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442 • Rush,
INVITED Denniss, and Graham

REVIEW
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Vascular Nitric Oxide and Oxidative Stress:

Determinants of Endothelial Adaptations to
Cardiovascular Disease and to Physical Activity
James W.E. Rush, Steven G. Denniss, and Drew A. Graham
Catalogue Data
Rush, J.W.E.; Denniss, S.G.; and Graham, D.A. (2005). Vascular nitric oxide and oxidative
stress: Determinants of endothelial adaptations to cardiovascular disease and to physical
For personal use only.
activity. Can. J. Appl. Physiol. 30(4): 442-474. © 2005 Canadian Society for Exercise
Physiology.
Key words: exercise, artery, reactive oxygen species, antioxidant, hypertension

Mots-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. A
universal feature of cardiovascular disease is dysfunction of the vascular endothelium, thus
disrupting 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 on
the sensitivity of target tissue to nitric oxide. Evidence supports a major contribution by
oxidative 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 exercise
training improves vascular endothelial function, especially when it has been impaired by
preexisting risk factors. Evidence is emerging to support a role for improved nitric oxide
bioavailability with training as a result of enhanced synthesis and reduced oxidative stress-
mediated destruction. Molecular targets sensitive to the exercise training effect include the
endothelial nitric oxide synthase and the antioxidant enzyme superoxide dismutase. How-
ever, many fundamental details of the cellular and molecular mechanisms linking exercise
The authors are with the Department of Kinesiology, University of Waterloo, Water-
loo, Ontario N2L 3G1.
442
Nitric Oxide and Oxidative Stress • 443
to altered molecular and functional endothelial phenotypes have yet to be discovered. The
working hypothesis is that some of the cellular mechanisms contributing to endothelial

dysfunction in cardiovascular disease can be targeted and reversed by signals associated
with regular increases in physical activity. The capacity for exercise training to regulate
vascular endothelial function, nitric oxide bioavailability, and oxidative stress is an ex-
ample of how lifestyle can complement medicine and pharmacology in the prevention and
management of cardiovascular disease.
La première cause de mortalité et de morbidité chez les Canadiens est la maladie
cardiovasculaire. La dysfonction de l’endothélium vasculaire, qui est la caractéristique
principale de la maladie, entraîne un dérèglement du contrôle de la vasodilatation, de la
perfusion des tissus, de l’hémostasie, et de la coagulation. Le maintien de la fonction et de
la 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’azote
et de la sensibilité des tissus ciblés par le monoxyde d’azote. Selon de solides études, la
destruction du monoxyde d’azote attribuable au stress par oxydation contribue à la
dysfonction de l’endothélium observée dans diverses conditions pathologiques dont
l’hypertension, le diabète, l’insuffisance cardiaque chronique, et l’athérosclérose. La pra-
tique régulière de l’activité physique (entraînement physique) réduit le risque de maladie
cardiovasculaire. De nombreuses études appuient la thèse que l’entraînement physique
améliore la fonction de l’endothélium vasculaire, notamment quand ce dernier a été déréglé
par des facteurs de risque en place. Il appert en outre que l’entraînement améliore la
biodisponibilité du monoxyde d’azote en favorisant la synthèse aux dépens de la dégradation
due au stress par oxydation. Les cibles moléculaires sensibles à l’entraînement physique
comprennent la monoxyde d’azote synthase endothéliale et la superoxyde dismutase.
Cependant, il reste à identifier les aspects fondamentaux des mécanismes moléculaires et
cellulaires reliant l’exercice physique aux diverses modifications moléculaires et
fonctionnelles des phénotypes endothéliaux. L’hypothèse de travail est la suivante: des
signaux associés à l’augmentation de la pratique régulière de l’activité physique contribuent
à 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, de
la 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 prévention
et le traitement de la maladie cardiovasculaire.
Introduction
In Canada, cardiovascular disease (CVD) causes ~35% of all deaths, contributes
significantly to morbidity, and accounts for ~20 billion dollars in annual direct and
indirect health care costs (Health Canada, 1997; Heart and Stroke Foundation of
Canada, 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 physical
inactivity as an independent CVD risk factor, habitual endurance exercise may
also have a beneficial effect on other independent risk factors including blood
444 • Rush, Denniss, and Graham
lipid profiles, blood pressure, body weight, and insulin sensitivity (American Heart
Association, 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, aerobic
physical 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 exercise
training; impairment of function accompanies a number of CVD states while im-
provement in endothelial function occurs with regular exercise. Over the past two
decades, a central role of endothelial cells in regulating vascular homeostasis has
been established through the discovery of certain endothelial-derived substances
that 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 endothelium
can alter vascular smooth muscle (VSM) cell contractile state (tone) and thus con-
trol blood pressure and tissue perfusion. The endothelium controls the vascular
thrombotic 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 platelet
activation, the clotting cascade, and the fibrinolytic system. Moreover, the endo-
thelium regulates vascular inflammatory and adhesion processes by producing
cytokines 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 a
healthy endothelial phenotype, in a pathophysiological CVD state the endothe-
lium may adopt an alternate phenotype wherein the balance is disrupted and
proconstrictory, proinflammatory, and prothrombotic signals prevail, leading to
vascular dysfunction.
Nitric oxide is a particularly important endothelial-derived substance in light
of 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 found
in persons with overt CVD or when one or more risk factors are present (Behrendt
and 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 et
al., 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 for
these improvements with chronic exercise have not been fully elucidated, though
recent data suggest that improvements in the management of vascular oxidative
stress 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 through
integrative studies in humans and animal models.
Assessment of Nitric Oxide-Dependent

