core" of the atherosclerotic lesion. Type IV lesions are defined by a relatively thin tissue separation of the lipid core from the arterial lumen, whereas type V lesions exhibit fibrous thickening of this structure, also known as the lesion "cap." These type IV and V lesions 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.
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C) PLAQUE MORPHOLOGY. The light microscopic appearance of a prototypical atherosclerotic plaque is depicted in Figure 2. Plaques contain a central lipid core that is most often hypocellular and may even include crystals of cholesterol that have formed in the aftermath of necrotic foam cells. In this late stage of lesion development, residual foam cells may be difficult to see but have often left the core with an abundant quantity of tissue factor (1033), an important activator of the clotting cascade. This lipid core is separated from the arterial lumen by a fibrous cap and myeloproliferative tissue that consists of extracellular matrix and smooth muscle cells. The junction between the cap and the morphologically more normal area of artery is known as the "shoulder" region of the atherosclerotic plaque. This area is typically more cellular than other areas of the plaque and may contain a variable composition of smooth muscle cells, macrophages, and even T cells. The shoulder region is most prone to rupture and, as shown in Figure 2, may even contain evidence of previously healed fissures.
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Using perfusion fixation techniques, Davies and Thomas (188) published a seminal study demonstrating that acute myocardial infarction and crescendo angina, two cardinal manifestations of atherosclerosis, were associated with atherosclerotic plaque rupture and fissuring in the artery with compromised blood flow. These observations suggested that clinical events were the consequence of an abrupt, catastrophic change in plaque morphology rather than a gradual narrowing of the lumen. Evidence for plaque rupture can also be found in patients dying from noncardiac causes (186), suggesting that plaque rupture is part of atherosclerotic lesion progression rather than a unique feature of clinical events from atherosclerosis.
Given that plaque rupture is implicated as a precipitating event in the clinical manifestations of atherosclerosis, a considerable effort has been directed at understanding the events involved in this process. Mature atherosclerotic plaques can be categorized as either stable or vulnerable to rupture. Stable plaques tend to be characterized by a smaller lipid core, a thick fibrous cap, and shoulder regions with few inflammatory cells, whereas vulnerable plaques contain considerable lipid in their core, a thin fibrous cap, and a robust population of macrophages and T cells in their shoulder regions. These differences in morphology suggest that vulnerable plaques may be weaker structurally and more likely to rupture in response to the physical forces of flowing blood. This contention is supported by experimental data linking an increased content of macrophages in lesions to structural weakness (539).
In summary, atherosclerosis is a major source of morbidity and mortality in the developed world that is characterized by LDL deposition in the arterial wall, a process that is stimulated by environmental and genetic factors such as tobacco use, diabetes, and hypertension. This LDL deposition occurs primarily within macrophages and ultimately begets the formation of well-defined lesions in the arterial intima. Such lesions then develop and are prone to rupture and, as a consequence, can precipitate the clinical events such as heart attack and stroke. Given the public health implications of this disease, it is not surprising that considerable effort has been devoted to understanding the molecular mechanisms of atherosclerosis and the factors that predispose individuals to clinical events. Thus we now turn our attention to contemporary theories of atherogenesis.
B. Hypotheses of Atherogenesis
Over the past 150 years, there have been numerous efforts to explain the complex events associated with the development of atherosclerosis. In this endeavor, three distinct hypotheses have emerged that are currently under active investigation. These hypotheses of atherosclerosis are not mutually exclusive but rather emphasize different concepts as the necessary and sufficient events to support the development of atherosclerotic lesions. For the purposes of this review, we refer to these paradigms as follows: 1) the response-to-injury, 2) the response-to-retention, and 3) oxidative modification.
