Summary. This review focuses on the morphologicalfeatures of atherosclerosis and the involvement ofoxidative stress in the initiation and progression of thisdisease. There is now consensus that atherosclerosisrepresents a state of heightened oxidative stresscharacterized by lipid and protein in the vascular wall.Reactive oxygen species (ROS) are key mediators ofsignaling pathways that underlie vascular inflammationin atherogenesis, starting from the initiation of fattystreak development, through lesion progression, toultimate plaque rupture. Plaque rupture and thrombosisresult in the acute clinical complications of myocardialinfarction and stroke. Many data support the notion thatROS released from nicotinamide adenine dinucleotidephosphate (NADPH) oxidase, myeloperoxidase (MPO),xanthine oxidase (XO), lipoxygenase (LO), nitric oxidesynthase (NOS) and enhanced ROS production fromdysfunctional mitochondrial respiratory chain, indeed,have a causatory role in atherosclerosis and othervascular diseases. Moreover, oxidative modifications inthe arterial wall can contribute to the arteriosclerosiswhen the balance between oxidants and antioxidantsshifts in favour of the former. Therefore, it is importantto consider sources of oxidants in the context ofavailable antioxidants such as superoxide dismutase(SOD), catalase (CAT), glutathione peroxidase (GPx),glutathione reductase and transferases thiol-disulfideoxidoreductases and peroxiredoxins. Here, we reviewalso the mechanisms in which they are involved in orderto accelerate the pace of the discovery and facilitatedevelopment of novel therapeutic approaches.

Key words: Reactive oxygen species, Antioxidantenzymes, Oxidative stress, Vascular damage

Introduction

Marchand introduced the term “atherosclerosis”,describing the association of fatty degeneration andvessel stiffening (Aschoff, 1933; Crowther, 2005). Thisprocess affects medium and large-sized arteries and ischaracterized by patchy intramural thickening of thesubintima that encroaches on the arterial lumen. Eachvascular bed may be affected by this process; theetiology, treatment and clinical impact of atherosclerosisvary from one vascular bed to another (Faxon et al.,2004). The earliest visible lesions of atherosclerosis arethe fatty streak, which is due to an accumulation of lipid-laden foam cells in the intimal layer of the artery. Withtime, the fatty streak evolves into a fibrous plaque, thehallmark of established atherosclerosis. Ultimately thelesion may evolve to contain large amounts of lipid; if itbecomes unstable, denudation of overlying endotheliumor plaque, or plaque rupture, may result in thromboticocclusion of the overlying artery.

Atherosclerosis, in fact, is a progressive diseasecharacterized by the accumulation of cholesteroldeposits in macrophages (foam cells) in large andmedium arteries. This deposition leads to a proliferationof certain cell types within the arterial wall, whichgradually impinge on the vessel lumen and impede bloodflow. These early lesions, called “fatty streak lesions”,can usually be found in the aorta in the first decade oflife, in the coronary arteries in the second decade, and inthe cerebral arteries in the third or fourth decades. Fattystreaks are not clinically significant, but they are theprecursors of more advanced lesions characterized bythe accumulation of lipid-rich necrotic debris andsmooth muscle cells (SMCs) (Lusis, 2000).Atherosclerosis manifests itself histological as arteriallesions known as plaques, which have been extensivelycharacterized. Plaques contain a central lipid core that ismost often hypocellular and may even include crystals ofcholesterol that have formed in the aftermath of necrotic

Review

Atherosclerosis and oxidative stress

F. Bonomini, S. Tengattini, A. Fabiano, R. Bianchi and R. RezzaniDivision of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy

Histol Histopathol (2008) 23: 381-390

Offprint requests to: Rita Rezzani, Division of Human Anatomy,Deparment of Biomedical Sciences and Biotecnology, University ofBrescia, V.le Europa 11, 25123, Brescia, Italy. e-mail:rezzani@med.unibs.it

http://www.hh.um.es

Histology andHistopathology

Cellular and Molecular Biology

foam cells. The lipid core is separated from the arteriallumen by a fibrous cape and myeloproliferative tissuethat consists of extracellular matrix and smooth musclecells (Stocker and Keaney, 2004). Plaques can becomeincreasingly complex, with calcification, ulceration atthe luminar surface, and hemorrhages from small vesselsthat grow into the lesion from the media of the bloodvessel wall. Although advanced lesions can growsufficiently large to block blood flow, the most importantclinical complication is an acute occlusion due to theformation of a thrombus or blood clot, resulting inmyocardial infarction or stroke. Age, gender, obesity,cigarette smoking, hypertension, diabetes mellitus anddyslipidemias are known atherogenic risk factors thatpromote the impairment of endothelial function, smoothmuscle function and vessel wall metabolism. These riskfactors are associated with an increased production offree radicals that are called ROS (Antoniades et al.,2003). ROS are metabolites of oxygen that, through theirhigh reactivity, are prone to participation in oxidation-reduction reactions. An increasing number of studieshave demonstrated that oxidative stress (dysregulation ofthe cellular redox state) plays a pivotal role in thepathogenesis of atherosclerosis, especially vascularendothelial dysfunction. Reactive oxygen species havedetrimental effects on vascular function through severalmechanisms. First, ROS, especially hydroxyl radicals,directly injure cell membranes and nuclei. Second, byinteracting with endogenous vasoactive mediatorsformed in endothelial cells, ROS modulate vasomotionand the atherogenic process. Third, ROS peroxidize lipidcomponents, leading to the formation of oxidizedlipoproteins (LDL), one of the key mediators of

atherosclerosis. Whereas native LDL does not causecholesterol ester accumulation in macrophages, LDLmodified by oxidation does. Oxidized LDL has alsobeen implicated in other mechanisms potentiallyinvolved in the development of atherosclerosis, such ascytotoxic or chemotactic actions on monocytes and theinhibition of macrophage motility (Inoue and Node,2006). Up-regulation of adhesion molecules, such asintracellular adhesion molecule-1 (ICAM-1), vascularcell adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1),integrins, and selectins (L, E, and P) will lead to theattachment and buildup of immune cells (monocytes,macrophages, platelets, and T lymphocytes) on theendothelial wall. This activation of the endotheliumleads to the release of pro-inflammatory cytokines,surface receptors, and proteinase enzymes including, butnot limited to, interleukins, interferons, monocytechemoattractant protein-1 (MCP-1), monocytechemoattractant protein-4 (MCP-4) and cyclooxygenase-2 (COX-2). The endothelium becomes more permeableto lipid particles and immune cells, which leads to asituation where macrophages engulf oxidized low-density LDL particles and other modified lipoproteins,thus becoming foam cells. Foam cells in turn lead to anincrease in inflammatory cytokine production (Fig. 1).This proliferating immune response activates thevascular smooth muscle cell (VSMC) layer to proliferateand migrate as well as leading to local vasoconstriction,collagen and matrix deposition, fibrous cap production,and immune cell and platelet recruitment and activation(Hennig et al., 2001). Here, we considered themorphological features of atherosclerosis and the

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Fig. 1. Oxidative stress inatherosclerosis. An increase offree radical production isassociated withatherosclerosis. Theseradicals provoke an oxidativemodification from low-densitylipoprotein (LDL) to oxidizedlow-density lipoprotein (ox-LDL). Circulating monocytesmigration to the subendothelialspace is stimulated by ox-LDLand also causes endothelialcell injury. The modified LDL istaken up by macrophageswhich become foam cells,leading to the formation ofatherosclerotic plaque.

involvement of oxidative stress in the initiation andprogression of this disease. In particular, we focused onpro-oxidant factors and on the main endogenousprotection mechanisms, such as antioxidant enzymes.

Morphological features of atherosclerosis

Atherosclerosis manifests itself histologically asarterial lesions known as plaques, which have beenextensively characterised into six major types of lesionsreflecting the early, developing and mature stages of thedisease (Stary et al., 1995) (Fig. 2). In the arterial treesites prone to the disease, adaptive thickening of theintima is among the earliest histological changes. Asmacrophages accumulate lipid, type II lesions form asnodular areas of lipid deposition that are also known as“fatty streaks”, and these represent lipid-filledmacrophages (i.e. foam cells). Continued foam cellformation and macrophage necrosis can produce type IIIlesions containing small extracellular pools of lipid.Type IV lesions are defined by a relatively thin tissueseparation of the lipid core from the arterial lumen,whereas type V lesions exhibit fibrous thickening of thisstructure, also known as the lesion “cap”. Recent datashowed that mature type VI lesions exhibit acomplicated architecture with plaque formation and

progression, culminating in their rupture.