Endothelial Vasomotor Function
Efforts to understand the vascular biology of NO• began with the discovery that
the endothelium plays a major role in controlling VSM tone. Using isolated rabbit
aorta 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 the
excised vascular segments. Disruption of the endothelium eliminated the relaxing
effect of Ach (Furchgott and Zawadzki, 1980). These researchers concluded that
endothelial cells were releasing an endothelium-derived relaxing factor (EDRF) in
response to Ach that was somehow responsible for arterial smooth muscle relaxation.
Subsequent studies by Furchgott (1988), Ignarro et al. (1987; 1988), Murad

and colleagues (Rapoport and Murad, 1983), Gryglewski et al. (1986), and Palmer
et 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 inactivated
by 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 beds
in 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 arterial
blood pressure (Rees et al., 1989).
Most experimental and clinical assessments of endothelial function make
use of either blood flow manipulations or drugs such as Ach to stimulate vasodilatory
and/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 next
several decades (for review, see Rubanyi, 1995) and shown to be endothelium-
dependent (Holtz et al., 1983; 1984; Smiesko et al., 1983; 1985). This dilatory
response can also be observed in vitro using isolated segments of either a conduit
or resistance vessel mounted on perfusion pipettes on a microscope stage, allow-
ing for precise manipulation of pressure and flow while continuously measuring
diameter (isolated vessel flow-mediated dilation technique; Figure 1B). Studies
using this methodology, together with pharmacological inhibitors of vasodilatory
pathways, 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).
A. Vascular Myography B. Isolated and Perfused Vessel
E. Soluble Markers of Vascular Function

C. Quantitative Coronary Angiography D. Forearm Blood Flow
Figure 1. Methods to assess endothelial function. Endothelial vasomotor function can

be assessed (A) in vitro using wire-mounted vessel rings by measuring isometric tension
development, or (B) in mounted, pipette-perfused vessel segments by monitoring changes
in 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 or
elevated 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 text
under Assessment of Nitric Oxide-Dependent Endothelial Vasomotor 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 through
an intracoronary arterial catheter, or in response to elevated blood flow caused by
vasodilator drug-induced dilation of downstream resistance vessels (quantitative
coronary 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 first
studies 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 owing
to a reduced NO• bioavailability in these arteries.
Although quantitative coronary angiography is considered the benchmark
for 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 accessible
peripheral 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, or
Doppler ultrasound to detect velocity (forearm blood flow technique; Figure 1D),
to quantify the change in forearm blood flow after intraarterial (brachial or radial
artery) 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 mechanisms
underlying 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 or
drug administration was introduced by Celermajer and colleagues in their assess-
ment of FMD in the human conduit brachial artery imaged using echo Doppler
ultrasound (forearm blood flow technique; Figure 1D; Celermajer et al., 1992;
Corretti et al., 2002). To create a flow stimulus through the brachial artery, a blood
pressure cuff placed about the arm or forearm is inflated to a suprasystolic pres-
sure to elicit arterial occlusion for several minutes. The resultant ischemia causes
downstream resistance vessels to dilate in response to the accumulation of meta-
bolic byproducts released by skeletal muscle. Subsequent rapid cuff deflation brings
about a rapid increase in blood flow through the brachial artery because of the
lowered downstream resistance. This response, termed reactive hyperemia, is char-
acterized by a peak brachial artery flow that is sustained for a few seconds before
slowly 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 brachial
artery to dilate, with a maximal response typically occurring 45 to 90 seconds after
cuff deflation.
The correlation between FMD assessed in the brachial artery and Ach-medi-
ated dilation in epicardial arteries (Anderson et al., 1995) indicates that peripheral
conduit 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 physiological
stimulus (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-noise
ratio (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 recent
studies 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• (Joannides
et 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 on
NO• (Bellien et al., 2003; Mullen et al., 2001). For the reasons described above
there 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 assessments
with blood markers of endothelial phenotype including proinflammatory/
prothrombotic biomarkers such as high-sensitivity C-reactive protein, vascular cell
adhesion 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; Willerson
and Ridker, 2004).
Factors Controlling NO• Bioavailability In Vivo

