Vascular Nitric Oxide and Oxidative Stress: Determinants ......Manson et al., 1999; Rosenthal et...

442 • Rush, Denniss, and Graham

442

INVITED REVIEW

The authors are with the Department of Kinesiology, University of Waterloo, Water-loo, Ontario N2L 3G1.

Vascular Nitric Oxide and Oxidative Stress:Determinants of Endothelial Adaptations toCardiovascular Disease and to Physical Activity

James W.E. Rush, Steven G. Denniss, and Drew A. Graham

Catalogue DataRush, J.W.E.; Denniss, S.G.; and Graham, D.A. (2005). Vascular nitric oxide and oxidativestress: Determinants of endothelial adaptations to cardiovascular disease and to physicalactivity. Can. J. Appl. Physiol. 30(4): 442-474. © 2005 Canadian Society for ExercisePhysiology.

Key words: exercise, artery, reactive oxygen species, antioxidant, hypertensionMots-clés: exercice, artère, espèces réactives de l’oxygène, antioxydant, hypertension

Abstract/Résumé

Cardiovascular disease is the single leading cause of death and morbidity for Canadians. Auniversal feature of cardiovascular disease is dysfunction of the vascular endothelium, thusdisrupting control of vasodilation, tissue perfusion, hemostasis, and thrombosis. Nitric ox-ide bioavailability, crucial for maintaining vascular endothelial health and function, de-pends on the processes controlling synthesis and destruction of nitric oxide as well as onthe sensitivity of target tissue to nitric oxide. Evidence supports a major contribution byoxidative stress-induced destruction of nitric oxide to the endothelial dysfunction that ac-companies a number of cardiovascular disease states including hypertension, diabetes,chronic heart failure, and atherosclerosis. Regular physical activity (exercise training) re-duces cardiovascular disease risk. Numerous studies support the hypothesis that exercisetraining improves vascular endothelial function, especially when it has been impaired bypreexisting risk factors. Evidence is emerging to support a role for improved nitric oxidebioavailability with training as a result of enhanced synthesis and reduced oxidative stress-mediated destruction. Molecular targets sensitive to the exercise training effect include theendothelial nitric oxide synthase and the antioxidant enzyme superoxide dismutase. How-ever, many fundamental details of the cellular and molecular mechanisms linking exercise

Nitric Oxide and Oxidative Stress • 443

to altered molecular and functional endothelial phenotypes have yet to be discovered. Theworking hypothesis is that some of the cellular mechanisms contributing to endothelialdysfunction in cardiovascular disease can be targeted and reversed by signals associatedwith regular increases in physical activity. The capacity for exercise training to regulatevascular endothelial function, nitric oxide bioavailability, and oxidative stress is an ex-ample of how lifestyle can complement medicine and pharmacology in the prevention andmanagement of cardiovascular disease.

La première cause de mortalité et de morbidité chez les Canadiens est la maladiecardiovasculaire. La dysfonction de l’endothélium vasculaire, qui est la caractéristiqueprincipale de la maladie, entraîne un dérèglement du contrôle de la vasodilatation, de laperfusion des tissus, de l’hémostasie, et de la coagulation. Le maintien de la fonction et dela santé de l’endothélium vasculaire, assuré par la biodisponibilité du monoxyde d’azote,dépend des processus de contrôle de la synthèse et de la dégradation du monoxyde d’azoteet de la sensibilité des tissus ciblés par le monoxyde d’azote. Selon de solides études, ladestruction du monoxyde d’azote attribuable au stress par oxydation contribue à ladysfonction de l’endothélium observée dans diverses conditions pathologiques dontl’hypertension, le diabète, l’insuffisance cardiaque chronique, et l’athérosclérose. La pra-tique régulière de l’activité physique (entraînement physique) réduit le risque de maladiecardiovasculaire. De nombreuses études appuient la thèse que l’entraînement physiqueaméliore la fonction de l’endothélium vasculaire, notamment quand ce dernier a été déréglépar des facteurs de risque en place. Il appert en outre que l’entraînement améliore labiodisponibilité du monoxyde d’azote en favorisant la synthèse aux dépens de la dégradationdue au stress par oxydation. Les cibles moléculaires sensibles à l’entraînement physiquecomprennent la monoxyde d’azote synthase endothéliale et la superoxyde dismutase.Cependant, il reste à identifier les aspects fondamentaux des mécanismes moléculaires etcellulaires reliant l’exercice physique aux diverses modifications moléculaires etfonctionnelles des phénotypes endothéliaux. L’hypothèse de travail est la suivante: dessignaux associés à l’augmentation de la pratique régulière de l’activité physique contribuentà cibler et à corriger les mécanismes cellulaires de la dysfonction endothéliale. L’entraîne-ment physique utilisé à des fins de régulation de la fonction de l’endothélium vasculaire, dela biodisponibilité du monoxyde d’azote, et du stress par oxydation est un exemple de complé-mentarité des saines habitudes de vie à la médecine et à la pharmacologie dans la préventionet le traitement de la maladie cardiovasculaire.

Introduction

In Canada, cardiovascular disease (CVD) causes ~35% of all deaths, contributessignificantly to morbidity, and accounts for ~20 billion dollars in annual direct andindirect health care costs (Health Canada, 1997; Heart and Stroke Foundation ofCanada, 1999). Major independent risk factors for CVD include hypertension,dyslipidemias, smoking, obesity, diabetes mellitus, and physical inactivity. Increas-ing habitual physical activity can reduce CVD risk. Indeed, a dose-response rela-tionship has been found between the amount of exercise performed and the reduc-tion in CVD mortality in middle-aged and elderly populations (Blair et al., 1995;Lee et al., 1995). The effect is multi-tiered; in addition to eliminating physicalinactivity as an independent CVD risk factor, habitual endurance exercise mayalso have a beneficial effect on other independent risk factors including blood


lipid profiles, blood pressure, body weight, and insulin sensitivity (American HeartAssociation, 1996; Booth et al., 2000; Health Canada, 1997; King et al., 1988;Manson et al., 1999; Rosenthal et al., 1983; Tran and Weltman, 1985; U.S. Dept.Health and Human Services, 1996; Williams, 1996; Wood et al., 1991). Thus, aerobicphysical activity is a potentially powerful intervention to prevent and/or counter-act the development of CVD.

Function of the vascular endothelium is affected by both CVD and exercisetraining; impairment of function accompanies a number of CVD states while im-provement in endothelial function occurs with regular exercise. Over the past twodecades, a central role of endothelial cells in regulating vascular homeostasis hasbeen established through the discovery of certain endothelial-derived substancesthat influence vascular physiology (Behrendt and Ganz, 2002; Verma et al., 2003;Widlansky et al., 2003).

