Redox Mechanisms in Blood Vessels

Cornelius F.H. Mueller, Karine Laude, J. Scott McNally and David G. Harrison
Arterioscler. Thromb. Vasc. Biol. 2005;25;274-278; originally published online Oct
28, 2004;
DOI: 10.1161/01.ATV.0000149143.04821.eb
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 ATVB In Focus
Redox Mechanisms in Blood Vessels
Series Editor: Kathy K. Griendling

Redox Mechanisms in Blood Vessels

Cornelius F.H. Mueller, Karine Laude, J. Scott McNally, David G. Harrison

Abstract—Reactive oxygen species have been implicated in the pathogenesis of virtually every stage of vascular lesion
formation, hypertension, and other vascular diseases. We are currently gaining insight into important sources of reactive
oxygen species in the vessel wall, including the NADPH oxidases, xanthine oxidase, uncoupled nitric oxide synthase,
and mitochondrial sources. Although various reactive oxygen species have pathological roles, some serve as important
signaling molecules that modulate vascular tone, growth, and remodeling. In the next several months, a series of articles
in Arteriosclerosis, Thrombosis, and Vascular Biology attempt to further elucidate how reactive oxygen species are
produced by vascular cells and the roles of these in vascular homeostasis. This series promises to provide a valuable
update on a wide variety of issues related to the biochemistry, molecular biology, and physiology of these important and
fascinating molecules. (Arterioscler Thromb Vasc Biol. 2005;25:274-278.)
Key Words: NADPH oxidase 䡲 superoxide 䡲 nitric oxide synthase 䡲 xanthine oxidase 䡲 mitochondria

I n the next several months, Arteriosclerosis, Thrombosis,

and Vascular Biology will publish a series of reviews
examining in depth the source and role of reactive oxygen
species (ROS) and oxidant stress in vascular disease. Disor-
ders such as hypercholesterolemia, hypertension, diabetes,
aging, and mechanical injury are associated with increased
ROS production.1–5 The fact that these conditions contribute
to ⬎33% of the deaths and 40% of the total medical expenses
in industrialized countries emphasizes the importance of
understanding how oxidative stress occurs and how ROS
affect the vascular system.6 Several enzyme systems have
recently been recognized to be sources of ROS in vascular
cells under pathological conditions. Experimental studies in
cells and experimental animals have indicated that ROS
mediate or enhance virtually every aspect of atherosclerotic
lesion formation. The best-characterized of these events is
oxidative modification of low-density lipoprotein, which can
occur via reaction of ROS with low-density lipoprotein or via
direct enzymatic modification by lipoxygenases.7 Beyond
this, ROS can promote inflammation,8 alter vasomotion,9
activate matrix metalloproteinases,10 induce apoptosis,11
cause platelet aggregation,12 and stimulate vascular smooth
muscle proliferation.13 All of these events are active in the
atherosclerotic lesion and are thought to contribute to vascu-
lar lesion formation.
See cover
It has been, only in the past decade, widely accepted that
vascular cells could produce ROS, and we are still learning a
great deal about the role of the various potential enzymes,
how they are regulated, and the role of oxidative stress in
vascular disease. A technical problem has been that vessels
do not produce ROS at levels equivalent to neutrophils;
therefore, the methods commonly used for neutrophils, such
as cytochrome c reduction, yield values that are only slightly
above the limit of detection. This problem was overcome by
the use of chemiluminescence techniques, such as lucigenin-
enhanced chemiluminescence, and this entire area of research
was dramatically advanced by the use of such methods. In the
late 1990s, several articles appeared in which the authors
showed that lucigenin-enhanced chemiluminescence could
artifactually produce superoxide; however, these “test tube”
experiments did not reflect the assay as used in intact
vessels.14 Subsequently, numerous methods including elec-

Original received September 27, 2004; final version accepted October 20, 2004.
From Emory University Division of Cardiology, Department of Medicine and the Atlanta Veterans Administration Hospital, Atlanta, Ga.
Correspondence to David G. Harrison, Division of Cardiology, Emory University, 1639 Pierce Drive, WMRB 319, Atlanta, GA 30322. E-mail
dharr02@emory.edu
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000149143.04821.eb

