ATVB In Focus

Redox Mechanisms in Blood Vessels

Series Editor: Kathy K. Griendling
Previous Brief Reviews in this Series:
• Mueller CFH, Laude K, McNally JS, Harrison DG. Redox mechanisms in blood vessels. 2005;25:274 –278.
• Gutterman DD, Miura H, Liu Y. Redox modulation of vascular tone: focus of potassium channel mechanisms of di-
lation. 2005;25:671– 678.
• Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. 2005;25:1102–1111.

Oxidative Enzymopathies and Vascular Disease

Jane A. Leopold, Joseph Loscalzo

Abstract—In the vasculature, reactive oxygen species (ROS) generated by both mitochondrial respiration and enzymatic
sources serve as integral components of cellular signaling and homeostatic mechanisms. Because ROS are highly
reactive biomolecules, the cellular redox milieu is carefully maintained by small-molecule antioxidants and antioxidant
enzymes to prevent the deleterious consequences of ROS excess. When this redox balance is perturbed, because of either
increased ROS production or decreased antioxidant capacity, oxidant stress is increased in the vessel wall and, if not
offset, vascular dysfunction ensues. A number of heritable polymorphisms of pro-oxidant enzymes, including
5-lipoxygenase, cyclooxygenase-2, nitric oxide synthase-3, and NAD(P)H oxidase, have been identified and found to
modulate ROS production and, thereby, the risk of atherothrombotic cardiovascular disease in individuals with these
Downloaded from http://ahajournals.org by on September 16, 2023

genetic polymorphisms. Similarly, heritable deficiency of the antioxidant enzymes catalase, glutathione peroxidases,
glutathione-S-transferases, heme oxygenase, and glucose-6-phosphate dehydrogenase favors ROS accumulation, and
has been associated with an increased risk of vascular disease. Individually, each of these polymorphisms imposes a state
of uncompensated oxidant stress on the vasculature and collectively comprise the oxidative enzymopathies. (Arterio-
scler Thromb Vasc Biol. 2005;25:1332-1340.)
Key Words: antioxidants 䡲 atherosclerosis 䡲 genetic polymorphism 䡲 reactive oxygen species

C ellular respiration in an oxygen-rich environment generates

abundant derivatives of partially reduced oxygen, collec-
tively termed reactive oxygen species (ROS). Under basal
conditions, ROS serve as an integral component of cellular
signaling pathways; however, when these highly reactive meta-
bolic products are in excess, they impose an oxidant stress on the
cellular environment, which, in turn, modifies biomolecules to
modulate cell and organism phenotype.1 To protect against
cellular oxidant stress and its adverse sequelae, adaptive enzy-
matic mechanisms have evolved to metabolize ROS into less
reactive forms and minimize their potential to produce oxidative
damage and cellular dysfunction.
The importance of maintaining a balance between ROS
production and metabolism is highlighted when there is
deficiency or dysfunction of vascular antioxidant enzymes.
This results in a net accumulation of ROS because of
decreased antioxidant capacity in the setting of ambient ROS
production by vascular sources. Recent interest has focused
on heritable polymorphisms of vascular antioxidant enzymes
as one mechanism by which antioxidant capacity is dimin-
ished. These polymorphisms have been associated with an
increased risk of vascular disease in both animal models and
human studies and collectively may be recognized as oxida-
tive enzymopathies.
Vascular Sources of ROS
Under basal conditions, superoxide anion, a 1-electron reduc-
tion product of oxygen, is the primary source from which

Original received December 30, 2004; final version accepted March 15, 2005.
From Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass.
Correspondence to Joseph Loscalzo, MD, PhD, Whitaker Cardiovascular Institute, Boston University School of Medicine, 700 Albany Street, CABR
W-507, Boston, MA 02118. E-mail jloscalz@bu.edu
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000163846.51473.09