By balancing the release of vasodilators such as nitric oxide (NO•), prosta-cyclin, and endothelium-derived hyperpolarizing factor (EDHF), and vasoconstric-tors such as thromboxane A2, endothelin-1, and angiotensin II, the endotheliumcan alter vascular smooth muscle (VSM) cell contractile state (tone) and thus con-trol blood pressure and tissue perfusion. The endothelium controls the vascularthrombotic state through the production of factors such as NO•, prostaglandins,tissue plasminogen activator, thrombomodulin, plasminogen activator inhibi-tor-1, tissue factor, and von Willibrand’s factor, which help to regulate plateletactivation, the clotting cascade, and the fibrinolytic system. Moreover, the endo-thelium regulates vascular inflammatory and adhesion processes by producingcytokines and adhesion molecules such as C-reactive protein, interleukins, mono-cyte chemotactic factor-1, tumor necrosis factors, adhesion molecules, and selectins.Whereas the balanced production of counter-regulatory substances maintains ahealthy endothelial phenotype, in a pathophysiological CVD state the endothe-lium may adopt an alternate phenotype wherein the balance is disrupted andproconstrictory, proinflammatory, and prothrombotic signals prevail, leading tovascular dysfunction.

Nitric oxide is a particularly important endothelial-derived substance in lightof its multiplex vascular functions. As well as being a potent vasodilator, NO•

inhibits the synthesis of proinflammatory cytokines and chemokines, the expres-sion of leukocyte adhesion molecules, the activation and aggregation of platelets,and the proliferation of VSM cells (Eberhardt and Loscalzo, 2000; Ganz and Vita,2003). Vascular endothelial NO•-dependent vasomotor dysfunction is often foundin persons with overt CVD or when one or more risk factors are present (Behrendtand Ganz, 2002; Celermajer et al., 1994; Ludmer et al., 1986; Verma et al., 2003;Vita et al., 1990; Widlansky et al., 2003). Studies of adaptations in NO• bioavail-ability (determined by synthesis and destruction of NO• and target tissue sensitiv-ity to NO•, details below) in CVD indicate that oxidative stress-mediated NO•

destruction is commonly associated with this loss of endothelial vasomotor func-tion (Cai and Harrison, 2000; Kojda and Harrison, 1999).

In contrast, regular physical activity (exercise training) can improve endo-thelial NO•-mediated vasomotor function and tissue blood flow control (Delp etal., 1993; Graham and Rush, 2004; Hambrecht et al., 1998; 2000; Higashi et al.,


1999a; Kingwell et al., 1996; Muller et al., 1994). The mechanisms responsible forthese improvements with chronic exercise have not been fully elucidated, thoughrecent data suggest that improvements in the management of vascular oxidativestress may provide a significant contribution to the enhanced NO• bioavailability.This review will outline the role of NO• and oxidative stress in endothelial vaso-motor function and adaptations to CVD and exercise training as identified throughintegrative studies in humans and animal models.

Assessment of Nitric Oxide-DependentEndothelial Vasomotor Function

Efforts to understand the vascular biology of NO• began with the discovery thatthe endothelium plays a major role in controlling VSM tone. Using isolated rabbitaorta mounted for isometric tension recordings in vitro (vascular myography; Fig-ure 1A), Furchgott and Zawadzki (1980) demonstrated that acetylcholine (Ach)caused relaxation so long as care was taken not to denude the endothelium of theexcised vascular segments. Disruption of the endothelium eliminated the relaxingeffect of Ach (Furchgott and Zawadzki, 1980). These researchers concluded thatendothelial cells were releasing an endothelium-derived relaxing factor (EDRF) inresponse to Ach that was somehow responsible for arterial smooth muscle relaxation.

Subsequent studies by Furchgott (1988), Ignarro et al. (1987; 1988), Muradand colleagues (Rapoport and Murad, 1983), Gryglewski et al. (1986), and Palmeret al. (1987; 1988) were instrumental in characterizing EDRF as NO•, and demon-strating that NO•: (a) is enzymatically synthesized from the amino acid L-arginine;(b) stimulates VSM soluble guanylate cyclase (sGC); and (c) is rapidly inactivatedby superoxide anion (O2-•). Moncada and contemporaries, using the arginine ana-logs NG-monomethyl-L-arginine (L-NMMA) and L- NG-arginine methyl ester (L-NAME) to inhibit EDRF/NO• production, demonstrated that loss of tonic EDRF/NO• synthesis resulted in significant vasoconstriction of a variety of vascular bedsin animals and humans (Amezcua et al., 1989; Lahera et al., 1991; Persson et al.,1990; Vallance et al., 1989; Wiklund et al., 1990), as well as an elevation in arterialblood pressure (Rees et al., 1989).

Most experimental and clinical assessments of endothelial function makeuse of either blood flow manipulations or drugs such as Ach to stimulate vasodilatoryand/or blood flow responses. Schertzenmayr (1933) provided the first experimen-tal evidence that increases in blood flow cause large arteries to dilate. This phe-nomenon, termed flow-mediated dilation (FMD), was confirmed over the nextseveral decades (for review, see Rubanyi, 1995) and shown to be endothelium-dependent (Holtz et al., 1983; 1984; Smiesko et al., 1983; 1985). This dilatoryresponse can also be observed in vitro using isolated segments of either a conduitor resistance vessel mounted on perfusion pipettes on a microscope stage, allow-ing for precise manipulation of pressure and flow while continuously measuringdiameter (isolated vessel flow-mediated dilation technique; Figure 1B). Studiesusing this methodology, together with pharmacological inhibitors of vasodilatorypathways, have shown that NO• released from the endothelium in response to el-evated flow is a major mechanism of FMD (Bagi et al., 2001; Sun et al., 2001).


Figure 1. Methods to assess endothelial function. Endothelial vasomotor function canbe assessed (A) in vitro using wire-mounted vessel rings by measuring isometric tensiondevelopment, or (B) in mounted, pipette-perfused vessel segments by monitoring changesin diameter by video microscopy. Human in vivo endothelial vasomotor function, as-sessed as a change in vessel diameter or flow in response to vasoactive drug infusion orelevated shear stress, can be measured (C) in the coronary circulation by quantitative an-giography, or (D) in the forearm circulation by plethysmography or Doppler ultrasound.Black arrowheads represent sites of catheter placement for infusion of vasoactive drugs;inset panels represent imaging of vessels and data acquisition. (E) Thrombotic, inflamma-tory, and damage/apoptotic aspects of human and animal endothelial function can be as-sessed by measuring blood-borne biomarkers such as high-sensitivity C-reactive protein(CRP), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule(ICAM), circulating endothelial microparticles (EMPs), and circulating endothelial pro-genitor cells (EPCs). For further details of these methods and their applications, see textunder Assessment of Nitric Oxide-Dependent Endothelial Vasomotor Function.

C. Quantitative Coronary Angiography D. Forearm Blood Flow

A. Vascular Myography B. Isolated and Perfused Vessel

E. Soluble Markers of Vascular Function


Quantitative angiography can be used to assess epicardial conduit artery func-tion in vivo by imaging changes in vascular diameter in response to graded con-centrations of endothelium-dependent vasodilators (e.g., Ach) delivered throughan intracoronary arterial catheter, or in response to elevated blood flow caused byvasodilator drug-induced dilation of downstream resistance vessels (quantitativecoronary angiography technique; Figure 1C). In this same type of invasive coro-nary catheterization protocol, the endothelial vasomotor function of coronary re-sistance vessels can be assessed by intracoronary Doppler ultrasound measure-ments of blood flow responses to vasoactive agents (Zeiher et al., 1991). The firststudies to demonstrate clinical endothelial dysfunction employed quantitative coro-nary angiography and showed that patients with coronary artery disease had “para-doxical vasoconstriction” in response to graded Ach infusions (Ludmer et al., 1986)and impaired dilation in response to elevated flow (Cox et al., 1989), likely owingto a reduced NO• bioavailability in these arteries.