1. The response-to-injury hypothesis
Early hypotheses of atherosclerosis included the "incrustation" hypothesis of Rokitansky (778), suggesting that intimal thickening was due to arterial fibrin deposition, and the "lipid transudation" hypothesis of Virchow (995) according to which lipid complexes with mucopolysaccharides caused atherosclerosis. A common theme to these early hypotheses was their dependence on passive deposition rather than an active cellular component. Ross and Glomset (782) offered a radical departure from this thinking by proposing the "response-to-injury" hypothesis of atherosclerosis. In this hypothesis, the proposed initial step in atherogenesis is endothelial denudation leading to a number of compensatory responses that alter the normal vascular homeostatic properties. For example, injury enhances endothelial adhesiveness for leukocytes and platelets and alters the local vascular anticoagulant milieu to a procoagulant one. Recruited leukocytes and platelets then release cytokines, vasoactive agents, and growth factors that promote an inflammatory response that is characterized by migration of smooth muscle cells into the intima and their proliferation to form an intermediate lesion.
Another component of the inflammatory response is the recruitment of macrophages into the arterial wall. These macrophages take up deposited LDL lipid to form lipid-laden "foam cells," the hallmark of an early atherosclerotic lesion. The process of lipid accumulation and foam cell formation perpetuates an inflammatory response that perpetuates macrophage and lymphocyte recruitment (454, 981). Continued inflammation allows for cellular necrosis, with a concomitant release of cytokines, growth factors, and proteolytic enzymes that sets the stage for autocatalytic expansion of the lesion to form a space-occupying collection in the intima not unlike an abscess that would form in other tissues. As the lesion enlarges it begins to encroach upon the lumen and, ultimately, blood flow is impaired.
This response-to-injury hypothesis was originally based on the notion of endothelium desquamation as a principal event initiating atherosclerosis (783, 784). More recently, it has become clear that endothelial desquamation is not common and that an intact endothelial cell layer covers developing atherosclerotic lesions. These facts, among others, promoted refinement of the initial hypothesis such that endothelial dysfunction is sufficient to initiate atherogenesis through increased endothelial permeability to atherogenic lipoproteins (781) (Fig. 3). This contention is not without its problems, however, as even normal artery segments exhibit rates of LDL entry that exceed the rate of LDL accumulation (124), suggesting that atherogenic lipoprotein entry into the arterial wall may not depend on endothelial dysfunction. In fact, the rate of LDL entry into the arterial wall is rather uniform, but the accumulation of atherogenic lipoproteins is concentrated in areas that are predisposed to future lesion development (820, 821). Such lesion-prone sites tend rather to demonstrate an enhanced retention of atherogenic apolipoprotein B-containing lipoproteins (238, 820, 821). Such observations have prompted alternative hypotheses for the initiation of atherosclerosis.
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This hypothesis submits that the lipoprotein retention is the inciting event for atherosclerosis (Fig. 4). Within 2 h of injecting LDL into rabbits, arterial retention of LDL and its microaggregates can be observed (667). The underlying mechanisms involved in this process are just now coming to light. It is estimated that 85% of subendothelial lipoprotein delivery is the result of transcytosis, and this process is restricted to particles <70 nm in diameter (852). This size restriction is important as it suggests that lipoprotein lipase activity is needed for triacylgycerol-rich lipoproteins to reach the subendothelial space (1110). The retention of lipoproteins within the arterial wall, however, appears tightly linked to components of the extracellular matrix. Apolipoprotein B-100, the single protein associated with LDL, is retained within the arterial wall in close association with arterial proteoglycans (118, 1091). This interaction is mediated by specific residues (3359–3369) (71) that, when mutated, protect experimental animals against the development of atherosclerosis (71, 857). Apolipoprotein B-48, which appears to be equally atherogenic as apolipoprotein B-100 in mice (990), also binds avidly to proteoglycans, and this interaction is mediated by residues 84–94 of apolipoprotein B-48 (250). Thus these data support an important role for proteoglycan binding in the retention of apolipoprotein B-containing lipoproteins in the early stages of atherosclerosis.