1332
 Leopold and Loscalzo
which converts hypoxanthine and xanthine to uric acid, is an additional source of ROS. As xanthine is converted to uric acid, 2 elec-
trons are donated to molybdenum (Mo) at the active site of the enzyme, thereby reducing it from Mo(VI) to Mo(IV). Finally, endothelial
nitric oxide synthase (eNOS), when substrate or cofactors are not replete, uncouples to generate superoxide in preference to NO. Q
indicates coenzyme Q; C, cytochrome C; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; FE, heme iron; BH4,
tetrahydrobiopterin.
Downloaded from http://ahajournals.org by on September 16, 2023
most other reactive oxidants are derived in the vasculature
and is generated by both metabolic and enzymatic sources, as
summarized in Figure 1. These sources include mitochondrial
respiration,2 xanthine oxidase,3 cyclooxygenases and lipoxy-
genases,4 NAD(P)H oxidases,5 and, when substrate or cofac-
tors are not replete, uncoupled nitric oxide synthase(s).6,7
Although these sources are primarily responsible for ambient
ROS production and basal cellular homeostatic function,
when vascular disease states ensue, redox balance in the
vessel wall is compromised because of increased ROS pro-
duction by these sources. When this is coupled with de-
creased antioxidant defense, as may occur in the setting of an
oxidative enzymopathy, the net result is an accumulation of
superoxide anions in the vessel wall, where they are free to
react and form a number of pathophysiologically relevant
reactive species. These species include hydrogen and lipid
peroxides, peroxynitrites and peroxynitrous acids, and hypo-
chlorite and hypochlorous acid (Figure 2), which, in turn,
may have deleterious consequences for vascular function.
Vascular Antioxidant Enzymes
To balance basal ROS production, the cellular redox milieu is
maintained by a number of key antioxidant enzymes that limit
intracellular and extracellular accumulation of deleterious
reactive oxygen metabolites. These antioxidant defense en-
zymes include the superoxide dismutases, which convert
superoxide anion into hydrogen peroxide; catalase, which
reduces hydrogen peroxide to water; the glutathione peroxi-
dases, which reduce hydrogen peroxide and lipid peroxides to
Oxidative Enzymopathies and Vascular Disease 1333
Figure 1. Metabolic and enzymatic
sources of superoxide in the vasculature.
Superoxide anion (䡠O2⫺) is formed by
several metabolic and enzymatic sources
within the cell. NADPH oxidase is com-
prised of multiple membrane-bound and
cytoplasmic subunits. The enzyme is ac-
tivated when the cytoplasmic subunits
p67 and p47 and the small G-protein rac
assemble with the membrane-bound
NOX (vascular homolog of gp91phox) and
p22phox. NADPH oxidase uses NADPH as
a substrate, and, in vascular cells, is
considered an important source of reac-
tive oxygen species (ROS) generation.
The lipoxygenases and cyclooxygenases
(COX) generate ROS indirectly by pro-
moting formation of inflammatory media-
tors. Arachidonic acid (AA) that is
cleaved from the membrane by phos-
pholipase A2 (PLA2) is then metabolized
by 5-lipoxygenase (5-LO) in the presence
of its accessory protein (FLAP) to form
leukotrienes (LTs). AA is also metabo-
lized by the cyclooxygenases to form
members of another family of inflamma-
tory mediators, the prostaglandins (PGs).
Mitochondria also generate superoxide
as electrons are transferred from com-
plex I to complex IV during normal cellu-
lar respiration. Xanthine oxidase (XO),
water and lipid alcohols, respectively; glutathione reductase,
which reduces glutathione disulfide to reduced glutathione;
the glutathione-S-transferases, which detoxify oxidants by
glutathiolation; the thiol-disulfide oxidoreductases and per-
oxiredoxins, which maintain protein thiol redox state; heme
Figure 2. Reactive species generated by superoxide in the vessel
wall. Once formed, superoxide may react with a number of other
species to generate pathophysiologically relevant reactive oxygen
and nitrogen species. Superoxide reacts with nitric oxide to yield
peroxynitirite (ONOO⫺), which, in the presence of hydrogen ions,
may form peroxynitrous acid (ONOOH). Superoxide may also
cause lipid peroxidation (LOOH) or, via the action of superoxide
dismutases (SOD), form hydrogen peroxide. Neutrophil myeloper-
oxidase (MPO), in turn, catalyzes the reaction of hydrogen perox-
ide and chloride (Cl⫺) to form hypochlorous acid (HOCl).
 1334 Arterioscler Thromb Vasc Biol. July 2005
oxygenase, which degrades heme to biliverdin and carbon
monoxide (and also releases free iron ions); and glucose-6-
phosphate dehydrogenase, which provides NADPH to serve
as a reducing equivalent and cofactor for other antioxidant
enzymes (Table 1).
Once formed, superoxide is dismutated to hydrogen per-
oxide by the activity of the superoxide dismutase (SOD)
family of antioxidant enzymes. Mammalian organisms pos-
sess 3 types of SOD: Cu,Zn-SOD, Mn-SOD, and extracellular
(EC)-SOD. Cu,Zn-SOD is located primarily in the cytosolic
compartment of all cells, although some activity has been
detected in lysosomes, peroxisomes, the nucleus, and the
intermembrane compartment of the mitochondrion. Mito-
chondria also contain Mn-SOD, which accounts for approx-
imately one-tenth of total cellular SOD activity, and, in
contrast to Cu,Zn-SOD, is pH-sensitive, manifesting decreas-
ing activity with increasing pH. EC-SOD, a copper-zinc–
containing SOD that is localized to the extracellular environ-
ment, is synthesized by vascular smooth muscle cells, binds
to extracellular matrix and heparan sulfate glycosaminogly-