Although quantitative coronary angiography is considered the benchmarkfor human endothelial vasomotor function testing, it is an invasive technique asso-ciated with risk and expense, and is therefore essentially restricted to use in pa-tients who have a clinical diagnostic need for cardiac catheterization. These limi-tations led to the development of surrogate indicators of coronary vascular endo-thelial vasomotor function utilizing less invasive technologies in easily accessibleperipheral arteries, such as those in the arm/forearm.

Human forearm resistance-vessel endothelial vasomotor function can be as-sessed using strain-gauge venous occlusion plethysmography to detect volume, orDoppler ultrasound to detect velocity (forearm blood flow technique; Figure 1D),to quantify the change in forearm blood flow after intraarterial (brachial or radialartery) infusion of vasoactive drugs. This approach is valuable in that it can pro-vide dose-response relationships and can be used to study the basic mechanismsunderlying endothelial vasomotor dysfunction with appropriate drugs/blockers.The major drawback of this technique is the requirement for arterial catheteriza-tion which increases invasiveness and risk, thus limiting its widespread use.

A solution to this limitation that does not require arterial catheterization ordrug administration was introduced by Celermajer and colleagues in their assess-ment of FMD in the human conduit brachial artery imaged using echo Dopplerultrasound (forearm blood flow technique; Figure 1D; Celermajer et al., 1992;Corretti et al., 2002). To create a flow stimulus through the brachial artery, a bloodpressure cuff placed about the arm or forearm is inflated to a suprasystolic pres-sure to elicit arterial occlusion for several minutes. The resultant ischemia causesdownstream resistance vessels to dilate in response to the accumulation of meta-bolic byproducts released by skeletal muscle. Subsequent rapid cuff deflation bringsabout a rapid increase in blood flow through the brachial artery because of thelowered downstream resistance. This response, termed reactive hyperemia, is char-acterized by a peak brachial artery flow that is sustained for a few seconds beforeslowly decaying back toward resting level as metabolites are washed out and arte-rioles regain their basal tone. The elevation in flow/shear stress causes the brachialartery to dilate, with a maximal response typically occurring 45 to 90 seconds aftercuff deflation.


The correlation between FMD assessed in the brachial artery and Ach-medi-ated dilation in epicardial arteries (Anderson et al., 1995) indicates that peripheralconduit arteries may serve as reasonable surrogate marker for coronary endothe-lial vasomotor responses. This observation has added to the legitimacy of the bra-chial artery FMD test as a practical and widely used technique for assessing endo-thelial vasomotor function in humans. Additional benefits include anatomical ac-cessibility, noninvasiveness, time- and cost-efficiency, and use of a physiologicalstimulus (flow) as opposed to pharmacological agonists. There are several techni-cal limitations to this approach, however, including the expertise required to im-age the brachial artery using Doppler ultrasound, a relatively poor signal-to-noiseratio (poor resolution relative to artery size), and no standardization among cen-ters with respect to an occlusion protocol to stimulate flow and subsequent dila-tion (Betik et al., 2003; Corretti et al., 2002).

The latter could be very important to interpretation of FMD data, as recentstudies suggest there may be protocol-dependent effects on the mechanism medi-ating FMD. For instance, studies assessing FMD in the brachial/radial artery fol-lowing arginine analog infusion have demonstrated that with brief periods of hy-peremia, conduit vessel dilation is almost exclusively mediated by NO• (Joannideset al., 1995; Lieberman et al., 1996; Mullen et al., 2001), whereas with more pro-longed periods of hyperemia the dilation appears to be much less dependent onNO• (Bellien et al., 2003; Mullen et al., 2001). For the reasons described abovethere is continued effort to develop better means of noninvasively assessing NO•-mediated endothelial vasomotor function (Ganz and Vita, 2003; Widlansky et al.,2003). There is also increasing emphasis on supplementing functional assessmentswith blood markers of endothelial phenotype including proinflammatory/prothrombotic biomarkers such as high-sensitivity C-reactive protein, vascular celladhesion molecules, circulating endothelial progenitor cells, and circulating en-dothelial microparticles, thought to result from endothelial damage/apoptosis (Fig-ure 1E; Horstman et al., 2004; Hristov et al., 2003; Verma et al., 2003; Willersonand Ridker, 2004).

Factors Controlling NO• Bioavailability In Vivo

Through 20 years of vascular cellular and molecular studies, the mechanism ofNO• action has been well defined and the factors that affect NO• bioavailability arebeing examined with a high degree of sophistication including gene/protein ma-nipulation and complex pharmacology. Through this integrative physiology ap-proach, molecular defects responsible for endothelial dysfunction and potentialtargets for improving endothelial function are being identified.

The generally accepted sequence of events of NO•-mediated vasodilation isas follows: in response to a number of physical and chemical stimuli to the endo-thelium, NO• is generated in the cytosol by the endothelial isoform of NO• syn-thase (eNOS; NOS3) and diffuses to underlying VSM cells. Within the media ofthe vessel wall, NO• may be transported as NO• per se or as an S-nitrosothiol formmoving between VSM cells by simple diffusion across cell membranes and throughcytosol and/or diffusion through gap junctions between VSM cells. The mecha-nism of NO• transport in the media of specific vessels likely depends on the num-


ber of layers of VSM cells that must be targeted simultaneously for vasodilation tooccur, and thus on the vessel caliber (i.e., arterioles vs. larger arteries). This com-plexity is not shown in Figure 2, for the sake of simplifying the illustration to thegeneral determinants of NO• bioavailability in all vessels. NO•-mediated activa-tion of sGC in the cytosol of VSM cells leads to cGMP accumulation, proteinkinase G (PKG) activation, and PKG-mediated phosphorylation of a number ofCa2+ regulatory and contractile proteins. The result is a lowering of VSM sarco-plasmic Ca2+ concentration (Cohen, 2000), relaxation of VSM, dilation of the bloodvessel, decreased vascular resistance, and increased flow through the vessel (Fig-ure 2).

Two important principles of cell signaling are identifiable in this simplifiedNO• mechanism of action: amplification of the original endothelial signal through

Figure 2. General determinants of NO• bioavailability as it relates to vasomotor func-tion. Both physical and chemical stimuli can activate eNOS acutely through various post-transcriptional mechanisms, determining the rate of NO• production (1). NO• that inter-acts with various reactive oxygen species, such as O2

-•, will be reduced to -ONOO and in-activated (2). Chemical (not shown) and enzymatic (SOD, GPx, catalase) antioxidant sys-tems can quench O2-•. NO• initiates VSM cell relaxation through a sGC-PKG cascade (3)in addition to direct NO• effects on VSM ion regulatory proteins (not shown). For detailsof these processes see text under Factors Controlling NO• Bioavailability In Vivo. Forsimplicity, this schematic shows the response of the first VSM cell encountered by the en-dothelium-derived NO•; transport of NO• between VSM cells is not shown.


second messenger and protein kinase systems; and redundancy in the multiplemolecular targets of PKG, all of which act to decrease cytoplasmic Ca2+ and thecontractile state of VSM. Although the amplification inherent in this signalingsystem allows for responsiveness to even nanomolar NO• concentrations, micro-molar concentrations of NO• are present physiologically (Malinski and Taha, 1992;Malinski et al., 1993). At physiological NO• concentrations there is also evidencefor cGMP-independent mechanisms of action mediated by the direct action of NO•

on redox-sensitive thiol groups of ion-regulatory proteins (Bolotina et al., 1994).The mechanism of NO• action identifies key components of the responsive-

ness or sensitivity of VSM to NO•. The factors influencing NO• synthesis anddestruction must also be considered in order to appreciate the many potential sitesfor molecular-level alterations that can result in depression or enhancement ofNO•-mediated vasodilation.