274
 Mueller et al
Known sources of reactive oxygen species (ROS) and potential
interactions. In vascular cells, predominant sources of ROS
include the NADPH oxidase, xanthine oxidase, uncoupled nitric
oxide synthase, and mitochondrial electron transport. The Nox
proteins are the catalytic component of the NADPH oxidase and
together with p22phox comprise the membrane cytochrome.
Shown also is the oxygenase domain of eNOS, which in the
absence of tetrahydrobiopterin (BH4) can produce large amounts
of superoxide. ROS produced from the NADPH oxidase can
promote oxidation of BH4 and increase the relative levels of
xanthine oxidase in the endothelium. Hydrogen peroxide and
lipid peroxides are also capable of stimulating the NADPH oxi-
dases to further produce superoxide.
tron spin resonance, dihydroethidium oxidation, other chemi-
luminescence methods, and even cytochrome c reduction
have been used to confirm the results observed with
lucigenin-enhanced chemiluminescence. Further, assays of
hydrogen peroxide often provide results that mirror these
measures of superoxide production in diseased vessels. All of
these have confirmed an increased ROS production by
vessels in the setting of disorders like hypercholesterolemia,
diabetes, and hypertension.15–17
Sources of ROS in Vascular Cells
This special series of ATVB provides an opportunity to review
our current understanding of how ROS are produced in the
vessel wall. Although there are numerous enzyme systems
that can potentially produce ROS in the vessel wall, 4 enzyme
systems seem to predominate. These include the NADPH
oxidases, xanthine oxidase, uncoupled NO synthase, and
mitochondrial sources. A recurring theme is that there ap-
pears to be substantial interplay between these sources, such
that activation of one can enhance activity of others (Figure).
This can lead to feed-forward processes, which further
augment ROS production and oxidant stress. These interac-
tions are further discussed in the following paragraphs.
NADPH Oxidases
The NADPH oxidase enzymes, also known as the Nox
enzymes, represent a major source of ROS in vascular cells.
The Nox proteins represent the catalytic subunits of these
enzymes and vary in terms of their mode of activation and
need for cofactor activation.18 Nox1 protein levels are quite
low in vascular cells, but can be induced by stimuli such as
Redox Mechanisms in Blood Vessels 275
PDGF, angiotensin II, and serum.18 Nox2, previously known
as gp91phox, is the large catalytic subunit of the phagocyte
cytochrome b558. It is expressed in endothelial and adventi-
tial cells of large vessels and in the vascular smooth muscle
cells of smaller vessels.19 –22 Nox4 is constitutively expressed
and constitutively active in vascular smooth muscle and
endothelial cells.23,24 Current understanding is that all the
Nox enzymes require p22phox, which serves as a docking
protein for other subunits and stabilizes the Nox proteins.25 A
variety of pathological stimuli, such as angiotensin II, stretch,
endothelin-1, thrombin, and catecholamines acutely activate
the NADPH oxidases in vascular smooth muscle and endo-
thelial cells. The biochemical pathway whereby angiotensin
II activates the NADPH oxidase involves activation of the
tyrosine kinase c-Src, transactivation of the EGF receptor,
and ultimately activation and translocation of the small g
protein Rac-1.26 Activation of Nox1 and Nox2 also require
translocation of cytoplasmic subunits, including p47phox,27 or
analogues of p47phox and p67phox termed NoxO1 and
NoxA1.28,29 Interestingly, pathophysiological stimuli such as
angiotensin II, hypercholesterolemia, growth factors, and
serum can also increase expression of several of the NADPH
oxidase subunits, including p22phox, Nox1, and Nox4, further
promoting an increase in ROS production.18 The biochemical
pathways regulating expression of these subunits have not
been elucidated. Nevertheless, activation and increased ex-
pression of these Nox-based oxidases are clearly very impor-
tant in modulating oxidant stress in vascular disease. These
issues are discussed in an upcoming review by Dr Francis
Miller. It should be noted that Dr Miller, working in collab-
oration with Dr Neal Weintraub, first showed that hydrogen
peroxide and lipid peroxides can stimulate activity of the
NADPH oxidases in vascular smooth muscle cells, leading to
a feed-forward increase in ROS production.30,31
Xanthine Oxidase
Another important source of ROS in mammalian cells is the
xanthine oxidoreductase. Xanthine oxidoreductase exists in 2
forms, as xanthine dehydrogenase (XDH) and as xanthine
oxidase (XO).32 XDH uses NAD⫹ to receive electrons from
hypoxanthine and xanthine, yielding NADH and uric acid. In
contrast, XO uses oxygen as an electron acceptor from these
same substrates to form O2. and hydrogen peroxide. The ratio
of XO to XDH in the cell is therefore critical to determine the