Plaque morphology

The light microscopical appearance is visible in Fig.3, showing histopathologic changes observed inapolipoproteins E-deficient (Apo-E°) mice. These miceare a useful tool for experimental studies, since theydevelop hypercholesterolemia and acceleratedatherosclerosis, and their atherosclerotic lesioncomposition is similar to human atherosclerotic lesions(Coleman et al., 2006; Rodella et al., 2007). The plaquesare characterised by a growing mass of extracellularlipids protruding into the lumen of the vessels; lipiddroplets are associated with the intimal cells of thevessels, especially in lipid-rich macrophages (“foamcells”). These early developing lesions typicallydeveloped acellular regions, characterized by necroticlipid cores. In several cases, the raised lesions arecovered by a collagenous cap with myeloproliferativetissue, which consists of extracellular matrix and SMCs.Substantial collagen deposition is associated with theVSMCs, many of which also show cytoplamic lipidinclusions. Elastin laminae are fragmented,discontinuous and commonly display reduced thickness(Rodella et al., 2007).

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Fig. 2. Early, developing andmature stages ofatherosclerosis. A showsearliest stage. B,C,D and Eshow developing stages of thedisease. F shows anadvanced stage thatculminates in plaque ruptureand thrombosis.

Plaque rupture

Over the last twenty years, the term ”plaque rupture”consists of a number of events responsible foratherosclerosis progression and considerable efforts havebeen directed at understanding the composition and thevulnerability of plaques, rather than the severity of thestenosis. In fact, mature atherosclerotic plaques havebeen subdivided in stable and vulnerable plaques.Vulnerable plaques generally have thin fibrous caps andincreased numbers of inflammatory cells (Lusis, 2000).Maintenance of the fibrous cap reflects matrixproduction and degradation, and products ofinflammatory cells are likely to influence both processes.For example, T cells produce interferon-γ (IFN-γ), whichinhibits the production of matrix by SMCs, andmacrophages produce various proteases that degradeextracellular matrix, including interstitial collagenase,gelatinases and stromolysin. Rupture frequently occursat the lesion edges, which are rich in foam cells,suggesting that factors, contributing to inflammation,may also influence thrombosis. In this regard, it isimportant to note that the incidence of myocardialinfarction and stroke increases during acute infections.

Moreover, the stability of atherosclerotic lesionsmay also be influenced by calcification andneovascularization, common features of advancedlesions. Intimal calcification is an active process inwhich pericyte-like cells secrete a matrix scaffold, whichsubsequently becomes calcified, akin to bone formation.The process is regulated by oxysterols and cytokines(Moultan and Folkman, 1999). The thrombogenicity ofthe lesion core is likely to depend on the presence oftissue factor, a key protein in the initiation of thecoagulation cascade. The production of tissue factors byendothelial cells (ECs) and macrophages is enhanced by

oxidized LDL, infection or the ligation of CD40 on ECsto CD40L on inflammatory cells. The expression ofother molecules mediating thrombosis, such asplasminogen activator, may also be important (Geller etal., 2000).

Oxidative stress

Oxidative stress: a definition

The notion of “oxidative stress” in biologicalsystems goes back to the early period of research onoxygen activation with an initial focus on oxygentoxicity and X-irradiation (Stocker and Keaney, 2004).Much of the relevant literature was reviewed in 1979 byChance et al. (1979) in an article on hydroperoxydemetabolism in mammalian organs. In the followingpaper, the concept of oxidative stress was developedprimarily by Sies (1985), with synonymous terms suchas “oxidant stress” and “pro-oxidant stress”, or therelated term “reductive stress” receiving comparativelyless emphasis. Sies described oxidative stress as a“disturbance in the pro-oxidant/oxidant balance infavour of the former” (Sies, 1985). This originaldenotation has been modified since to the more refineddefinition of “imbalance between oxidants andantioxidants in favour of the oxidants, potentiallyleading to damage” (Sies, 1991). This more carefuldefinition accounts for more important operationalconsiderations. For example, an oxidative challenge or aloss of antioxidants alone does not constitute oxidativestress. However, if increased formation of oxidant(s) isaccompanied by a loss of antioxidant(s) and/oraccumulation of oxidized forms of the antioxidant(s),oxidative stress is approached. The refined definitionalso conceptually distinguishes oxidative stress from