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3. The oxidative modification hypothesis
The oxidative modification hypothesis, discussed in more detail in section III, focuses on the concept that LDL in its native state is not atherogenic. However, LDL modified chemically is readily internalized by macrophages through a so-called "scavenger receptor" pathway (317). Exposure to vascular cells in medium that contains transition metals also results in modification of LDL such that it serves as a ligand for the scavenger receptor pathway (395). It is now clear that one mechanism whereby cells in vitro render LDL a substrate for the scavenger receptor pathway is via oxidation of LDL lipids and the resulting modification of apolipoprotein B-100 (886). These observations form the basis for the oxidative modification hypothesis of atherosclerosis (Fig. 5), in which LDL traverses the subendothelial space of lesion-prone arterial sites. During this process, LDL lipids are subject to oxidation and, as a consequence, apolipoprotein B-100 lysine groups are modified so that the net negative charge of the lipoprotein particle increases (347). This modification of apolipoprotein B-100 renders LDL susceptible to macrophage uptake via a number of scavenger receptor pathways producing cholesterol ester-laden foam cells (349). It is this accumulation of foam cells that forms the nidus of a developing atherosclerotic lesion.
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In summary, the aforementioned hypotheses of atherosclerosis have each attempted to explain the complex cellular events of atherosclerosis along a common theme. The response-to-injury hypothesis focuses on vascular injury as the inciting event in atherosclerosis with the entire spectrum of the disease representing an attempt to heal an ongoing vascular insult. In contrast, the response-to-retention hypothesis uses lipoprotein-matrix interactions as the critical event in early atherosclerosis, whereas the oxidative modification hypothesis requires oxidation of LDL lipids. Although each hypothesis points to its own critical initiating event, there are many common features among these competing hypotheses. For example, each involves a significant component of inflammation, a known feature of atherosclerosis (556). Each hypothesis also includes LDL as a central element, an important point as reduction in LDL cholesterol is among the most effective means of treating atherosclerosis. Perhaps the most unique, however, is the oxidative modification hypothesis as it alone proposes a particular importance of oxidative events and redox reactions in the genesis of vascular disease. The concept of oxidative events in atherosclerosis has changed considerably since the early days of the oxidative modification hypothesis, and it is this subject that will form the basis for the remainder of this review.
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II. REDOX REACTIONS IN THE VASCULATURE |
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The notion of "oxidative stress" in biological systems goes back to the early period of research on oxygen activation with an initial focus on oxygen toxicity and X-irradiation (300). Much of the relevant early literature was reviewed in 1979 by Chance et al. (143) in an article on hydroperoxide metabolism in mammalian organs. In the following, the concept of oxidative stress was developed primarily by Sies (847–849), with synonymous terms such as "oxidant stress" and "pro-oxidant stress," or the related term "reductive stress" receiving comparatively less emphasis.
Sies described oxidative stress as a "disturbance in the pro-oxidant/antioxidant balance in favor of the former" (847). This original denotation has been modified since to the more refined definition of "imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage" (848). This more careful definition accounts for some important operational considerations. For example, an oxidative challenge or a loss of antioxidants alone does not constitute oxidative stress. However, if increased formation of oxidant(s) is accompanied by a loss of antioxidant(s) and/or accumulation of oxidized forms of the antioxidant(s), oxidative stress is approached. The refined definition also conceptually distinguishes oxidative stress from oxidative damage. Thus even a severe oxidative assault that is accompanied by a loss of antioxidants may not necessarily result in oxidative damage (848). For example, biological systems are characterized by adaptive responses that may compensate, and perhaps even overcompensate, the oxidative stress that may manifest itself as a situation of increased redox environment (898).
The refined definition of oxidative stress and its underlying redox chemistry involving reduction-oxidation reactions implies that any form of "tipping the balance" causes an "imbalance." This has led to the concept of reductive stress to describe a situation where the balance is altered in favor of reductants (1027). Reductive stress can be intimately linked to oxidative stress. For example, an overproduction of reducing equivalents such as NAD(P)H may result in increased redox cycling of substances that can undergo repetitive rounds of oxidation/reduction, ultimately leading to the increased generation of superoxide anion radical (O2–·) and secondary oxidants. This has been implicated in the formation of ROS by hypoxia-like metabolic imbalances (951).