Through 20 years of vascular cellular and molecular studies, the mechanism of
NO• action has been well defined and the factors that affect NO• bioavailability are
being 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 potential
targets for improving endothelial function are being identified.
The generally accepted sequence of events of NO•-mediated vasodilation is
as 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 of
the vessel wall, NO• may be transported as NO• per se or as an S-nitrosothiol form
moving between VSM cells by simple diffusion across cell membranes and through
cytosol 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-
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 details
of these processes see text under Factors Controlling NO• Bioavailability In Vivo. For
simplicity, 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.
ber of layers of VSM cells that must be targeted simultaneously for vasodilation to
occur, 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 the
general determinants of NO• bioavailability in all vessels. NO•-mediated activa-
tion of sGC in the cytosol of VSM cells leads to cGMP accumulation, protein
kinase G (PKG) activation, and PKG-mediated phosphorylation of a number of
Ca2+ regulatory and contractile proteins. The result is a lowering of VSM sarco-
plasmic Ca2+ concentration (Cohen, 2000), relaxation of VSM, dilation of the blood
vessel, decreased vascular resistance, and increased flow through the vessel (Fig-
ure 2).
Two important principles of cell signaling are identifiable in this simplified
NO• mechanism of action: amplification of the original endothelial signal through
second messenger and protein kinase systems; and redundancy in the multiple
molecular targets of PKG, all of which act to decrease cytoplasmic Ca2+ and the
contractile state of VSM. Although the amplification inherent in this signaling

system 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 evidence
for 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 and
destruction must also be considered in order to appreciate the many potential sites
for molecular-level alterations that can result in depression or enhancement of
NO•-mediated vasodilation.
ENDOTHELIAL NO• SYNTHESIS
Synthesis of NO• in endothelial cells is mediated by eNOS (Figure 2). In response