amount of ROS produced by these enzymes. Conversion of
XDH to XO is stimulated by inflammatory cytokines, like
tumor necrosis factor-␣, and also by oxidation of critical
cysteine residues by oxidants, such as peroxynitrite.33,34
Recently, we have shown in bovine and mouse aortic endo-
thelial cells that the relative levels of these is markedly altered
by the presence of a functioning NADPH oxidase, such that
in cells with an absence of the NADPH oxidase, the levels of
XO are extremely low.35,36 In more recent studies, we have
found that this is mediated by hydrogen peroxide released
from the NADPH oxidase. Thus, this regulation represents a
second situation in which cellular production of ROS from
the NADPH oxidase further begets ROS production.
There is substantial debate as to whether XDH is ex-
pressed. Immunohistochemical studies of normal human
276 Arterioscler Thromb Vasc Biol. February 2005
tissues have failed to demonstrate XDH in endothelial cells or
other cardiovascular tissues.36 In contrast, there is evidence
that XO can produce ROS and affect endothelial function in
humans in the setting of pathology,15,37–39 possibly caused by
stimulation of XO expression by inflammatory cytokines or
other stimuli in these conditions. For example, Sohn et al
have shown that hypoxia induces XO activity in human
umbilical vein endothelial cells.40 There is substantial interest
in the concept that the XO present in endothelial cells
originates from other organs and that the enzyme is probably
taken-up via heparin binding sites.15,41,42 Whatever the source
of XO, its vascular activity correlates inversely with endo-
thelial function in patients with heart failure and in subjects
with atherosclerosis.15,43 Dr Margaret Tarpey discusses these
concepts in greater depth in her upcoming review of xanthine
oxidoreductase.
Uncoupled Endothelial Nitric Oxide Synthase
A third major contributor to vascular ROS generation is
uncoupled endothelial nitric oxide synthase (eNOS). The
normal product of this enzyme is nitric oxide (NO); however,
in the absence of either L-arginine or tetrahydrobiopterin
(BH4), the NO synthases are incapable of transferring elec-
trons to L-arginine and begin to use oxygen as a substrate for
O2. formation.44 Uncoupling of eNOS has been demonstrated
in various pathophysiological conditions including diabetes,17
hypercholesterolemia,45 and hypertension.46 In DOCA-salt
hypertension, oxidation of BH4 occurs as a result of ROS
(particularly peroxynitrite) produced by the NADPH oxidase.
Oral treatment of mice that have DOCA-salt hypertension
with BH4“re-couples” eNOS, leading to increased vascular
NO production, decreased O2. production, and lowering of
blood pressure.46 Thus, uncoupling of eNOS caused by
oxidation of BH4 represents a third mechanism whereby ROS
produced by the NADPH oxidase can stimulate another
enzyme to produce additional ROS in a self-perpetuating
fashion.
Related to this situation, eNOS is subject to regulation by
H2O2, which acutely activates the enzyme and, over the
longer-term, increases its expression. Thus, increased expres-
sion of eNOS in the setting of oxidant stress can lead to a
situation in which high levels of the uncoupled enzyme can
generate even greater amounts of O2. . It should be stressed
that not all eNOS becomes uncoupled, such that some of the
enzyme continues to produce NO, leading to a condition
favoring production of peroxynitrite. Dr Thomas Münzel
discusses NOS uncoupling in much greater detail in his
upcoming review.
Mitochondrial Electron Transport
In other organs and tissues, the mitochondrial electron transport
chain represents the predominant source of O2. and consequently
H2O2. It has been estimated that between 1% and 4% of the
oxygen reacting with the respiratory chain is incompletely
reduced to O2. . Defects in mitochondria DNA can be inherited or
can develop as a result of disease, and have been proposed to
augment ROS production by these organelles.47,48 Interestingly,
exposure of endothelial or vascular smooth muscle cells to
exogenous peroxynitrite or H2O2 leads to mitochondrial DNA
damage.49 This represents yet another mechanism whereby
oxidant stress could beget further oxidant stress.
In the vessel wall, the precise contribution of mitochondria
to the total ROS production, however, remains unclear. Part
of this problem relates to the fact that specific antagonists are
not well-characterized. For example, rotenone is often used to
inhibit mitochondrial radical production50,51 but, in fact, is
capable of having the opposite effect.31,52 Furthermore, inhi-
bition of mitochondrial function can dramatically alter many
other aspects of cell metabolism, making results from such
interventions difficult to interpret. Nevertheless, there is
ample evidence that mitochondria play an important role in
vascular ROS production. Stimuli such as high glucose,
cyclic strain, leptin, and cigarette smoking have been shown