ENDOTHELIAL NO• SYNTHESIS

Synthesis of NO• in endothelial cells is mediated by eNOS (Figure 2). In responseto a number of physical (e.g., flow/shear stress) and chemical (e.g., Ach) stimuli,cell signaling events lead to elevation in endothelial cell cytosolic Ca2+, whichbinds to the regulatory protein calmodulin (CaM). In turn, Ca2+-CaM binds to andactivates eNOS.

The acute regulation of endothelial NO• synthesis is much more complexthan the Ca2+ -CaM influences alone and involves: availability of substrate (L-arginine), endogenous inhibitor (asymmetric dimethyl arginine), and cofactors(FAD, FMN, NADP+/NADPH and tetrahydrobiopterin); non-Ca2+-dependent eNOSactivation; proteins that regulate eNOS localization and activity through protein-protein interactions (HSP-90, caveolin); and covalent modifications of eNOS in-cluding phosphorylation/ dephosphorylation by multiple kinases/phosphatases andacylation (Cohen, 2000; Dillon and Vita, 2000; Feron et al., 1999; Feron and Michel,2000; Forstermann et al., 1995; Garcia-Cardeña et al., 1998; Hambrecht et al.,2003; Sessa et al., 1994; Wang and Marsden, 1995). Elucidating the details oflocalization and phosphorylation effects on eNOS are topics of recent reviews(Boo and Jo, 2003; Fleming and Busse, 2003; Govers and Rabelink, 2001; Shaul,2002). In addition to the acute factors, chronic regulation of eNOS is achieved inpart through adaptations in its level of expression, which is modulated by a num-ber of stimuli. There are also excellent reviews of the regulation of eNOS activityand expression (Boo and Jo, 2003; Li et al., 2002).

Recent results add to the understanding of vascular NO• production by illus-trating that NO• released from neuronal nitric oxide synthase (nNOS) localized inneurons lining coronary and pial arteries can mediate flow- and agonist-induceddilations in eNOS knockout mice (Huang et al., 2002; Lamping et al., 2000; Menget al., 1998). This suggests a compensatory interaction between eNOS and nNOSthat could offset eNOS deficiencies. However, as the convincing results are cur-rently limited to knockout models lacking the eNOS gene, it will require muchmore work to clarify the relative role of nNOS-derived NO• in vasomotor controlin vessels with competent eNOS, and its physiological and pathophysiologicalimportance.


DESTRUCTION OF NO• BY OXIDATIVE STRESS

Although the term oxidative stress is frequently used to indicate an excess expo-sure to reactive oxygen species (ROS), it should be kept in mind that oxidativestress is a relative term. While increasing evidence suggests that low levels ofROS contribute to normal physiological cell signaling (Buetler et al., 2004), ex-cessive production or impaired buffering of ROS leads to increased oxidative stressthat can have pathophysiological consequences (Buetler et al., 2004; Griendling etal., 2000; Kunsch and Medford, 1999; Taniyama and Griendling, 2003).

Local NO• degradation in the artery wall in vivo occurs predominantly throughthe interaction of NO• with ROS, such as O2-•, to form peroxynitrite (-ONOO)(Figure 2). Since the rate of interaction between NO• and O2

-• is diffusion-limited,a fine balance between NO• and ROS must be maintained in the vascular wall inorder to preserve adequate NO• bioavailability (Darley-Usmar et al., 1995) (Fig-ure 2). Support for a major contribution of oxidative stress to the regulation of NO•

has come from several fronts: exposure to endogenous or exogenous O2-• reduces

endothelium-dependent dilation to Ach (Gryglewski et al., 1986; Katusic andVanhoutte, 1989); experimental inhibition of superoxide dismutase (SOD) impairsagonist-evoked endothelium-dependent NO•-mediated dilation (Carneado et al.,2002; Mugge et al., 1991a; Omar et al., 1991; Wambi-Kiesse and Katusic, 1999);inclusion of SOD or chemical antioxidants in vitro protects NO• against O2-•(Laursen et al., 1997; Zalba et al., 2000); and enhancement of vascular wall SODby gene or protein transfer in vivo restores the NO• action previously impaired byoverproduction of ROS (Chu et al., 2003; Fennell et al., 2002; Mugge et al., 1991b;Schnackenberg et al., 1998).

The conclusion stemming from these and other observations is that oxida-tive stress-induced destruction of NO• is a major mechanism in the regulation ofNO• bioavailability. It is thus important to consider the chemical and enzymaticpro- and antioxidant influences in the vascular wall.

In vascular endothelial and smooth muscle cells the main pro-oxidant influ-ences are the enzymes NAD(P)H oxidase, xanthine oxidase, and eNOS (Cai andHarrison, 2000; Darley-Usmar et al., 1995; Dillon and Vita, 2000; Kojda andHarrison, 1999), with NAD(P)H oxidase recognized as the predominant source ofO2

-• (Figure 2; Griendling et al., 1994; 2000; Mohazzab-H et al., 1994; Mohazab-H and Wolin, 1994; Pagano et al., 1995; Rajagopalan et al., 1996)

The NAD(P)H oxidase enzyme complex is present in both endothelial andVSM cells, although its molecular composition varies slightly between cell types.Activation of this enzyme involves recruitment of at least three regulatory cytoso-lic subunits to the heterodimeric membrane-bound catalytic complex to form theholoenzyme complex. A recent and excellent review of the structure and functionof this enzyme and its role in CVD provides much more detail (Griendling et al.,2000).

Several chemical and enzymatic antioxidant systems exist in the vascularenvironment. Cellular chemical antioxidants include glutathione and other thiols,as well as antioxidant vitamins such as vitamins C and E, and β-carotene. Enzy-matic antioxidants in the vascular wall include three isoforms of SOD: the cytoso-lic Cu/Zn-dependent SOD-1 isoform and the mitochondrial Mn-dependent SOD-2


isoform, both of which are present in endothelial cells and VSM cells, and the Cu/Zn-dependent SOD-3 isoform (ecSOD) located in the extracellular matrix. In ad-dition, the H2O2-reducing enzymes glutathione peroxidase (GPx) and catalase arepresent in both endothelial and VSM cells (Figure 2).

Superoxide dismutase is essential for normal NO• function because of itsaction in converting O2-• to H2O2, thus limiting the interaction of NO• and O2

-•(Darley-Usmar et al., 1995). Much less is known regarding the roles of GPx andcatalase in controlling NO• bioavailability. Recent evidence suggests that H2O2may contribute to the chronic regulation of NO• bioavailability, however, as itinduces eNOS expression at both transcriptional and posttranscriptional levels incultured bovine endothelial cells (Drummond et al., 2000).