to 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+, which
binds to the regulatory protein calmodulin (CaM). In turn, Ca2+-CaM binds to and
activates eNOS.
The acute regulation of endothelial NO• synthesis is much more complex
than 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 eNOS
activation; 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 and
acylation (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 of
localization 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 in
part 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 activity
and 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 in
neurons lining coronary and pial arteries can mediate flow- and agonist-induced
dilations in eNOS knockout mice (Huang et al., 2002; Lamping et al., 2000; Meng
et al., 1998). This suggests a compensatory interaction between eNOS and nNOS
that could offset eNOS deficiencies. However, as the convincing results are cur-
rently limited to knockout models lacking the eNOS gene, it will require much
more work to clarify the relative role of nNOS-derived NO• in vasomotor control
in vessels with competent eNOS, and its physiological and pathophysiological
importance.
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 oxidative
stress is a relative term. While increasing evidence suggests that low levels of
ROS contribute to normal physiological cell signaling (Buetler et al., 2004), ex-
cessive production or impaired buffering of ROS leads to increased oxidative stress
that can have pathophysiological consequences (Buetler et al., 2004; Griendling et
al., 2000; Kunsch and Medford, 1999; Taniyama and Griendling, 2003).
Local NO• degradation in the artery wall in vivo occurs predominantly through
the 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 in
order 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 and
Vanhoutte, 1989); experimental inhibition of superoxide dismutase (SOD) impairs
agonist-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 SOD
by gene or protein transfer in vivo restores the NO• action previously impaired by
overproduction 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 of
NO• bioavailability. It is thus important to consider the chemical and enzymatic
pro- 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 and
Harrison, 2000; Darley-Usmar et al., 1995; Dillon and Vita, 2000; Kojda and
Harrison, 1999), with NAD(P)H oxidase recognized as the predominant source of
O2-• (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 and
VSM 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 the
holoenzyme complex. A recent and excellent review of the structure and function
of this enzyme and its role in CVD provides much more detail (Griendling et al.,
2000).
Several chemical and enzymatic antioxidant systems exist in the vascular
environment. 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 are
present in both endothelial and VSM cells (Figure 2).
Superoxide dismutase is essential for normal NO• function because of its
action 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 and
catalase in controlling NO• bioavailability. Recent evidence suggests that H2O2
may contribute to the chronic regulation of NO• bioavailability, however, as it
induces eNOS expression at both transcriptional and posttranscriptional levels in
cultured 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 and
impaired 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; Hattori
et al., 1991; Horie et al., 1998; Jackson et al., 1998; Kinlay et al., 1999; Kinouchi
et 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 of
NO•-mediated endothelial vasomotor function has prognostic value for clinical
cardiovascular events including myocardial infarction and ischemic stroke
(Behrendt and Ganz, 2002; Ganz and Vita, 2003; Verma et al., 2003; Widlansky et
al., 2003). Considering this, a detailed analysis of vascular NO• bioavailability and
how 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 depressed
synthesis 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 supports
oxidative stress-induced destruction of NO• as a major mechanism of the reduced
NO• bioavailability leading to vascular dysfunction in CVD. Indeed, elevated ROS
has been causatively linked to endothelial vasomotor dysfunction in atherosclero-
sis, hypertension, diabetes, and chronic heart failure (CHF) (Berry et al., 2000; Cai
and Harrison, 2000; Crabos et al., 1997; Darley-Usmar et al., 1995; Dillon and
Vita, 2000; Fujita et al., 1995; Fukui et al., 1997; Gil-Longo et al., 1996; Griendling
A. B.
Healthy phenotype
Antioxidant therapy,
Endothelial Homeostasis
exercise training, and
Endothelium-dependent
pharmacological control
Vessel Relaxation
of CVD risk factors
Sedentary lifestyle,
oxidative stress,
hypertension, and other
CVD risk factors
Pathological phenotype
Agonist or Shear Stress
Figure 3. Healthy and pathological states of endothelial vasomotor function. Solid curve on
the graph in Panel A depicts a healthy dose-response relationship between an agonist or shear
stress 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 and
endothelium-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 risk
factors, each to some degree associated with elevated oxidative stress. With exercise training,
antioxidant therapy, and/or pharmacological control of CVD risk factors, oxidative stress may
be 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 depicted
in Panel A. The “measuring stick” in Panel B illustrates that between the extremes of healthy
and 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 bioavailability
and vascular oxidative stress.
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-
thelium-dependent vasodilation assessed in vitro using vascular myography (Mugge

et 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, while
subsequent studies have revealed an involvement of macrophage and VSM cell
O2-• 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 endothelial
vasomotor function in hypercholesterolemia (Huang and Keaney, 2000; Figure 3).
For instance, Mugge and co-workers doubled the aortic wall SOD enzyme activity
in hypercholesterolemic rabbits using polyethylene glycol-conjugated SOD injec-
tions, and this reversed endothelium-dependent NO•-mediated aortic vasomotor
dysfunction (Mugge et al., 1991b). Furthermore, intraarterial infusion of the chemi-
cal antioxidant, vitamin C, at 1-10 mM improved endothelial vasomotor function
in the forearm resistance arteries of human hypercholesterolemic patients assessed
in vivo using strain-gauge venous occlusion plethysmography (Ting et al., 1997).
Conversely, oral consumption of chemical antioxidants at more reasonable
dietary concentrations daily for one month (1,000 mg of vitamin C; 800 IU of
vitamin E; 30 mg of β-carotene) had no effect on vasomotor function, although the
treatment was associated with a reduction in the susceptibility of LDL cholesterol
to oxidation ex vivo (Gilligan et al., 1994). Notably, this latter study demonstrates
that 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 vascular
health 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 et
al., 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 dilatory
response in arteries from hypertensive animals; treatment with liposome-encapsu-
lated SOD for 8 days increased aortic SOD activity by ~30% and partially prevented
angiotensin-II-induced O2-• release, elevation of blood pressure, and endothelial va-
somotor dysfunction assessed by vascular myography (Laursen et al., 1997). In
addition, the SOD mimetic tempol (4-hydroxy-2,2,6,6,-tetramethyl piperidinoxyl)
reduces blood pressure and renal vascular resistance while preserving renal blood
flow in spontaneously hypertensive rats (SHR; Schnackenberg et al., 1998).
In some but not all reports, treatment of hypertensive humans with chemical
antioxidants can partially restore endothelial vasomotor function and reduce blood
pressure (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 relatively
small dosage of vitamin C taken chronically (500 mg/day for 1 month) was able to
significantly reduce blood pressure in otherwise healthy patients with essential
hypertension. However, experiments assessing forearm vascular function using
FMD and strain-gauge venous occlusion plethysmography report that it took a
relatively large (pharmacological) dosage of vitamin C delivered intraarterially to
improve endothelial vasomotor function in the forearm arteries of hypertensive