to damage aortic mitochondrial DNA and alter mitochondrial
enzyme activity. Dr Paul Schumacker’s review examines the
role of mitochondria in vascular oxidant stress further.
Diverse Properties of ROS
It is important to note that one should not lump all ROS together
when considering the topic of oxidant stress and vascular
disease. Some ROS, such as superoxide O2. the hydroxyl radical
(HO), peroxy-radicals (ROO䡠), and NO have unpaired electrons
in their outer orbital. Others, such as H2O2 and peroxynitrite, do
not have unpaired electrons but are oxidants. These can have
very different roles. Superoxide reacts with NO in a diffusion
limited fashion, leading to formation of peroxynitrite and loss of
the beneficial actions of NO. This phenomenon is thought to be
responsible for reduced endothelium-dependent vasodilatation in
many conditions, and likely contributes to hypertension by
removing the vasodilator effect of NO. Peroxynitrite is a very
potent oxidant and can oxidize lipids and thiols and react with
iron sulfur centers in a variety of enzymes. Whereas scavenging
O2. improves endothelium-dependent vasodilatation, it might not
reduce atherosclerosis, because O2. scavenging does not elimi-
nate other ROS and, in fact, can enhance production of H2O2
(Scheme 1).53
Scheme 1:
Scheme 1 is important because there is increasing evidence
that H2O2 plays an important role in atherosclerosis. As an
example, Tribble et al showed that lesion formation was not
diminished in mice overexpressing Cu/Zn SOD and, in fact,
was increased in direct relation to aortic Cu/Zn SOD
activity.54
Another aspect of ROS is that they are not uniformly
deleterious and can have important signaling properties
within the vessel wall.55 Hydrogen peroxide, in particular, is
relatively stable and uncharged so that it can easily diffuse
between cells. Recently, it has become clear that endoge-
nously produced H2O2 can act as an endogenous hyperpolar-
izing factor in both human and mouse resistance vessels.56,57
Dr Gutterman discusses this further in his upcoming review.
 Mueller et al
Hydrogen peroxide has been shown to inhibit phosphatases,
and activate guanylate cyclase, and to stimulate NO produc-
tion and alter gene expression. The mechanisms involved in
these signaling events are varied. An important reaction is
oxidation of critical cysteines to sulfenic acids, which in turn
can serve as precursors to sulfonic acid and/or disulfide
formation. These modifications can alter function of a num-
ber of enzymes, including phosphatases, glyceraldehyde-3-
phosphate dehydrogenase, and peroxiredoxin.58 – 60 Recent
evidence suggests that interactions between H2O2 and peroxi-
dases, especially myeloperoxidase, lead to formation of
oxidizing and nitrosating species that are particularly athero-
genic. Importantly, modification of proteins by these radicals
alters their enzymatic function. Nitration of apolipoprotein
A1 dramatically impairs its ability to participate in reverse
cholesterol transport from lipid-laden macrophages, and thus
reduces atheroprotective properties of high-density lipopro-
tein.61 Dr Hazen discusses this in detail in his review of
myeloperoxidase.
Finally, it is essential that we develop a greater understand-
ing of all the compensatory responses to prolonged oxidant
stress. Recent studies from our groups have supported the
concept that increased ROS production has minimal effects
on vascular function and hemodynamics at baseline but can
augment the response pathogenic stimuli. We have produced
mice with smooth muscle-targeted overexpression of the
NADPH oxidase subunit p22phox. Overexpression of this
subunit resulted in a concomitant increase in expression of
Nox1 and enhanced vascular O2. and H2O2 production.62 At
baseline, these mice were normotensive and had normal
vascular reactivity and histology. In response to angiotensin
II, however, augmented hypertension and increased thickness
of the vascular media develop in these animals.63 In addition,
vascular injury enhances atheroma formation in these ani-
mals.64 Further characterization of these mice indicated that
they have increased endothelial NO synthase expression, NO
production, and expression of the extracellular superoxide
dismutase, which likely compensate for their enhanced vas-
cular ROS production. We have concluded from these studies
that increased vascular ROS production has minimal effects
in the absence of stimuli such as angiotensin II or mechanical
injury, but the presence of such stimuli markedly augments
pathological responses. In this regard, the compensatory
responses to oxidative stress are likely critically important,
and disease probably does not occur until these mechanisms
fail. It remains a challenge to understand all of these com-
pensatory mechanisms, why they fail, and what can be done
to preserve their function.
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