cans on the endothelial surface,8 and serves to minimize the
superoxide-dependent inactivation of endothelial-derived
nitric oxide.
Hydrogen peroxide that results from the action of SODs (or
the action of oxidases, such as xanthine oxidase) is reduced to
water by catalase and the glutathione peroxidases (GPx).
Catalase exists as a tetramer comprised of 4 identical mono-
mers that each contain a heme group at the active site.
Degradation of hydrogen peroxide is accomplished via a
Downloaded from http://ahajournals.org by on September 16, 2023
conversion between 2 conformations of catalase: ferricatalase
(iron coordinated to water) and compound I (iron complexed
with an oxygen atom). Catalase also binds NADPH as a
reducing equivalent to prevent oxidative inactivation of the
enzyme (formation of compound II) by hydrogen peroxide as
it is reduced to water.9,10
The GPxs, a family of tetrameric enzymes that contain the
unique amino acid selenocysteine within their active sites, use
low-molecular-weight thiols such as glutathione (GSH) to
reduce hydrogen and lipid peroxides to their corresponding
alcohols. At present, 4 isoforms of GPx have been identified
and reasonably well-characterized. GPx-1 (cellular GPx) is
ubiquitous and reduces hydrogen peroxide as well as fatty
acid peroxides, but not esterified peroxyl lipids. Esterified
lipids are reduced by membrane-bound GPx-4 (phospholipid
hydroperoxide GPx), which can use several different low-
molecular-weight thiols as reducing equivalents.9 GPx-2
(gastrointestinal GPx) is localized to gastrointestinal epithe-
lial cells, where it serves to reduce dietary peroxides. GPx-3
(extracellular GPx) is the only member of the GPx family that
resides in the extracellular compartment and is believed to be
the most important extracellular antioxidant enzyme in mam-
mals. Because of the low concentration of GSH in extra-
cellular fluid, however, the precise nature of the GPx-3
reducing cosubstrate is unknown; glutaredoxin and thiore-
doxin have been proposed as candidate reductants.11,12
Thioredoxin, via a series of coupled reactions catalyzed by
thioredoxin reductase and NADPH, also functions as an
antioxidant molecule in the cytosol of vascular endothelial
cells and smooth muscle cells of medial arteries. The active
site of thioredoxin forms a disulfide bond with cysteine
groups within proteins. These oxidized cysteine groups, in
turn, are reduced by thioredoxin reductase in a reaction that
uses NADPH as a reducing equivalent. Peroxiredoxin, a
thioredoxin peroxidase, confers antioxidant properties on the
system by scavenging hydrogen peroxide and requires sulf-
hydryl groups as a cosubstrate. A second thioredoxin system
has been identified within mitochondria with sequence ho-
mology for the catalytic site of cytosolic thioredoxin; how-
ever, little is known about its function.13
Common to these antioxidants is the requirement of
NADPH as a reducing equivalent. NADPH maintains cata-
lase in the active form and is used as a cofactor by thioredoxin
as well as glutathione reductase, which converts oxidized
glutathione (GSSG) to GSH, a cosubstrate for the GPxs.
Intracellular NADPH, in turn, is generated by the reduction of
NADP⫹ by glucose-6-phosphate dehydrogenase (G6PD), the
first and rate-limiting enzyme of the pentose phosphate
pathway, during the conversion of glucose-6-phosphate to
6-phosphogluconolactone. By generating NADPH, G6PD is a
critical determinant of cytosolic glutathione buffering capac-
ity (GSH/GSSG) and, therefore, can be considered an essen-
tial, regulatory antioxidant enzyme. The role of G6PD as an
antioxidant enzyme has long been recognized in erythrocytes,
and we have previously demonstrated that G6PD is essential
to maintain redox balance in vascular endothelial and smooth
muscle cells.14 –17 Table 1 lists these and other key antioxidant
enzymes in the vasculature, their biochemical functions, and
unique properties.
Genetic Determinants of ROS Excess and
Vascular Disease Risk
The presence of both ROS-generating and antioxidant en-
zyme systems suggests that ROS excess, caused by increased
production or decreased antioxidant capacity, results in a state
of uncompensated oxidant stress that promotes vascular
dysfunction. In fact, many heritable polymorphisms of en-
zyme sources of ROS and of antioxidant enzymes that alter
redox balance have been identified and several appear to be
associated with an increased risk of atherothrombotic cardio-
vascular disorders (Table 2).
To date, heritable polymorphisms of the pro-oxidant en-
zymes 5-lipoxygenase, cyclooxygenase-2, nitric oxide
synthase-3, and NAD(P)H oxidase have been identified and
shown to confer an increased risk of vascular disease on
individuals who carry them. Similarly, heritable deficiencies
of the antioxidant enzymes catalase, GPxs, glutathione-S-
transferases, heme oxygenase, and G6PD that increase ROS
accumulation have also been associated with vascular dys-
function and disease.
The functional consequences of these heritable polymor-
phisms and enzyme deficiencies have been studied in both
animal models and human subjects. Although animal models
provide a unique opportunity to examine the effects of altered
gene expression on vascular function, results from these
studies must be interpreted with caution as the biological
microenvironment often represents an extreme form of enzy-
matic deficiency or dysfunction. Similarly, observations
made in patients that link heritable polymorphisms and
 Leopold and Loscalzo Oxidative Enzymopathies and Vascular Disease 1335