patients (Duffy et al., 2001; Sherman et al., 2000; Solzbach et al., 1997; Taddei et
al., 1998).
Thus, as was the case for the chemical antioxidant treatment experiments
with hypercholesterolemic patients, these studies demonstrate that nonpharma-
cological doses of chemical antioxidants in humans may be insufficient to combat
vascular oxidative stress-induced endothelial vasomotor dysfunction associated
with hypertension—though they may exert other vascular health benefits such as
reducing blood pressure itself in certain cases. These results are consistent with
those of the seven large-scale primary and secondary prevention trials conducted
in humans in the last decade demonstrating little or no benefit of chronic chemical
antioxidant dietary supplementation at reasonable doses in preventing or treating
CVD (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 and
worthwhile 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 this
regard, cell-permeable mimetics of SOD are a promising possibility (Muscoli et
al., 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 to
temper oxidative stress-induced reductions in NO• bioavailability and vasomotor
dysfunction and thus contribute to the prevention and treatment of CVD.
Sedentary lifestyle is also a CVD risk factor. Recent investigations of the
vascular endothelial adaptations to exercise confirm that, in general, endothelial
function 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 et
al., 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 response
to 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 the
very cellular mechanisms that contribute to endothelial dysfunction in well-
defined cases of CVD could be targeted and reversed by signals associated with
regular 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 training
in otherwise healthy sedentary subjects, and in those with preexisting endothelial
dysfunction 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; Higashi
et al., 1999a; 1999b; Kingwell et al., 1996; Laughlin et al., 1998; 2001; Muller et
al., 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 blood
flow response to intrabrachial artery Ach infusion in sedentary humans, was ob-
served to be ~40% lower in hypertensives vs. normotensives. However, 12 weeks
of low-intensity. aerobic exercise training (brisk walking 6 bouts/week, 30 min/
bout @ ~55% VO2max) 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 hypertension
confirm 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 exercise
training demonstrated similar Ach-induced vasodilatory responses as those from
WKY rats (Graham and Rush, 2004). Thus, exercise training can reverse endothe-
lial vasomotor dysfunction associated with hypertension, at least in some vessel
types and experimental conditions (Figure 3).
In coronary artery disease patients, exercise training at a heart rate of ~110