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Fig. 3. Histology of an atherosclerotic palque in aorta stained with verhoeff’s elastin stain (A) and Masson’s trichrome stain (B). The asterisk indicatesthe plaque. x 1,000

oxidative damage. Thus even an oxidative assault that isaccompanied by a loss of antioxidants may notnecessarily result in oxidative damage. For example,biological systems are characterized by adaptiveresponses that may compensate and perhaps evenovercompensate the oxidative stress, which maymanifest itself as a situation of increased redoxenvironment. The refined definition of oxidative stressand its underlying redox chemistry, involving reduction-oxidation reactions, implies that any form of “tipping thebalance” causes an “imbalance”. This has led to theconcept of reductive stress to describe a situation wherethe balance is altered. Healthy vascular cells metabolizeoxygen and generate in favour of reductans. Reductivestress can be intimately linked to oxidative stress. Forexample, an overproduction of reducing equivalents,such as NAD(P)H, may result in increased redox cyclingof substances that can undergo repetitive rounds ofoxidation/reduction, ultimately leading to the increasedgeneration of superoxide anion radical (O2–) andsecondary oxidants. This has been implicated in theformation of ROS by hypoxia-like metabolic imbalances(Tilton et al., 1997).

With the increased appreciation of interplay betweenROS and reactive nitrogen species (RNS), includingtheir responses in cells, the term “nitrosative stress” hasalso been introduced. Nitrosative stress, defined as anincrease in S-nitrosated compounds, associated with adecrease in intracellular thiols, may be associated with anumber of biological responses, some of which are ofparticular interest to vascular physiology andpathophysiology.

Because of the apparent importance of ROS invascular disease, there is substantial interest in theenzymatic sources that contribute to production of freeradicals in vascular tissues. Several enzyme systemsseem to be important in this process, including XO, theNADPH- oxidase, MPO, LPO, mitochondrial sourcesand NOS (Harrison et al., 2003).

Oxidants and markers of oxidants events

A free radical can be defined as any species capableof independent existence that contains one or moreunpaired electrons. In biological systems, a variety ofradicals can be generated (Table 1) with their reactivitydepending on their nature and the molecule(s)encountered. If two radicals meet, they can join theirunpaired electrons to form a covalent bond in reactionsthat are often kinetically fast and that lead to nonradicalproducts. An example relevant to the vessel wall is thevery fast reaction of O2

– with NO to form peroxynitrite(ONOO–) (reaction 1)

O2–· + · NO Õ ONOO– (1)

Alternatively, a radical may add to a nonradicalmolecule or abstract a hydrogen atom from C-H, O-H, orS-H bond of nonradical molecules. These types of

radical reaction are common in biological systems wheremost molecules are nonradical species. The moleculespotentially affected include low-molecular-weightcompounds like antioxidants and cofactors of enzymes,lipids, proteins, nucleic acids and sugars. In this case, anew radical is generated and this can set up a chainreaction.

A typical example of such a chain reaction is theprocess of lipid peroxidation that may be initiated by, forexample, a hydroxyl radical (·OH) abstracting ahydrogen atom from a fatty acid side chain (LH)containing carbon atoms with bisallylic hydrogens(reaction 2). The resulting carbon-centered radical(LOO·) (reaction 3) which itself can propagate the chainby reacting with a neigh-boring lipid molecule togenerate another L· and lipid hydroperoxide (LOOH)(reaction 4). In this fashion, many molecules of LOOHmay be generated for each initiating radical.