With the increased appreciation of interplay between ROS and reactive nitrogen species (RNS), including their responses in cells, the term nitrosative stress has also been introduced (364). Nitrosative stress, defined as increase in S-nitrosated compounds associated with a decrease in intracellular thiols, may be associated with a number of biological responses, some of which are of particular interest to vascular physiology and pathophysiology (1044, 1112).
B. Oxidants and Markers of Oxidant Events
1. Free radicals or 1e-oxidants
A free radical can be defined as any species capable of independent existence that contains one or more unpaired electrons (353). In biological systems, a variety of radicals can be generated (Table 1) with their reactivity depending on their nature and the molecule(s) encountered. If two radicals meet, they can join their unpaired electrons to form a covalent bond in reactions that are often kinetically fast and that lead to nonradical products. An example relevant to the vessel wall is the very fast reaction of O2–· with ·NO to form peroxynitrite (ONOO–) (reaction 1):
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A typical example of such a chain reaction is the process of lipid peroxidation that may be initiated by, for example, a hydroxyl radical (·OH) abstracting a hydrogen atom from a fatty acid side chain (LH) containing carbon atoms with bisallylic hydrogens (
) (reaction 2). The resulting, carbon-centered radical (L·) adds rapidly to O2 to generate a lipid peroxyl radical (LOO·) (reaction 3) that itself can propagate the chain by reacting with a neighboring lipid molecule to generate another L· and lipid hydroperoxide (LOOH) (reaction 4). In this fashion, many molecules of LOOH may be generated for each initiating radical.
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-tocopherol (-TOH). In this case, the -tocopheroxyl radical (-TO·) is produced and, for LOO·, a molecule of LOOH (reaction 5):
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A radical may be an oxidizing agent, accepting a single electron from a nonradical, or a reducing agent, donating a single electron to a nonradical. As implied above and like other reactions, free radical reactions are governed by thermodynamic and kinetic principles. A thermodynamic parameter commonly applied in free radical chemistry is the reduction potential that determines the feasibility of a compound X to chemically reduce compound Y. Buettner (104) has compiled a useful list of biologically relevant standard reduction potentials that predict the direction of reactions. Accordingly, the ascorbate/ascorbyl radical system is, for example, capable of reducing the -TO·, H+/-TOH system (reaction 6) that has a more positive standard reduction potential.
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In addition to radicals, several nonradical oxidants are important when considering oxidative modifications in the vessel wall (Table 2). Arguably, the most abundant of these is hydrogen peroxide (H2O2) derived from the action of oxidases such as glucose oxidase on O2, or from the dismutation of O2–· (reaction 7):
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7.5 (488), in biological systems the formation of ONOO– likely results in a powerfully oxidizing environment, comparable to that resulting from generation of ·OH. Nonradical oxidants like ONOOH (947) and HOCl (371–373) appear to react preferentially with proteins rather than lipids (998). Cysteine and methionine residues are the preferred targets, followed by tyrosine, tryptophan, and phenylalanine residues; lysine residues can also be oxidized and, due to their relative abundance, may account for a majority of the ONOOH or HOCl that reacts with proteins (714, 1046). This preference for a reaction with proteins differentiates nonradical from radical oxidants, the latter commonly initiating lipid peroxidation (see above).
In the case of methionine residues, methionine sulfoxide is produced as the predominant species. In comparison, thiol oxidation by HOCl gives rise to a complex array of products, including disulfides (RSSR), sulfenic (RSOH), sulfinic (RSO2H), and sulfonic acids (RSO3H), as well as intra- and intermolecular sulfenamides (RSN-), sulfinamides [RS(O)N-], and sulfonamides [RS(O2)N-] (279, 750, 1047). The reaction with aromatic amino acid residues is studied best for tyrosine, with 3-chloro- and 3-nitrotyrosine being chemically characteristic oxidation products of HOCl and ONOOH, respectively (46, 377), although aldehydes can also be formed (see sect. IIC4). An example of formation of 3-nitrotyrosine is the specific nitration of tyrosine residue 430 of bovine prostacyclin synthase by ONOO– that results in a decrease in enzymatic activity (814).