bpm (80% of maximal achievable heart rate in these patients, most of whom were
taking β-blockers), 6 bouts per day for 10 minutes each bout over a study period of
4 weeks, improved endothelium-dependent dilation both in epicardial vessels and
in 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 a
30% increase in coronary blood flow reserve. Similarly, feeding a high fat/high
cholesterol diet to miniature swine resulted in a blunting of endothelium-depen-
dent vasodilation in isolated coronary arteries, and exercise training was shown to
attenuate these functional effects (Thompson et al., 2004; Woodman et al., 2004).
Six months of moderate exercise training in CHF patients (70% individual
maximal heart rate, ~25 min/day, 5 days/week)
. resulted in improved exercise ca-
pacity associated with a 25% increase in VO2max and a 200% increase in the femoral
artery flow response to Ach, indicating improved endothelial-dependent dilation
of human skeletal muscle resistance vessels (Hambrecht et al., 1998). Animal studies
have 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) that
in turn compromises peripheral tissue perfusion (Drexler and Lu, 1992; Kiuchi et
al., 1993; Ueno et al., 1994).
The impairment in peripheral artery endothelium-dependent dilation is highly
correlated with the degree of exercise intolerance and the severity of CHF in both
animals and humans (Demopoulos et al., 1997; Drexler and Lu, 1992; Hambrecht
et al., 1998; Katz et al., 1997; Kobayashi et al., 2003; Linke et al., 2001; Nakamura
et al., 1996; Vona et al., 2004; Walsh et al., 2003). In contrast, chronic physical
activity 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 of
training-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 and
Rush, 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 of
adaptations in prostanoid and EDHF pathways to physical activity and disease are
less clear at present because, relative to the NO• system, these other endothelium-
dependent dilatory pathways have received much less research attention. This should
not be confused, however, with a lack of importance of these pathways in the
explanation of vascular adaptations to CVD and to exercise. For instance, recent
data indicates that the endothelium-dependent dilatory responses in coronary ar-
teries of hyperlipidemic pigs are improved after exercise training as a combined
result of both enhanced NO• bioavailability and reduced prostanoid constrictor
availability (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 endothelium
is the possible nonuniversality of the responses. Thus, observed effects of exercise
training 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 the
relationship 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 be
quite distinct from those that characterize the steady-state, fully-adapted trained
phenotype, and thus it may not be simply a matter of the degree of adaptation that
contrasts the early responses from the stably-trained phase responses.
Isolated vessel segment and vessel ring experiments demonstrate increased
sensitivity and maximal effect of NO•-mediated endothelium-dependent dilation
in some but not all vessel types and calibers after exercise training (Chen and
Chiang, 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; Yen
et al., 1995). Increases in vascular eNOS protein levels may play a role in the
exercise-induced improvements in NO• bioavailability. For instance, prolonged
exercise 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 and
Laughlin, 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 pigs
exercise trained for several weeks do not exhibit evidence of functional or bio-
chemical improvements in NO•-dependent dilation (Laughlin et al., 2001; Oltman
et 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 and
biochemically with upregulation of NOS and NO• action in response to chronic
exercise 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 series
of 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 and
enhanced 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 et
al., 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. In
light of the presence of several shear stress response elements in the eNOS gene
promotor region (Venema et al., 1994), one likely contributor to the exercise-in-
duced eNOS response is the elevated flow/shear stress associated with increased
blood flow during the exercise bouts. Shear stress has been shown to increase
eNOS mRNA and protein in vivo and in cell culture (Nishida et al., 1992; Noris et
al., 1995; Topper et al., 1996; Uematsu et al., 1995), and in models of increased
flow in vivo (Miller and Vanhoutte, 1988; Nadaud et al., 1996). In addition, it was
recently demonstrated that elevated shear stress in isolated, perfused arterioles is
capable 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 conduit
coronary arteries.
After the initial weeks of exercise training, however, the conduit coronary
arteries undergo structural adaptations that result in increased diameter (Bove and
Dewey, 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 the
restoration of eNOS (Laughlin, 1995; Laughlin et al., 2001; Thompson et al., 2004)
and NO•-dependent dilation (Oltman et al., 1995; Rogers et al., 1991; Thompson
et 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, as
there are profound improvements in forearm artery endothelium dilatory responses
after 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 of
vascular cell oxidative stress can influence NO• bioavailability and function. Thus
it 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 of
accelerated 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 suggesting
that exercise has positive effects on vascular antioxidant enzyme pathways (Rush
et al., 2000; 2003). Another recent study demonstrated that extracellular SOD-3 is
also increased as a result of exercise training, and that this response depends in
part on NO•-mediated SOD-3 gene expression (Fukai et al., 2000). Furthermore,
endothelial cell culture studies have recently demonstrated that H2O2 is a potent
inducer 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 paracrine
control of eNOS and antioxidant enzymes by NO• and ROS involves extensive
biochemical cross-talk and is an exciting new area of interest in the context of
exercise 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 contributors
to exercise training-induced responses of vascular antioxidant enzymes, as flow/
shear stress-induced increases SOD-1 mRNA in cultured human aortic endothelial
cells and in isolated, perfused porcine coronary arterioles have been observed (Inoue
et 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 the
improved NO•-mediated endothelium-dependent function that accompanies exer-
cise training in some vascular beds and in some levels of the arterial tree (Delp et
al., 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 branch
orders of arterioles) within a vascular bed are all important to understanding the
effects of exercise in vascular wall plasticity, the control of oxidative stress and
NO• bioavailability, and ultimately the role of exercise in the treatment and pre-
vention of CVD. The observations of exercise-induced increases in SOD-1 and