LH + OH Õ L· + H2O (2)L· + O2 Õ LOO· (3)LOO· + LH Õ L· + LOOH (4)

Whereas the highly reactive ·OH abstracts H atomsalmost with discrimination, less reactive radicals, suchas LOO· preferentially abstract H atoms from moleculeswith weaker bonds, such as the chromanol O-H bondcontained in α-tocopherol (α-TOH). The α-tocopheroxilradical (α-TO·) is produced and, for LOO·, a moleculeof LOOH (reaction 5):

LOO· + α-TOH Õ LOOH + α-TO·

A radical may be an oxidizing agent, accepting asingle electron from nonradical, or a reducing agent,donating a single electron to a nonradical. Buettner(1993) has compiled a useful list of biologically relevantstandard reduction potentials that predict the directionsof reactions. Accordingly, the ascorbate/ascorbyl radicalsystem is, for example, capable of reducing the α-TO· /H+ α -TOH system (reaction 6) that has a more positivestandard reduction potential.

H+ + ascorbate– + α-TO· Õ α-TOH· + ascorbyl radical

Oxidants

In addition to radicals, several nonradical oxidantsare important when considering oxidative modificationsin the vessel wall (Table 1). Arguably, the most abundantof these is hydrogen peroxide (H2O2) derived from theaction of oxidases such as glucose oxidase on O2 or fromthe dismutation of O2

– (reaction 7):

2O2– · + 2H+ Õ H2O2 + O2

As in the case for radicals, the reactivity of thedifferent radical species varies. Hydrogen peroxide isgenerally a weak oxidant, although it can directly

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oxidize thiol (-SH) groups, for example, at the active siteof enzymes like glyceraldehyde-3-phosphatedehydrogenase. Nonradical oxidants like ONOOHH andhypochlorous acid (HOCl) appear to react preferentiallywith proteins rather than lipids. A major biological targetfor ONOO– is carbon dioxide (CO2). The reaction ofONOO– with CO2 is complex and produces metastablespecies that promote nitration, nitrosation and oxidationreaction.

Sources of oxidants and markers of oxidative enzymes

Several lines of evidence implicate ROS and RNS inatherogenesis. The advent of gene targeting topredictably modify genes has generated valuable animalmodels and allowed a genetic approach to gaininformation on how oxidants affect disease. Thedevelopment of sensitive analytical methods also enablesresearchers to obtain direct chemical evidence forspecific reaction pathways that promote oxidative eventsand lesion formation in vivo, and to relate theaccumulation of specific oxidized lipid and protein todifferent stages of atherosclerosis. Understanding theprocesses that lead to the different oxidative events isimportant in developing strategies to effectively inhibitsuch processes and hence, potentially, atherosclerosis.Within the vessel wall, the different oxidants canoriginate principally from cellular and extracellularsources, and from enzymatic and nonenzymatic pathsthat are reviewed briefly in the following section.

XO

XO is an iron sulfur molybdenum flavoprotein withmultiple functions and present in high concentrations inECs of capillary and sinusoids. It exists in two forms,xanthine dehydrogenase and XO, of which the former ispredominant. It generates O2

– by catalyzinghypoxanthine and xanthine to uric acid. Underpathophysiologic conditions, this is another major sourceof vascular oxidative stress (Dröge, 2002). XO exists inplasma and endothelial cells but not in smooth muscle

cells (SMCs) (Harrison et al., 2003). The role for XO in atherosclerosis is further

corroborated by the following observations(Spiekermann et al., 2003):- In the coronary arteries of patients with coronary arterydisease (CAD), electron spin resonance studies showsignificant activation of XO.- In these same patients, endothelial XO is inverselyproportional and positively related to the effect ofvitamin C on endothelium-dependent vasodilatation.- In asymptomatic young individuals with familialhypercolesterolemia, the increase of vascular XOactivity is an early event.

NAD(P)H oxidases

It has long been known that phagocytes, includingneutrophils, monocytes and macrophages contain aplasma membrane-bound, multicomponent oxidase thatutilizes electrons derived from NADPH to reducemolecular oxygen to O2

–·. While O2–· is principally a

reducing agent, it can give rise to secondary productsthat include strong oxidants.