TABLE 1. Key Vascular Antioxidant Enzymes

Enzyme Activity Comments
SODs8
Cu,Zn-SOD Dismutation of ·O2⫺ into H2O2 and O2 Cytosolic, nuclear, and lysosomal localization
⫺
Mn-SOD Dismutation of ·O2 into H2O2 and O2 Mitochondrial localization
EC-SOD Dismutation of ·O2⫺ into H2O2 and O2 Synthesized by VSMC; bound to matrix and EC
proteoglycans
Catalase9 Reduction of H2O2 into H2O and O2 Principal peroxisome localization
GPxs 11, 12
GPx-1 Reduction of H2O2 to H2O and LOOH to LOH, Ubiquitous GSH is obligate co-substrate
GPx-3 Reduction of H2O2 to H2O and LOOH to LOH Essential extracellular antioxidant enzyme; glutaredoxin
and thioredoxin can serve as co-substrates
GPx-4 Reduction of H2O2 to H2O and PLOOH to PLOH and Utilizes several different low-molecular-weight thiols as
Chol-OOH to Chol-OH reducing co-substrates
GSSG reductase68 Reduction of GSSG to GSH NADPH is essential co-substrate; co-localizes with
peroxidases
GSH-S-transferase69 Metabolism of xenobiotics by GSH conjugation Extensive enzyme family
Thiol-Disulfide Oxidoreductases 13,68

Protein–disulfide isomerase
Thioredoxin
Thioredoxin reductase
Glutaredoxin
Downloaded from http://ahajournals.org by on September 16, 2023
Catalysis of protein disulfide bond formation
Reduction of protein disulfides to thiols; reduction of
hydrogen and lipid peroxides; has peroxynitrite
reductase activity
Reduction of oxidized thioredoxin and PDI disulfides
to (vicinal) dithiols
Catalysis of protein disulfide reduction and
glutathiolation of protein cysteinyl groups
Endoplasmic reticulum and plasma membrane localization
Cytosolic localization
Mitochondria
Sec-containing enzyme; NADPH as reducing co-substrate
GSH required for reduction of active site disulfide to vicinal
dithiol

Peroxiredoxin Reduction of H2O2 to H2O and LOOH to LOH Three classes of enzyme based on active-site cysteinyl
residues; thiol reductant not yet identified, may involve
thioredoxin system
Heme oxygenase70 Metabolizes heme to biliverdin, CO, and free iron;
the latter is subsequently bound by ferritin
Methionine sulfoxide reductase71 Reduces methionine sulfoxide to methionine Two isoforms, one of which contains Sec; uses thioredoxin
for reducing equivalents
Glucose-6-phosphate Exclusive source of cytosolic NADPH Ubiquitous; important for antioxidant defense in all cells
dehydrogenase14–16,64