The preceding information suggests that oxidative stress-induced destruc-tion of NO• contributes to the endothelial vasomotor dysfunction. A tempered abil-ity of antioxidant enzyme systems to buffer O2

-• results in NO• destruction andimpaired NO• bioavailability whereas supplemental SOD and chemical antioxi-dant treatments can buffer excess O2

-• production and restore endothelium-depen-dent dilation (Carneado et al., 2002; Fukui et al., 1997; Gokce et al., 1999; Hattoriet al., 1991; Horie et al., 1998; Jackson et al., 1998; Kinlay et al., 1999; Kinouchiet al., 1991; Laursen et al., 1997; Mugge et al., 1991b; Nakazono et al., 1991; Raja-gopalan et al., 1996; Taddei et al., 1998; Ting et al., 1996; 1997; Zalba et al.,2000). With the main vascular pro- and antioxidant influences introduced, atten-tion will now shift to specific evidence supporting oxidative stress-induced reduc-tions in NO• bioavailability as a pathophysiological mechanism in CVD.

Reduced NO• Bioavailability in Cardiovascular Disease

A transition in endothelial cell phenotype toward increased vascular constriction,thrombosis, and inflammation occurs at the earliest stages of CVD (Figure 3).Numerous recent prospective and retrospective studies have shown that testing ofNO•-mediated endothelial vasomotor function has prognostic value for clinicalcardiovascular events including myocardial infarction and ischemic stroke(Behrendt and Ganz, 2002; Ganz and Vita, 2003; Verma et al., 2003; Widlansky etal., 2003). Considering this, a detailed analysis of vascular NO• bioavailability andhow it is manipulated by risk factors and various interventions is essential to un-derstanding CVD.

Although there are undoubtedly contributions of diminished VSM respon-siveness to NO• (Adachi et al., 2002; Adams et al., 1998; Bauersachs et al., 1998;Creager et al., 1990; Kojda et al., 1998; Weisbrod et al., 1997), and depressedsynthesis of NO• resulting from multiple deficiencies in the NO• synthesis path-way (Blair et al., 1999; Crabos et al., 1997; Creager et al., 1992; Feron et al., 1999;Stroes et al., 1997; Yoshizumi et al., 1993), the overwhelming evidence supportsoxidative stress-induced destruction of NO• as a major mechanism of the reducedNO• bioavailability leading to vascular dysfunction in CVD. Indeed, elevated ROShas been causatively linked to endothelial vasomotor dysfunction in atherosclero-sis, hypertension, diabetes, and chronic heart failure (CHF) (Berry et al., 2000; Caiand Harrison, 2000; Crabos et al., 1997; Darley-Usmar et al., 1995; Dillon andVita, 2000; Fujita et al., 1995; Fukui et al., 1997; Gil-Longo et al., 1996; Griendling


et al., 1994; Huang and Koller, 1996; Kojda and Harrison, 1999; Konishi and Su,1983; Laursen et al., 1997; Mayhan et al., 1987; Mohazzab-H et al., 1994; Raja-gopalan et al., 1996; Rudd et al., 2000; Taddei et al., 1993; Treasure et al., 1992).

Elevations in the expression and activity of NAD(P)H oxidase are likely in-volved in the vascular pathogenesis of coronary artery disease, hypertension, diabe-tes, and CHF (Cai and Harrison, 2000; Fukui et al., 1997; Griendling et al., 1994;Rajagopalan et al., 1996; Zalba et al., 2000), since the enzyme is regulated by atten-dant changes in cytokines, hormones, and mechanical forces (De Keulenaer et al.,1998a; 1998b; Griendling et al., 1994; Holland et al., 1998; Marumo et al., 1997).

CORONARY ARTERY DISEASE

O2-• production and oxidative NO• destruction is elevated several-fold in the aortic

wall of hypercholesterolemic and atherosclerotic animals (Mugge et al., 1991b;Ohara et al., 1993) and this is associated with impairment in NO•-mediated, endo-

Figure 3. Healthy and pathological states of endothelial vasomotor function. Solid curve onthe graph in Panel A depicts a healthy dose-response relationship between an agonist or shearstress and endothelium-dependent vessel relaxation. Dashed curve in Panel A depicts an at-tenuated, pathological dose-response relationship between the same agonist or shear stress andendothelium-dependent vessel relaxation. The reduced endothelium-dependent vessel relax-ation may be indicative of an attenuated vascular homeostasis caused by a pathological endo-thelial phenotype brought about by a sedentary lifestyle, hypertension, and/or other CVD riskfactors, each to some degree associated with elevated oxidative stress. With exercise training,antioxidant therapy, and/or pharmacological control of CVD risk factors, oxidative stress maybe alleviated resulting in a healthy endothelial phenotype, a more robust vascular homeostasis,and a greater agonist or shear stress-to-vessel relaxation dose-response relationship as depictedin Panel A. The “measuring stick” in Panel B illustrates that between the extremes of healthyand pathological endothelial function there is a continuous dynamic range of endothelial phe-notype, function, and homeostasis. The position in this continuum assumed at a given time de-pends in part on the balance of lifestyle and other factors affecting nitric oxide bioavailabilityand vascular oxidative stress.

End

othe

lium

-dep

ende

ntV

esse

l Rel

axat

ion

A. B.

Agonist or Shear Stress

Pathological phenotype

Healthy phenotype

Sedentary lifestyle,oxidative stress,hypertension, and otherCVD risk factors

Antioxidant therapy,exercise training, andpharmacological controlof CVD risk factors

End

othe

lial H

omeo

stas

is


thelium-dependent vasodilation assessed in vitro using vascular myography (Muggeet al., 1991b; Figure 3). A landmark study by Ohara et al. (1993) identified endo-thelial cells as the major contributor of excess O2

-• in hypercholesterolemia, whilesubsequent studies have revealed an involvement of macrophage and VSM cellO2

-• production in the later stages of atherosclerosis (Miller et al., 1998).Several investigations have shown that providing a more robust vascular

enzymatic/chemical antioxidant capacity can improve NO•-mediated endothelialvasomotor function in hypercholesterolemia (Huang and Keaney, 2000; Figure 3).For instance, Mugge and co-workers doubled the aortic wall SOD enzyme activityin hypercholesterolemic rabbits using polyethylene glycol-conjugated SOD injec-tions, and this reversed endothelium-dependent NO•-mediated aortic vasomotordysfunction (Mugge et al., 1991b). Furthermore, intraarterial infusion of the chemi-cal antioxidant, vitamin C, at 1-10 mM improved endothelial vasomotor functionin the forearm resistance arteries of human hypercholesterolemic patients assessedin vivo using strain-gauge venous occlusion plethysmography (Ting et al., 1997).

Conversely, oral consumption of chemical antioxidants at more reasonabledietary concentrations daily for one month (1,000 mg of vitamin C; 800 IU ofvitamin E; 30 mg of β-carotene) had no effect on vasomotor function, although thetreatment was associated with a reduction in the susceptibility of LDL cholesterolto oxidation ex vivo (Gilligan et al., 1994). Notably, this latter study demonstratesthat nonpharmacological doses of chemical antioxidants in humans may not al-ways be sufficient to combat vascular oxidative stress-induced endothelial vaso-motor dysfunction in hypercholesterolemia, though they may exert other vascularhealth benefits such as reducing circulating oxidized LDL cholesterol.