The NADPH oxidases have emerged as veryimportant sources of ROS in vascular cells. Animportant aspect of this enzyme system is that it isregulated by a variety of pathophysiologic stimulirelevant to atherosclerosis (Harrison et al., 2003).Angiotensin II, platelet derived growth factor (PDGF),and tumor necrosis factor α (TNF-α) may increase ROSin the atherosclerotic lesion by stimulating the localvascular myocytes to produce ROS. Subsequently, ROSmay contribute to LDL oxidation, local MCP-1production, upregulation of adhesion molecules andmacrophage recruitment, endothelial dysfunction, andextracellular matrix remodeling through collagendegradation and eventually plaque rupture (Griendling etal., 2000).

MPO

It is a heme-containing enzyme that generates HOCl

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Vascular disease and ROS

Table 1. Free redicals generation and cellular antioxidants in biological systems.

Examples of free radicals in Examples of nonradical oxidants of potential Cellular antioxidantsbiological systems relevance in oxidative stress

Carbon centered radical Hydrogen peroxide Cu, Zn-SODSuperoxide anion Hypochlorite Mn-SODHydroperoxyl radical Hypochlorus acid CatalasePeroxyl radical Ozone Glutathione peroxidaseAlkoxyl radical Singlet oxygen Glutathione reductaseHydroxyl radical Oxoperoxonitrate or peroxynitrite Thiol-disulfide oxidoreductasesNitric oxide Peroxinitrous acid PeroxiredoxinNitrogen dioxide Alkylperoxynitrites Methionine sulfoxide reductasesThiyl radical Nitryl chloride Heme oxygenasePerthiyl radical Nitronium ion FerritinTransition-metal ions Nitrosothiols GSH

and chlorinated biomolecules, which are thereforeconsidered specific markers of oxidation reactionscatalyzed by the enzyme. There is a growing body ofevidence suggesting that MPO might play a role indifferent mechanisms that damage the human arterywall. MPO is an enzyme that is stored within theazurophile granules of neutrophils and monocytes,which generate modified and (or) oxidized (lipo)proteins via the production of HOCl from H2O2 andchloride. MPO, HOCl-modified proteins, and HOCl-modified LDL (HOCl-LDL) are present in humanatherosclerotic lesions, where they are located both invascular cells and extracellular spaces (Daugherty et al.,1994). Recent clinical studies have shown that a MPOdeficiency, or a low level of blood MPO, had beneficialeffects against cardiovascular damage in patientspresenting with this deficiency (Kutter et al., 2000;Zhang et al., 2001). Two other reports demonstrated thatthe MPO serum level is a predictor of risk in patientswith acute coronary syndromes or chest pain (Baldus etal., 2003; Brennan et al., 2003). The oxidation of LDLby MPO mainly leads to modifications of apo-lipoproteins with the formation of chlorotyrosine,dityrosine, and nitrotyrosine (Heinecke, 2003), thelipidic moiety attack being more dependent on pH andthe presence of preformed hydroperoxide (Spickett et al.,2000). Shishehbor et al. (2003) reported such changes inplasma proteins from hypercholesterolemic subjects withno known coronary artery disease. In addition, weobserved that MPO can use the H2O2 produced byendothelial cells to oxidize LDL in vitro at their surface(Zouaoui Boudjeltia et al., 2004).

LO

Another important ROS-generating system isrepresented by the LO; they are iron-containingdioxygenases that catalyze the sterospecific insertion ofmolecular oxygen into polyunsaturated fatty acids togive rise to a complex family of biologically activelipids, including prostaglandins, thromboxanes andleukotrienes. Prostaglandins and thromboxanes comprisemetabolites from arachidonate and similar fatty acids.The LO are non-heme iron-containing dioxygenases thatcatalyze stereospecific insertion of molecular oxygeninto polyunsaturated fatty acids, yielding a family ofbiologically active lipids, including prostaglandins,tromboxanes and leucotrienes that contribute toinflammatory reactions and can increase vascularpermeability. LO require low levels of “seedingperoxides” to oxidize inactive Fe++ to active Fe+++

enzyme, and are probably affected by the “peroxidetone” of cells (Stocker and Keaney, 2005). Leucocyte-type 12/15-LO and its products have been implicated inatherogenesis. The first direct proof in vivo of a role forthe lipoxygenase pathway was provided by Cyrus andcolleagues (1999) in Apo E null mice that were madegenetically deficient in 12/15-lipoxygenase and thatwere found to have significantly less atherosclerosis as a

result. Deletion of the 12/15-lipoxygenase gene in LDLreceptor null mice and in macrophages of mice that wereboth LDL receptor null and also deficient in the apoB-editing catalytic polypeptide-1 enzyme resulted indecreased atherogenesis in both mouse models.Conversely, overexpression of the 12/15-lipoxygenasegene in the endothelium of LDL receptor null miceaccelerated atherosclerosis (Navab et al., 2004).