enzyme deficiencies to vascular disease risk are subject to
scrutiny as they often demonstrate only association and not
causality. Furthermore, disease status may be modulated by
both environmental factors and therapeutic interventions to
obscure the full effects of antioxidant enzyme deficiency.
Nevertheless, here we review the results of relevant and
complementary studies performed in animal models and
human subjects to demonstrate the role of genetic determi-
nants of ROS excess and vascular disease risk.
Pro-Oxidant Enzymes
In murine models, Mehrabian et al18 demonstrated that
5-lipoxygenase (5-LO), a key regulatory enzyme in the
oxidative biosynthesis of pro-inflammatory leukotrienes, is a
major determinant of susceptibility to atherosclerosis. In
these studies, they found that resistance to atherosclerosis in
mice resulted from decreased 5-LO mRNA and protein levels
produced by two amino acid substitutions (Ile645Val and
Val646Ile) in the 5-LO cDNA sequence. The functional
importance of these amino acid substitutions was confirmed
by kinetic studies of mutated human 5-LO cDNA; these
mutations resulted in a decrease in Vmax to ⬍20% of control
and a relative 5-LO activity in vitro of 11% to 19% of
control.19 In vivo, absence of 5-LO in atherosclerotic prone
low-density lipoprotein receptor-null mice resulted in a ⬎26-
fold reduction in aortic lesion formation compared with mice
that expressed 5-LO.
In human subjects, histological examination of pathologi-
cal specimens from patients with atherosclerotic vascular
disease confirmed abundant 5-LO expression in plaques that
localized to macrophages and inflammatory cells.20 These
studies, in turn, led investigators to identify 5-LO poly-
morphisms associated with an increased risk of atherosclero-
sis. For example, Dwyer et al21 showed that variant 5-LO
genotypes—tandem promoter repeats of Sp-1 binding mo-
tifs—identified a subpopulation of individuals with increased
carotid intima-media thickness, as a marker of atherosclero-
sis, and that diets rich in n-6 fatty acids interact positively
with these genetic variants to promote leukotriene-mediated
inflammatory responses. These investigators also found that
 1336 Arterioscler Thromb Vasc Biol. July 2005

TABLE 2. Enzyme Genotypes and Vascular Disease

Enzyme
Pro-oxidant Enzymes
5-Lipoxygenase18–21
5-Lipoxygenase activating peptide
22
Cyclooxygenase 223,24
NO synthase-3 25–27
NAD(P)H oxidase28–34
Antioxidant Enzymes
Catalase35–39
Mn-SOD41
GPx-142–49
GPx-350–55
GSH-S-transferase56–58
Heme oxygenase-159–62
Glucose-6-phosphate
dehydrogenase14–17, 63–67
Downloaded from http://ahajournals.org by on September 16, 2023
Genotype Disease Risk
Tandem Sp-1 motifs in promoter Carotid intima-media thickness
4-SNP polymorphic haplotypes Myocardial infarction
Stroke
⫺765G–⬎C promoter polymorphism Protection from stroke
⫺786C–⬎T promoter polymorphism Coronary heart disease
27-bp VNTR in intron 4 Hypertension
Glu298Asp polymorphism Subarachnoid hemorrhage
Flow-mediated dilation
Carotid intima-media thickness
Coronary artery disease
p22phox ⫺930A3G promoter polymorphism Hypertension
p22phox His72Tyr polymorphism Cerebrovascular disease
Coronary heart disease
GA insertion in exon 2 Hyperhomocystinemia, increased vascular oxidant stress,
G insertion in exon 2 diabetes mellitus
T3G substitution in intron 7
Ala16Val polymorphism Carotid intima-media thickness
Polyalanine sequence (Ala6) polymorphism in exon 1 Coronary heart disease
7-SNP promoter haplotype Stroke, cerebral venous thrombosis
M1*0 and T1*1 alleles Coronary heart disease, peripheral vascular disease
⫺413T3A promoter polymorphism Protection from coronary heart disease, hypertension
(GT)n repeats, n⬎26 Restenosis, coronary heart disease
A⫺ variants (Asn126Asp & Val68Met, Asn126Asp & Endothelial dysfunction, hypertension, diabetes mellitus
Arg227Leu, Asn126Asp & Leu323Pro)