The local environment also governs the likelihood with which 2e-oxidants react with amino acids in protein side chains. For example, vicinal thiol and methionine residues are particularly sensitive to oxidation, as exemplified by H2O2-mediated oxidation of the carboxy-terminal vicinal methionine residues in calmodulin (1083). Similarly, a local environment that promotes ionization of the thiol (Cys-SH) group a cysteine residue, even at neutral pH, to the thiolate anion (Cys-S–) enhances its susceptibility to oxidation. This may be achieved via neighboring amino acids to lower the pKa value of cysteine residues, or via complex formation with metal ions. Examples of the latter relevant to oxidative stress in the vessel wall are the zinc thiolate (ZnS4) cluster of endothelial nitric oxide synthase (eNOS) (757) and the cysteine-switch domain of prometalloproteinases (984) that are sensitive to ONOOH (1112) and HOCl, respectively (278).
Two-electron oxidants like HOCl and ONOOH avidly react with certain prosthetic groups, such as heme and iron sulfur centers of proteins (8), often in preference to reaction with amino acid side chains, as in the case of ONOO–-induced inactivation of inducible NOS in the presence of calmodulin (418). As mentioned, lipids are poor targets compared with proteins, although chlorinated species have been characterized as products upon reaction with HOCl (978, 1049).
A major biological target for ONOO– is carbon dioxide (CO2) (reviewed in Ref. 869). The reaction of ONOO– with carbon dioxide is complex and produces metastable species that promote nitration (i.e., addition of a nitro group -NO2), nitrosation (addition of a nitrogen monoxide group -NO), and oxidation reaction. This probably occurs via the intermediate nitrosoperoxycarbonate (ONOOCO2–) that homolyzes to form a pair of caged radicals (CO3–· ·NO2) that can then diffuse apart to become free radicals. Alternatively, the radicals may combine to nitrocarbonate (O2NOCO2) that then decomposes to nitrite and CO2. Therefore, in the presence of carbon dioxide, the redox chemistry of ONOO– is mediated largely by ·NO2 and CO3–·; in the absence of carbon dioxide it is mediated by ·NO2 and ·OH (487). Nitrogen dioxide (·NO2) is a strongly oxidizing radical that can initiate a variety of oxidative pathways (486). For example, ·NO2 initiates and promotes lipid peroxidation including that of LDL (115), and it gives rise to nitrotyrosine formation (327, 486). However, ·NO2 is not only derived from ONOO–, as other sources, including enzymatic ones, have been identified (see sect. IIC4). Therefore, systems that simultaneously generate ·NO and O2–· have the potential to generate ONOO– and a complex mixture of oxidants including the radical oxidant ·NO2 that can readily oxidize a number of targets in the vessel wall, including LDL.
C. Sources of Oxidants and Markers of Oxidative Events
Several lines of evidence implicate ROS and RNS in atherogenesis. The advent of gene targeting to predictably modify genes has generated valuable animal models and allowed a genetic approach to gain information on which oxidants affect disease. The development of sensitive analytical methods also enables researchers to obtain direct chemical evidence for specific reaction pathways that promote oxidative events and lesion formation in vivo, and to relate the accumulation of specific oxidized lipid and protein to different stages of atherosclerosis. Understanding the processes that lead to the different oxidative events is important in developing strategies to effectively inhibit such processes and hence potentially atherosclerosis. Within the vessel wall, the different oxidants can originate principally from cellular and extracellular sources, and from enzymatic and nonenzymatic paths that are reviewed briefly in the following sections. The primary consideration given here will be on oxidants of plausible physiological relevance, noting those with the strongest support.
It has long been known that phagocytes, including neutrophils, monocytes, and macrophages, contain a plasma membrane-bound, multicomponent oxidase that utilizes electrons derived from NADPH to reduce molecular oxygen to O2–· in a reaction that is insensitive to the mitochondrial poison KCN. While O2–· is principally a reducing agent, it can give rise to secondary products that include strong oxidants.