HYPERTENSION

Both human and animal models of hypertension are associated with elevated O2-•

production and NO•-mediated vascular dysfunction (Duffy et al., 1999; 2001;Laursen et al., 1997; Mehta et al., 1994; Schnackenberg et al., 1998; Sherman etal., 2000; Solzbach et al., 1997; Taddei et al., 1993; 1998; Figure 3). As in hypercholes-terolemia, manipulation of artery SOD content affects the NO•-mediated dilatoryresponse in arteries from hypertensive animals; treatment with liposome-encapsu-lated SOD for 8 days increased aortic SOD activity by ~30% and partially preventedangiotensin-II-induced O2-• release, elevation of blood pressure, and endothelial va-somotor dysfunction assessed by vascular myography (Laursen et al., 1997). Inaddition, the SOD mimetic tempol (4-hydroxy-2,2,6,6,-tetramethyl piperidinoxyl)reduces blood pressure and renal vascular resistance while preserving renal bloodflow in spontaneously hypertensive rats (SHR; Schnackenberg et al., 1998).

In some but not all reports, treatment of hypertensive humans with chemicalantioxidants can partially restore endothelial vasomotor function and reduce bloodpressure (Duffy et al., 1999; 2001; Sherman et al., 2000; Solzbach et al., 1997;Taddei et al., 1998). A study by Duffy et al. (1999) demonstrated that a relativelysmall dosage of vitamin C taken chronically (500 mg/day for 1 month) was able tosignificantly reduce blood pressure in otherwise healthy patients with essentialhypertension. However, experiments assessing forearm vascular function usingFMD and strain-gauge venous occlusion plethysmography report that it took arelatively large (pharmacological) dosage of vitamin C delivered intraarterially to


improve endothelial vasomotor function in the forearm arteries of hypertensivepatients (Duffy et al., 2001; Sherman et al., 2000; Solzbach et al., 1997; Taddei etal., 1998).

Thus, as was the case for the chemical antioxidant treatment experimentswith hypercholesterolemic patients, these studies demonstrate that nonpharma-cological doses of chemical antioxidants in humans may be insufficient to combatvascular oxidative stress-induced endothelial vasomotor dysfunction associatedwith hypertension—though they may exert other vascular health benefits such asreducing blood pressure itself in certain cases. These results are consistent withthose of the seven large-scale primary and secondary prevention trials conductedin humans in the last decade demonstrating little or no benefit of chronic chemicalantioxidant dietary supplementation at reasonable doses in preventing or treatingCVD (for review, see Shihabi et al., 2002).

Recognizing the established role of oxidative stress in endothelial dysfunc-tion, from an antioxidant supplementation perspective it seems necessary andworthwhile to put significant effort into the development of more powerful chemi-cal antioxidants in order for dietary, nutraceutical, or pharmacological supplemen-tation to be effective in treating this root cause of vascular pathophysiology. In thisregard, cell-permeable mimetics of SOD are a promising possibility (Muscoli etal., 2003). An alternative and parallel strategy is to establish physiological or phar-macological mechanisms to enhance the endogenous vascular enzymatic antioxi-dant capacity and/or to dampen the enzymatic pro-oxidant capacity in order totemper oxidative stress-induced reductions in NO• bioavailability and vasomotordysfunction and thus contribute to the prevention and treatment of CVD.

Sedentary lifestyle is also a CVD risk factor. Recent investigations of thevascular endothelial adaptations to exercise confirm that, in general, endothelialfunction is poorer in sedentary individuals than in those who exercise regularly(Higashi et al., 1999a; Kingwell et al., 1996; Figure 3). There is substantial evi-dence that increased NO• bioavailability could be of key importance to the im-proved endothelial vasomotor function that results from exercise training (Delp etal., 1993; Delp and Laughlin, 1997; Graham and Rush, 2004; Hambrecht et al.,1998; 2000; Higashi et al., 1999a; 1999b; Kingwell et al., 1996; Muller et al.,1994; Rush et al., 2000; 2003; Sessa et al., 1994; Woodman et al., 1997; 1999).However, the mechanisms controlling changes in NO• bioavailability in responseto exercise training are not completely understood. The potential roles of adapta-tions in eNOS and oxidative stress in the functional adaptations of arteries to exer-cise training will now be considered. The working hypothesis is that some of thevery cellular mechanisms that contribute to endothelial dysfunction in well-defined cases of CVD could be targeted and reversed by signals associated withregular increases in physical activity.

Adaptations Associated With Exercise Training

Numerous studies using humans and other animal models have demonstrated im-proved NO•-dependent endothelial function as a result of aerobic exercise trainingin otherwise healthy sedentary subjects, and in those with preexisting endothelialdysfunction associated with conditions such as hypertension, coronary artery dis-ease/hyperlipidemia, and CHF (Chen et al., 1996; Delp et al., 1993; Delp and


Laughlin, 1997; Graham and Rush, 2004; Hambrecht et al., 1998; 2000; Higashiet al., 1999a; 1999b; Kingwell et al., 1996; Laughlin et al., 1998; 2001; Muller etal., 1994; Parker et al., 1994; Sessa et al., 1994; Walsh et al., 2003; Wang et al.,1993; Yen et al., 1995; Figure 3).

Resistance artery endothelial function, assessed as the maximal forearm bloodflow response to intrabrachial artery Ach infusion in sedentary humans, was ob-served to be ~40% lower in hypertensives vs. normotensives. However, 12 weeksof low-intensity aerobic exercise training (brisk walking 6 bouts/week, 30 min/bout @ ~55% V

.O2max) improved the response in hypertensive patients to ~85%

of sedentary normotensive values (Higashi et al., 1999a). The same training pro-gram also improved the Ach-induced forearm blood flow response in normoten-sive subjects by ~35% (Higashi et al., 1999a). Animal models of hypertensionconfirm this observation; maximal Ach-induced dilation of aortic rings from sed-entary SHR was approximately half as much as the dilation of rings from seden-tary, normotensive Wistar Kyoto rats (WKY) in myography experiments, but aor-tic rings from SHR that had undergone 6 weeks of moderate intensity exercisetraining demonstrated similar Ach-induced vasodilatory responses as those fromWKY rats (Graham and Rush, 2004). Thus, exercise training can reverse endothe-lial vasomotor dysfunction associated with hypertension, at least in some vesseltypes and experimental conditions (Figure 3).

In coronary artery disease patients, exercise training at a heart rate of ~110bpm (80% of maximal achievable heart rate in these patients, most of whom weretaking β-blockers), 6 bouts per day for 10 minutes each bout over a study period of4 weeks, improved endothelium-dependent dilation both in epicardial vessels andin coronary resistance vessels (Hambrecht et al., 2000). This resulted in an aver-age doubling of the peak flow velocity in response to intracoronary Ach, and a30% increase in coronary blood flow reserve. Similarly, feeding a high fat/highcholesterol diet to miniature swine resulted in a blunting of endothelium-depen-dent vasodilation in isolated coronary arteries, and exercise training was shown toattenuate these functional effects (Thompson et al., 2004; Woodman et al., 2004).