levels of the inflammatory marker C-reactive protein were
increased 2-fold in patients with the variant genotype. More
recently, Helgadottir et al22 showed that a promoter haplotype
comprised of 4 linked polymorphisms in the 5-LO activating
peptide, an accessory protein that facilitates presentation of
substrate arachidonate to 5-LO, conferred ⬇2-fold increased
risk of myocardial infarction and stroke in an Icelandic
population. Interestingly, stimulated neutrophils isolated
from patients who were carriers of the at-risk haplotype and
had a myocardial infarction synthesized more inflammatory
leukotrienes than neutrophils isolated from those patients
who were not carriers.
Common promoter variants in the cyclooxygenase 2 (COX-2)
gene have also been shown to influence the risk for cardiovas-
cular disease in human subjects. This is not surprising because
COX-2 generates prostanoids, which modulate inflammatory
responses, and induces production of macrophage matrix
metalloproteinase-2 and matrix metalloproteinase-9, which have
been implicated in plaque destabilization and rupture. Papafili et
al23 demonstrated a novel promoter polymorphism in the COX-2
gene (⫺765G3 C) that localizes to the putative binding site for
Sp1 and reduces promoter activity by 30% compared with the
⫺756G allele. In patients undergoing elective coronary artery
bypass grafting surgery, a greater increase in C-reactive protein
levels was seen postoperatively in patients with the ⫺756G
allele. Cipollone et al24 studied the association between this
polymorphism and first myocardial infarction or ischemic stroke
in 864 patients and 864 hospitalized controls. These investiga-
tors found that the prevalence ratios for myocardial infarction or
stroke were 0.48 (95% CI, 0.36 to 0.68) and 0.33 (95% CI, 0.24
to 0.55) for the heterozygous (⫺756 GC) and homozygous
(⫺756 CC) polymorphisms, respectively. They also noted that
expression of both COX-2 and matrix metalloproteinases in
atherosclerotic plaques obtained by carotid endarterectomy was
significantly less in individuals carrying the C allele than those
carrying the G allele. Thus, this polymorphism appears to confer
protection from ischemic atherosclerotic events compared with
the wild type promoter.
The endothelial nitric oxide synthase gene is highly poly-
morphic, and some of these polymorphisms appear to have
functional significance, either for gene expression or enzyme
activity.25 Several human studies have investigated the po-
tential association between these polymorphisms and the risk
of atherothrombotic vascular disease, but offer conflicting
results because of the low population prevalence of the
individual polymorphisms and the small sample sizes exam-
ined. Recently, Casas et al26 performed a meta-analysis of 26
case-control studies involving ⬎23 000 subjects focusing on
the Glu298Asp, the ⫺786T3 C, and the intron-4 variable
nucleotide tandem repeat (4, minor allele; 5, major allele)
polymorphisms in the endothelial nitric oxide synthase gene.
They found that both the Glu298Asp and intron-4 variable
nucleotide tandem repeat polymorphisms conferred a signif-
icant, albeit moderate, increase in the risk of ischemic heart
disease (odds ratio⫽1.31, 95% CI, 1.13 to 1.51; and odds
ratio⫽1.34, 95% CI, 1.03 to 1.75, respectively). This in-
creased risk may occur because the 298Asp isoform of the
enzyme appears to have a shorter half-life than the 298Glu
 Leopold and Loscalzo
isoform,25 and individuals with homozygosity for the intron-4
minor allele have been shown to have a small, but significant,
decrease in levels of plasma nitrogen oxides,27 suggesting that
both polymorphisms result in decreased levels of bioavailable
nitric oxide.
Several polymorphisms in the gene coding for the p22phox
subunit of NAD(P)H oxidase with functional consequences
have been described in humans. The ⫺930A3 G promoter
polymorphism, which has been associated with hypertension,
increases p22phox expression, NADPH oxidase activity, and
ROS formation.28,29 The C242T polymorphism, which results
in a His72Tyr substitution at the putative heme binding site,
appears to confer an increased risk of ischemic stroke (odds
ratio⫽1.81, 95% CI, 1.15 to 2.86) in a Japanese population30
and progression of coronary atherosclerosis in a Western
population.31 Notwithstanding a statistically significant asso-
ciation between this coding region polymorphism and athero-
thrombotic risk, functional studies suggest that this amino
acid substitution actually reduces the superoxide-generating
activity of NADPH oxidase.32 Interestingly, when the effects
of the C242T, A640G, and ⫺930A3 G polymorphisms on
neutrophil ROS production were examined, superoxide pro-
duction was decreased by the C242T polymorphism only; the