SOD-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 aortic
vasodilation 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 impact
of exercise-induced increases in vascular antioxidant enzyme levels and reduc-
tions in pro-oxidant enzyme levels need to be defined more precisely, and a more
thorough assessment must be made of the changes in all the parameters affecting
ROS production and management in response to regular physical activity.
Conclusion
Sedentary lifestyle and other cardiovascular disease risk factors are associated with
dysfunction of the vascular endothelium. Oxidative stress-mediated destruction of
nitric oxide appears to be a common mechanism mediating this dysfunction. Exer-
cise training can improve endothelial function and reverse dysfunction associated
with cardiovascular disease. Exercise training-induced improvements in endothe-
lial function are associated with increased nitric oxide bioavailability. This holds
great promise for rigorously describing one of the molecular mechanisms by which
exercise improves endothelial function and cardiovascular health. The challenges
that lie ahead include identifying the specific molecular adaptations that result in
an improved functional endothelial phenotype as a result of exercise training. Pre-
liminary evidence suggests an interaction of eNOS regulatory adaptations with
modifications 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 are
elucidated, 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 in
this and other exercise research, a better evidence-based model of the mechanisms
of physical activity and lifestyle in the prevention and treatment of cardiovascular
disease 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-Canada

Research Chair in Integrative Vascular Biology. S.G. Denniss and D.A. Graham are
supported by NSERC doctoral scholarships.
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Received August 13, 2004; accepted in final form October 11, 2004.

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442 • Rush,

INVITED Denniss, and Graham

Vascular Nitric Oxide and Oxidative Stress:

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

Key words: exercise, artery, reactive oxygen species, antioxidant, hypertension

working hypothesis is that some of the cellular mechanisms contributing to endothelial

Assessment of Nitric Oxide-Dependent

Subsequent studies by Furchgott (1988), Ignarro et al. (1987; 1988), Murad

A. Vascular Myography B. Isolated and Perfused Vessel

E. Soluble Markers of Vascular Function

C. Quantitative Coronary Angiography D. Forearm Blood Flow

Figure 1. Methods to assess endothelial function. Endothelial vasomotor function can

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-

peripheral arteries, such as those in the arm/forearm.

Factors Controlling NO• Bioavailability In Vivo

Figure 2. General determinants of NO• bioavailability as it relates to vasomotor func-

contractile state of VSM. Although the amplification inherent in this signaling

ENDOTHELIAL NO• SYNTHESIS

Synthesis of NO• in endothelial cells is mediated by eNOS (Figure 2). In response

DESTRUCTION OF NO• BY OXIDATIVE STRESS

Reduced NO• Bioavailability in Cardiovascular Disease

of CVD risk factors

Agonist or Shear Stress

ation may be indicative of an attenuated vascular homeostasis caused by a pathological endo-

CORONARY ARTERY DISEASE

thelium-dependent vasodilation assessed in vitro using vascular myography (Mugge

that nonpharmacological doses of chemical antioxidants in humans may not al-

improve endothelial vasomotor function in the forearm arteries of hypertensive

macological mechanisms to enhance the endogenous vascular enzymatic antioxi-

Adaptations Associated With Exercise Training

In coronary artery disease patients, exercise training at a heart rate of ~110

vention of CVD. The observations of exercise-induced increases in SOD-1 and

SOD-3 in vascular cells could be particularly interesting in the case of hyperten-

Engineering Research Council of Canada (NSERC). J.W.E. Rush is the CIHR-Canada

Biochem. Pharmacol. 50: 1321-1332.

lipoprotein oxidation and impaired endothelium-dependent vasodilation on patients

with hypercholesterolemia. J. Am. Coll. Cardiol. 24: 1611-1617.

and Oshima, T. (1999a). Regular aerobic exercise augments endothelium-dependent

Circ. Res. 83: 691-696.

Exerc. Sport Sci. Rev. 23: 135-166.

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Effects of NG-nitro-L-arginine methyl ester on renal function and blood pressure.

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