Mitochondrial sources

Mitochondria provide energy (adenosinetriphosphate, ATP) to the cell through oxidativephosphorylation. Oxidative phosphorylation is theprocess by which ATP is formed as electrons aretransferred from NADH or FADH2 to molecular oxygen.This occurs through a series of electron transport carrierslocalized in the inner mitochondrial membrane (Singhand Jialal, 2006). It is becoming increasingly clear thatunder pathological conditions, mitochondrial oxidativephosphorylation can become uncoupled and results inthe generation of O2

•–. The paradigm that mitochondrial ROS are key

determinants of myocardial dysfunction has beenaddressed by a number of investigators in bothexperimental animal systems and humans (Madamanchiet al., 2005). Mitochondrial ROS are clearly associatedwith early atherosclerotic lesion development, andemerging evidence indicates that many cardiovascularsyndromes are associated with some evidence formitochondrial dysfunction, although a causal role has yetto be established in vivo (Singh and Jialal, 2006).

NOS

NOS are a family of enzymes that catalyze theoxidation of L-arginine to L-citrulline and the potentvasodilator NO•. In the context of the vasculature andatherosclerosis, endothelial NOS (eNOS) and inducibleNOS (iNOS) are most relevant. The activity of eNOS, asopposed to iNOS and strong evidence, suggests thateNOS plays an important role in protection of the vesselwall from atherosclerosis, yet the role of iNOS invascular pathology is variable and poorly defined inatherosclerosis (Singh and Jialal, 2006). Under normalconditions, eNOS requires tetrahydrobiopterin (BH4),bound near this heme group, to transfer electrons to aguanidino nitrogen of L-arginine to form nitric oxide. Inthe absence of either L-arginine or BH4, eNOS canproduce O2

•– and H2O2. This phenomenon has beenreferred to as NOS uncoupling. Uncoupling of eNOS inthe endothelium may lead to oxidative stress andendothelial dysfunction via at least 3 mechanisms. First,the enzymatic production of NO• is diminished, allowingthe radicals that it normally might react with to attackother cellular targets. Second, the enzyme begins toproduce O2

–, contributing to oxidative stress. Finally, itis likely that eNOS can become partially uncoupled,such that both O2

–• and NO• are produced

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simultaneously. Under this circumstance, eNOS maybecome a peroxynitrite generator, leading to a dramaticincrease in oxidative stress (Cai and Harrison, 2000).

The redox equilibrium between NO and oxidativestress has a profound impact on expression of genes inthe vessel wall, related to progression and vulnerabilityof atherosclerotic lesions as well as on parameters ofinflammation and cell apoptosis. Oxidation is essentialfor life; however, it is deleterious for the vasculature ifoxidative processes are out of control, as seen inendothelial dysfunction and subsequent atherosclerosis.Experimental findings indicate that in atherosclerosis theactivity of endothelial NO synthase is reduced. However,total NO production might be enhanced, since NO is notonly produced by endothelial NO synthase, but alsothrough neuronal NO synthase, and more importantly, byinducible NO synthase in macrophages and other celltypes in the atherosclerotic plaque (Schächinger andZeiher, 2002).

Antioxidant enzyme systems

Oxidative modifications within the arterial wall thatmay initiate and/or contribute to atherogenesis likelyoccur when the balance between oxidants andantioxidants shifts in favour of the former. Therefore, itis important to consider sources of oxidants in thecontext of available antioxidants. Many substancesprevent, or significantly delay, the oxidation of othersubstrates. However, antioxidant is defined as asubstance that it protects. With regard to atherosclerosis,vascular antioxidants need to protect against 2e-oxidants, both within and outside cells. In the context ofthe oxidative modification hypothesis, antioxidantprotection of LDL in the extracellular space deservesfocus, as oxidized LDL has many potentialproatherogenic activities and the cellular accumulationof oxidized LDL is considered a hallmark ofatherosclerosis (Steinberg et al., 1989). In addition,cellular antioxidants are probably important in thecontext of the presence of heightened oxidative stresswithin the vessel wall and the known effect of oxidativeevents on key cellular activities such as ·NO-relatedbioactivities. The following review briefly describes theantioxidant defenses in arterial wall cells andlipoproteins that counteract oxidative modifications.