A640G, and ⫺930A3 G polymorphisms had no effect on
respiratory burst.33 Despite these findings, other studies have
failed to relate this polymorphism to vascular disease; the
prevalence was similar in patients with or without angio-
graphic evidence of atherosclerosis and was not associated
with impaired endothelium-dependent vasodilator
Downloaded from http://ahajournals.org by on September 16, 2023
responses.34
With the recent cloning of unique vascular isoforms of this
enzyme (NOXs) and identification of NOX isoforms in
vascular endothelial and smooth muscle cells, analysis of
these related genes for variants that may be associated with an
increased risk atherothrombotic disease awaits study.
Antioxidant Enzymes
Inherited catalase deficiency is a rare autosomal recessive
disorder that has been characterized uniquely in families of
Hungarian, Swiss, and Japanese origin.35 The phenotypes of
these individuals are generally normal with one reported
exception: Japanese subjects with profound deficiency (acata-
lasemia) have oral ulcerations.36 A recent report identified a
series of mutations in a Hungarian kindred with catalase
deficiency (GA insertion in exon 2, G insertion in exon 2, and
T3 G substitution in intron 7) associated with adverse lipid
profiles and a high incidence (12.7%) of familial diabetes
mellitus. In addition, Hungarian hypocatalasemic patients
were found to have higher plasma levels of homocysteine and
lower levels of folate,37 suggesting that these patients are at
greater risk for cardiovascular disease. Common functional
polymorphisms in the catalase promoter have been identified
in a Swedish population,38 although their relationship to
vascular disease risk has not yet been determined. Interest-
ingly, in an isolated Chinese population, a variant within the
catalase promoter region has been associated with essential
hypertension;39 however, this study did not measure catalase
activity and, therefore, the significance of this finding is
unknown.
Oxidative Enzymopathies and Vascular Disease 1337
No convincing relationship has been reported between
Cu,Zn-SOD or EC-SOD deficiency and atherothrombotic
disease risk, either in animal models or in human population
studies. In fact, eliminating EC-SOD in atherosclerosis-prone
apolipoprotein E–null mice does not appear to influence
atheroma burden at all.40 In contrast, a potentially functional
polymorphism in the signal sequence of Mn-SOD
(Ala163 Val) has been identified in humans and appears to
be a minor determinant of carotid atherosclerosis as demon-
strated by increased carotid intima-media thickness.41
Glutathione peroxidase-1 (GPx-1) is a ubiquitous antioxi-
dant enzyme whose deficiency has been shown to promote
endothelial dysfunction, heart failure, and abnormal structural
changes in the vasculature and myocardium.42– 45 Interest-
ingly, hyperhomocystinemia appears to enhance vascular
oxidant stress and atherothrombosis, in part by suppressing
expression of the GPx-1 gene; overexpressing GPx-1 in an
animal model of hyperhomocystinemia rescues this adverse
phenotype.46 Recently, Blankenberg et al47 have shown a
strong dose-dependence for erythrocyte GPx-1 activity as an
independent determinant of risk for cardiovascular events:
patients in the highest quartile of enzyme activity had a
hazard ratio of 0.29 (95% CI, 0.15 to 0.58) compared with
those in the lowest quartile. Importantly, no genotyping was
performed in this trial. Another recent trial by Winter et al48
analyzed the association between GPx-1 genotypes and the
risk of angiographically significant coronary artery disease.
These investigators focused on a polyalanine sequence poly-
morphism in exon 1 of the GPx-1 gene, which produces 3
alleles with 5, 6, or 7 alanine repeats. They studied 207 men
with angiographic evidence of coronary artery disease com-
pared with 146 controls and found that after adjusting for age,
body mass index, and tobacco use, men with at least one
6-alanine allele had a significantly increased risk of coronary
artery disease with an odds ratio of 2.07 (95% CI, 1.08 to
3.99). In a study of diabetic Japanese patients, investigators
identified an additional polymorphism, Pro198Leu, which
was associated with increased carotid intima-media thickness,
prevalence of cardiovascular disease, and peripheral vascular
disease. They also found that this polymorphism, when
associated with the 6-alanine allele, resulted in a 40%
decrease in GPx activity.49
Deficiency of glutathione peroxidase-3 (GPx-3), an essen-
tial extracellular peroxidase, has been associated with de-
creased nitric oxide bioavailability and increased platelet-
dependent thrombosis. Early work by our group linked a
reduction in plasma GPx-3 activity with increased platelet