Six months of moderate exercise training in CHF patients (70% individualmaximal heart rate, ~25 min/day, 5 days/week) resulted in improved exercise ca-pacity associated with a 25% increase in V

.O2max and a 200% increase in the femoral

artery flow response to Ach, indicating improved endothelial-dependent dilationof human skeletal muscle resistance vessels (Hambrecht et al., 1998). Animal studieshave confirmed that CHF is accompanied by impairment of endothelium-depen-dent vasodilation of systemic vessels (Buikema et al., 1993; Drexler and Lu, 1992;Kaiser et al., 1989; Kiuchi et al., 1993; Lindsay et al., 1992; Mulder et al., 1996;Nasa et al., 1996; Ontkean et al., 1991; Teerlink et al., 1993; Varin et al., 1999) thatin turn compromises peripheral tissue perfusion (Drexler and Lu, 1992; Kiuchi etal., 1993; Ueno et al., 1994).

The impairment in peripheral artery endothelium-dependent dilation is highlycorrelated with the degree of exercise intolerance and the severity of CHF in bothanimals and humans (Demopoulos et al., 1997; Drexler and Lu, 1992; Hambrechtet al., 1998; Katz et al., 1997; Kobayashi et al., 2003; Linke et al., 2001; Nakamuraet al., 1996; Vona et al., 2004; Walsh et al., 2003). In contrast, chronic physicalactivity improves endothelial vasomotor function in numerous vascular beds in


CHF patients/animals (Hambrecht et al., 1998; Hornig et al., 1996; Katz et al.,1997; Kobayashi et al., 2003; Linke et al., 2001; Maiorana et al., 2000; Varin et al.,1999; Walsh et al., 2003; Wang et al., 1997; Figure 3).

Improved NO• bioavailability underlies many of the above listed cases oftraining-induced improvements in endothelium-dependent dilation, since the ad-aptations can be reduced or eliminated with NOS inhibitors (Chen and Chiang,1996; Chen et al., 1996; Delp et al., 1993; Delp and Laughlin, 1997; Graham andRush, 2004; Hambrecht et al., 1998; Higashi et al., 1999a; 1999b; Hornig et al.,1996; Kingwell et al., 1996; Koller et al., 1995; Muller et al., 1994; Parker et al.,1994; Varin et al., 1999; Wang et al., 1993; 1997; Yen et al., 1995). The roles ofadaptations in prostanoid and EDHF pathways to physical activity and disease areless clear at present because, relative to the NO• system, these other endothelium-dependent dilatory pathways have received much less research attention. This shouldnot be confused, however, with a lack of importance of these pathways in theexplanation of vascular adaptations to CVD and to exercise. For instance, recentdata indicates that the endothelium-dependent dilatory responses in coronary ar-teries of hyperlipidemic pigs are improved after exercise training as a combinedresult of both enhanced NO• bioavailability and reduced prostanoid constrictoravailability (Thompson et al., 2004; Woodman et al., 2004).

Potential Mechanisms

An important principle to be established prior to discussion of the potential mecha-nisms responsible for exercise-induced functional adaptations of the endotheliumis the possible nonuniversality of the responses. Thus, observed effects of exercisetraining on vascular endothelial function and gene/protein expression likely de-pend not only on the characteristics of the exercise training (mode, intensity, dura-tion) but also on the vascular bed examined (coronary, cerebral, skeletal muscle,etc.), and the position in the arterial tree (conduit artery, smaller artery, arteriole,and branch order of arteriole). These issues have been highlighted previously (e.g.,Laughlin et al., 1996; 1998; 2003b; 2003c). An additional consideration is therelationship of functional and molecular/cellular adaptations to structural adapta-tions that also occur in a given vessel and the interaction this has with duration-dependent effects. Early adaptations to physical activity in a given vessel may bequite distinct from those that characterize the steady-state, fully-adapted trainedphenotype, and thus it may not be simply a matter of the degree of adaptation thatcontrasts the early responses from the stably-trained phase responses.

Isolated vessel segment and vessel ring experiments demonstrate increasedsensitivity and maximal effect of NO•-mediated endothelium-dependent dilationin some but not all vessel types and calibers after exercise training (Chen andChiang, 1996; Chen et al., 1996; Delp et al., 1993; Delp and Laughlin, 1997; Gra-ham and Rush, 2004; Koller et al., 1995; Laughlin et al., 1998; 2001; 2003b; 2003c;Muller et al., 1994; Oltman et al., 1995; Parker et al., 1994; Varin et al., 1999; Yenet al., 1995). Increases in vascular eNOS protein levels may play a role in theexercise-induced improvements in NO• bioavailability. For instance, prolongedexercise training for a period of several weeks increased eNOS levels and im-proved NO•-mediated vasomotor activity of rat aorta and pig coronary arteries and


arterioles (Chen and Chiang, 1996; Chen et al., 1996; Delp et al., 1993; Delp andLaughlin, 1997; Graham and Rush, 2004; Griffin et al., 1999; Laughlin et al., 2001;Muller et al., 1994; Parker et al., 1994; Woodman et al., 1997; Yen et al., 1995).

In contrast, conduit coronary arteries and aortic endothelial cells from pigsexercise trained for several weeks do not exhibit evidence of functional or bio-chemical improvements in NO•-dependent dilation (Laughlin et al., 2001; Oltmanet al., 1995; Rush et al., 2003; Thompson et al., 2004; Woodman et al., 2004).Earlier studies (Sessa et al., 1994; Wang et al., 1993) had concluded that dog con-duit coronary arteries and aortic endothelial cells did respond functionally andbiochemically with upregulation of NOS and NO• action in response to chronicexercise training. However, these conclusions were based on a 10- to 14-day train-ing period (Sessa et al., 1994; Wang et al., 1993), and recent evidence from a seriesof studies on pigs demonstrates that the responses of eNOS expression and NO•-dependent dilation of coronary conduit arteries and aortic endothelial cells is dif-ferent in short-term vs. long-term training; early functional improvements andenhanced eNOS expression are lost later in the training period (Griffin et al., 1999;Laughlin et al., 2001; 2003a; Oltman et al., 1995; Rush et al., 2003; Thompson etal., 2004; Woodman et al., 2004).

One explanation for this phasic response of coronary conduit arteries to ex-ercise training relates to the role of shear stress in controlling eNOS expression. Inlight of the presence of several shear stress response elements in the eNOS genepromotor region (Venema et al., 1994), one likely contributor to the exercise-in-duced eNOS response is the elevated flow/shear stress associated with increasedblood flow during the exercise bouts. Shear stress has been shown to increaseeNOS mRNA and protein in vivo and in cell culture (Nishida et al., 1992; Noris etal., 1995; Topper et al., 1996; Uematsu et al., 1995), and in models of increasedflow in vivo (Miller and Vanhoutte, 1988; Nadaud et al., 1996). In addition, it wasrecently demonstrated that elevated shear stress in isolated, perfused arterioles iscapable of increasing eNOS mRNA in as little as 4 hours (Woodman et al., 1999).Thus it is quite conceivable that shear stress is a mediator of the early (days) re-sponses to exercise training, both functional and eNOS upregulation, in conduitcoronary arteries.