Enzymatic antioxidants

The classic antioxidant enzymes are largely cell-associated proteins whose function is to maintain areducing tone within cells (Table 1); they may also beinvolved in the maintenance of extracellularantioxidants. Enzymatic antioxidants principally includeSOD, CAT, GPx, glutathione reductase, transferasesthiol-disulfide oxidoreductases, and peroxiredoxins.Many of these enzymatic antioxidants are present innormal arteries, most likely within vascular wall cells asextracellular fluid is largely devoid of enzymatic

antioxidants (Hamilton et al., 2004).

SOD

SODs are a major cellular defense system againstsuperoxide in all vascular cells. These enzymes containredox metal in the catalytic center, and dismutasesuperoxide radicals to hydrogen peroxide and oxygen.Three different isoforms of SOD have been identified:the mitochondrial manganese-containing SOD (MnSOD,SOD2), the cytosolic copper/zinc-containing SOD(CuZnSOD, SOD1), and the extracellular SOD (ecSOD,SOD3) (Hamilton et al., 2004; Wassmann et al., 2004).

CAT

CAT is an intracellular antioxidant enzyme that ismainly located in cellular peroxisomes and to someextent, in the cytosol, which catalyzes the reaction ofhydrogen peroxide to water and molecular oxygen in a2-step reaction. CAT is very effective in high-leveloxidative stress and protects cells from hydrogenperoxide produced within the cell. The enzyme isespecially important in the case of limited glutathionecontent or reduced glutathione peroxidase activity andplays a significant role in the development of toleranceto oxidative stress in the adaptive response of cells(Wassmann et al., 2004).

GPx

Reduced glutathione plays a major role in theregulation of the intracellular redox state of vascularcells by providing reducing equivalents for manybiochemical pathways. GPx is a selenium-containingantioxidant enzyme that effectively reduces hydrogenperoxide and lipid peroxides to water and lipid alcohols,respectively, and in turn oxidizes glutathione toglutathione disulfide. The GPx/glutathione system isthought to be a major defense in low-level oxidativestress (Wassmann et al., 2004).

Thiol-disulfide oxidoreductases

The protein disulfide isomerases are a group ofenzymes important in the correct folding of proteinsduring their synthesis, in the control of the redox state ofexisting exofacial protein thiols or reactive disulfidebonds, and in the “ordered movement” of ·NO across theplasma membrane (Stocker and Keaney, 2004). They areproduced, and at least some of them may be secreted, byvarious cells, raising the possibility that they alsocontribute to the antioxidant defense in extracellularfluid, although it is unknown whether they can mediatethe reduction of lipoprotein lipid hydroperoxides.

Peroxiredoxin

Peroxiredoxins are a family of antioxidant enzymes

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that comprises several members located in the cytosol,mitochondria, peroxisomes and plasma membrane ofcells as well as in plasma. The enzymes generally exhibitperoxidase activity and appear to serve a variety offunctions associated with different biological processes,such as cell proliferation, differentiation and geneexpression (Fujii and Ikeda, 2002).

Conclusion

The growing evidence from data of experimentaland clinical studies suggests that oxidative stress isimplicated in atherosclerotic diseases. However, a betterunderstanding of ROS production mechanisms andsignaling pathways in vascular pathophysiology is aprerequisite for effective pharmacological interventionsfor vascular disease. Moreover, the knowledge of thecomplexity of cellular redox reactions and the utilizationof natural antioxidants targeted to specific subcellularorganelles will likely be avenues for future researchtoward the broader use of pharmacological therapies inthe treatment and prevention of atherosclerosis.

Acknowledgements. The authors thank Miss. Stefania Castrezzati forher excellent technical assistance.

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Accepted August 30, 2007

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