The phagocyte NADPH oxidase (Nox) consists of four major units: p22phox (CYBA gene product) and gp91phox (CYBB gene product), the two subunits of the membrane-spanning cytochrome b558, and two cytosolic components, p47phox (NOXO2 gene product) and p67phox (NOXA2 gene product) (25). Other proteins associate or copurify with the oxidase and/or participate in its assembly/activation or inactivation. Phagocyte NADPH oxidase needs to be activated to produce O2–·, a process that begins with the phosphorylation of p47phox leading to its association with cytochrome b558(25). Translocation of the G protein Rac to cytochrome b558 is also involved (706) but independent of both p47phox and p67phox translocation (397). Rac1 is the predominant protein expressed in monocytes, in contrast to Rac2 in neutrophils (137). Once activated appropriately, phagocytic cells produce large amounts of O2–· over relatively short periods that are involved in host defense.
In analogy to phagocytes, adventitial fibroblasts, vascular smooth muscle cells, and endothelial cells contain a membrane-associated NAD(P)H oxidase that utilizes NADH or NADPH as the electron donor to generate O2–· via 1e-reduction of molecular oxygen (reaction 8) (for reviews, see Refs. 333, 766).
 | (8) |
There is evidence that NAD(P)H oxidase activity represents a major source of ROS in the vasculature (631, 692, 752), and there is some controversy regarding the orientation and cellular location of these enzymes. Whereas in phagocytes electrons are transferred across the membrane to extracellular oxygen, in vascular smooth muscle cells, O2–· and H2O2 appear to be produced predominantly inside the cells, and addition of NAD(P)H to the cells augments O2–· generation (333). For endothelial cells, there is evidence for extracellular release of O2–· as indicated using cell-impermeable "trapping" agents (972).
There are also differences in the protein components of the NAD(P)H oxidases in different vascular cells. Fibroblast and endothelial cells resemble phagocytes in that they contain mRNAs for gp91phox, p22phox, p47phox, and p67phox, whereas vascular smooth muscle cells appear to lack gp91phox and gp67phox (333, 766). Instead, these cells contain homologs of gp91phox, the expression of which increases cellular O2–· production (918). A number of homologs including Nox1, -4, and -5, have been identified in vascular cells (147, 864), with endothelial cells expressing very low levels of Nox1, intermediate levels of Nox2, and abundant Nox4 mRNA (6, 864). In contrast, vascular smooth muscle cells express predominantly Nox4 and to a lesser extent Nox1 with negligible amounts of Nox2 (864). There also appear to be distinctions in Nox expression based on the particular type of blood vessel, as Nox2 is relatively more abundant in human aortic smooth muscle cells derived from resistance compared with conductance arteries (955). In addition, there is increasing evidence that Nox1 and Nox4 are located in different cellular compartments (398).
In contrast to phagocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts exhibit low basal activity for O2–· generation (691), suggestive of a constitutively active oxidase. Consistent with this, Nox2 in endothelial cells appears to be present as a preassembled, intracellular complex including the p22phox, p47phox, and p67phox subunits (547). Similar to the situation in phagocytes, however, NAD(P)H oxidases in vascular cells can also be activated by stimuli such as angiotensin II, thrombin, platelet-derived growth factor, tumor necrosis factor-, interleukin-1, and for endothelial cells, mechanical forces (including shear stress) and vascular endothelial growth factor (972). Activation appears to involve the association of additional proteins with the oxidase components (972). In the case of angiotensin II-induced production of ROS by vascular smooth muscle cells, the early phase (30 s) is protein kinase C dependent, whereas the prolonged phase (30 min) is dependent on Rac, Src, and phosphatidylinositol 3-kinase (828). In endothelial cells, angiotensin II rapidly induces ROS production through serine phosphorylation of p47phox, and its enhanced binding to p22phox (548). More recently, homologs of p47phox and p67phox have been identified and proposed to participate in the activation of the Nox family oxidases (929). However, even under stimulated conditions, the amounts of O2–· produced by vascular cells are only a fraction of those generated by activated phagocytes