After the initial weeks of exercise training, however, the conduit coronaryarteries undergo structural adaptations that result in increased diameter (Bove andDewey, 1985; Kramsch et al., 1981; Laughlin, 1995; Laughlin and McAllister,1992; Leon and Bloor, 1968; Windecker et al., 2002; Wyatt and Mitchell, 1978)and a consequent reduction in the shear stress signal associated with a given exer-cise-induced elevation in blood flow (Laughlin, 1995). This is consistent with therestoration of eNOS (Laughlin, 1995; Laughlin et al., 2001; Thompson et al., 2004)and NO•-dependent dilation (Oltman et al., 1995; Rogers et al., 1991; Thompsonet al., 2004; Woodman et al., 2004) to control levels in the steady-state, fully-trained state after their initial elevation in the first days of training.

Shear stress is not the only factor accounting for the exercise effect. In addi-tion to the local and bed-specific signals, it is clear that there must be some neuro-humoral influence on the vascular endothelium adaptations to exercise training, asthere are profound improvements in forearm artery endothelium dilatory responsesafter leg-specific exercise training protocols that do not elicit major forearm blood


flow responses during the exercise bout itself (Linke et al., 2001; Maiorana et al.,2000; Walsh et al., 2003). As the field advances it is likely that the neural, humoral,autocrine, paracrine, metabolic, and mechanical signals coordinating vascular ad-aptations to chronic physical activity will be identified and characterized.

It is not clear whether simple changes in eNOS levels are necessary or suffi-cient enough to be responsible for exercise training-induced improvements in NO•

bioavailability and NO•-mediated function. As has been established in the discus-sion of the effects of CVD on NO• bioavailability, it appears that regulation ofvascular cell oxidative stress can influence NO• bioavailability and function. Thusit is possible that favorable adjustments in vascular cell oxidative stress could ac-count in part for improvements in NO• bioavailability and NO•-mediated vasomo-tor function that result from chronic exercise training.

Although acute exercise transiently increases oxidative stress because ofaccelerated ROS generation (Ji, 1995; Liu et al., 2000), recent preliminary evi-dence suggests that exercise training can result in an increased availability of en-zymatic antioxidant defenses in vascular tissue (Fukai et al., 2000; Rush et al.,2000; 2003). Increased mRNA, protein, and enzymatic activity of SOD-1 in coro-nary arterioles, aortic endothelium, and whole aortic homogenates from exercise-trained pigs compared to sedentary controls are among the recent data suggestingthat exercise has positive effects on vascular antioxidant enzyme pathways (Rushet al., 2000; 2003). Another recent study demonstrated that extracellular SOD-3 isalso increased as a result of exercise training, and that this response depends inpart on NO•-mediated SOD-3 gene expression (Fukai et al., 2000). Furthermore,endothelial cell culture studies have recently demonstrated that H2O2 is a potentinducer of eNOS expression (Drummond et al., 2000), and it is therefore conceiv-able that the elevations in ROS accompanying exercise training bouts could con-tribute to eNOS expression via this mechanism. Thus, the autocrine and paracrinecontrol of eNOS and antioxidant enzymes by NO• and ROS involves extensivebiochemical cross-talk and is an exciting new area of interest in the context ofexercise training (Drummond et al., 2000; Fukai et al., 2000; Rush et al., 2000;Woodman et al., 1997).

Physical signaling mechanisms cannot be ignored as possible contributorsto exercise training-induced responses of vascular antioxidant enzymes, as flow/shear stress-induced increases SOD-1 mRNA in cultured human aortic endothelialcells and in isolated, perfused porcine coronary arterioles have been observed (Inoueet al., 1996; Woodman et al., 1999). The noted increases in vascular cell antioxi-dant enzymes (Fukai et al., 2000; Rush et al., 2000; 2003) suggest a role in theimproved NO•-mediated endothelium-dependent function that accompanies exer-cise training in some vascular beds and in some levels of the arterial tree (Delp etal., 1993; Hambrecht et al., 2000; Higashi et al., 1999a; 1999b; Muller et al., 1994;Sessa et al., 1994; Wang et al., 1993).

Identification of the cellular factors that control vascular dose-response ef-fects to exercise, the differential adaptations among vascular beds, and the re-sponse of different types of arterial vessels (large and small arteries and branchorders of arterioles) within a vascular bed are all important to understanding theeffects of exercise in vascular wall plasticity, the control of oxidative stress andNO• bioavailability, and ultimately the role of exercise in the treatment and pre-


vention of CVD. The observations of exercise-induced increases in SOD-1 andSOD-3 in vascular cells could be particularly interesting in the case of hyperten-sion, since this risk factor is known to be associated with increased O2

-• produc-tion from NADPH oxidase in vascular cells (Azumi et al., 1999; Berry et al., 2000;Fukui et al., 1997; Görlach et al., 2000; Griendling et al., 1994; Laursen et al.,1997; Mohazzab-H et al., 1994; Nakazono et al., 1991; Rajagopalan et al., 1996;Tschudi et al., 1996).

In addition, recent preliminary data also support a possible reduction in aor-tic NADPH oxidase expression associated with improved NO•-dependent aorticvasodilation in exercise-trained vs. sedentary SHR (Graham and Rush, 2004). Thus,a possible basis for improvements in blood pressure management and in NO•-mediated endothelial function resulting from regular aerobic exercise could in-volve improved balance of O2-• and NO•. However, the precise functional impactof exercise-induced increases in vascular antioxidant enzyme levels and reduc-tions in pro-oxidant enzyme levels need to be defined more precisely, and a morethorough assessment must be made of the changes in all the parameters affectingROS production and management in response to regular physical activity.

Conclusion

Sedentary lifestyle and other cardiovascular disease risk factors are associated withdysfunction of the vascular endothelium. Oxidative stress-mediated destruction ofnitric oxide appears to be a common mechanism mediating this dysfunction. Exer-cise training can improve endothelial function and reverse dysfunction associatedwith cardiovascular disease. Exercise training-induced improvements in endothe-lial function are associated with increased nitric oxide bioavailability. This holdsgreat promise for rigorously describing one of the molecular mechanisms by whichexercise improves endothelial function and cardiovascular health. The challengesthat lie ahead include identifying the specific molecular adaptations that result inan improved functional endothelial phenotype as a result of exercise training. Pre-liminary evidence suggests an interaction of eNOS regulatory adaptations withmodifications in the expression level of antioxidant enzymes, although many de-tails remain to be discovered.

Even when more of the specifics regarding the molecular mechanisms areelucidated, identifying the universality of adaptations throughout the vascular tree,in different vascular beds, and in different preexisting physiological and patho-physiological states in humans and a variety of useful animal models present sig-nificant but surmountable challenges to our complete understanding of the mo-lecular etiology of functional benefits of exercise training. As progress is made inthis and other exercise research, a better evidence-based model of the mechanismsof physical activity and lifestyle in the prevention and treatment of cardiovasculardisease will emerge.

Acknowledgments

Work in the authors’ laboratory is supported by the Canadian Institutes of Health Research(CIHR), the Heart and Stroke Foundation of Ontario, and the Natural Sciences and


Engineering Research Council of Canada (NSERC). J.W.E. Rush is the CIHR-CanadaResearch Chair in Integrative Vascular Biology. S.G. Denniss and D.A. Graham aresupported by NSERC doctoral scholarships.

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Received August 13, 2004; accepted in final form October 11, 2004.

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