activation and cerebrovascular arterial thrombosis.50,51 More
recently, we identified a promoter haplotype consisting of 7
tightly linked polymorphisms that reduce promoter activity,52
an effect that was more pronounced under hypoxic condi-
tions.53 This promoter haplotype was associated with an
increased risk of arterial thrombotic stroke in the young with
an odds ratio of 2.1 (95% CI, 1.1 to 3.9)54 and with an odds
ratio of 4.6 (95% CI, 1.6 to 13.1) for the risk of cerebral
venous thrombosis.55
The glutathione-S-transferases (GST) are xenobiotic-
metabolizing enzymes that are involved in the detoxification
of reactive electrophiles, including those found in tobacco
 1338 Arterioscler Thromb Vasc Biol. July 2005
smoke. Although the relationship between polymorphic ge-
notypes of these enzymes and cancer risk has been appreci-
ated for some time, only recently has their association with
coronary artery disease been recognized. In particular, pa-
tients with the GSTM1*0 null allele were found to have the
highest levels of the inflammatory markers, C-reactive pro-
tein, fibrinogen, von Willebrand factor, intercellular adhesion
molecule-1 and vascular cell adhesion molecule-1,56 as well
as greater levels of DNA damage present in atherosclerotic
plaques.57
A recent meta-analysis found that the GSTM1*0 null allele
and the GSTT1*1 functional allele are associated with an
increased risk of coronary heart disease in smokers (odds
ratio⫽2.30, 95% CI, 1.40 to 9.00; and odds ratio⫽2.50, 95%
CI, 1.30 to 4.80, respectively), and the GSTT1*1 functional
allele is associated with an increased risk of peripheral arterial
disease (odds ratio⫽3.60, 95% CI, 1.40 to 9.00).58
Because of its ability to detoxify heme and generate
vasoprotective levels of carbon monoxide, heme oxygenase
has been studied as a potential atheroprotective gene. Recent
data from a Japanese population suggest that the ⫺413T3 A
promoter polymorphism in the heme oxygenase-1 gene is
associated with an increased atherothrombotic risk.59 The A
allele appears to enhance gene expression and protect against
ischemic heart disease, whereas in contrast the T allele
increases the risk of myocardial infarction (odds ratio⫽1.42,
95% CI, 1.01 to 1.77) and angina pectoris (odds ratio⫽1.86,
95% CI, 1.08 to 2.41). Recent studies have shown that short
tandem repeats (⬍25 GT) in the promoter were associated
Downloaded from http://ahajournals.org by on September 16, 2023
with a reduced risk of restenosis after femoropopliteal angio-
plasty (relative risk⫽0.43, 95% CI, 0.24 to 0.71),60 whereas
long tandem repeats (⬎25 GT) conferred an increased risk of
restenosis after coronary stenting (odds ratio⫽3.74, 95% CI,
1.61 to 8.70) and adverse cardiac events during 6 months of
follow-up (odds ratio⫽3.26, 95% CI, 1.58 to 6.72).61 This
promoter polymorphism has also been associated with hyper-
tension in a population of Japanese women, but not men;
women with the AA genotype had an increased frequency of
hypertension (odds ratio⫽1.59, 95% CI, 1.14 to 2.20) and use
of antihypertensive agents compared with those with the AT
or TT genotype.62
Glucose-6-phosphate dehydrogenase (G6PD), the princi-
pal source of cytosolic reducing equivalents necessary for
thiol redox balance in all cells, is a highly polymorphic gene
with many functional variants.63 G6PD deficiency is the most
common enzymopathy in humans, with ⬎400 million cases
worldwide. To date, ⬇440 biochemical variants have been
identified with ⬇120 different mutations characterized. Most
of these mutations are single or double missense mutations or,
much less commonly, small deletions in the gene. Selective
pressure exists for these mutations, all of which reduce
activity, because erythrocytes with reduced G6PD activity are
more susceptible to oxidative damage, which renders cells
parasitized by plasmodium species more readily cleared by
the reticuloendothelial system than cells with a normal
complement of enzyme activity.14
Until very recently, G6PD deficiency was believed to be an
important pro-oxidant mechanism only in erythrocytes. In-
creasing evidence from several groups, however, supports the
view that deficiency of this important antioxidant enzyme
leads to increased oxidative cell injury in neurons, pancreatic
beta cells, embryonic stem cells, and, importantly, vascular
cells, including vascular smooth muscle cells15 and endothe-
lial cells.16,17 Furthermore, G6PD deficiency appears to be
related to a decreased potential for angiogenesis64 and wors-
ening ventricular function with ischemia-reperfusion injury in
animal models.65
The findings of these in vitro and in vivo studies are
supported by one recent study on the risk of hypertension and
diabetes in G6PD-deficient individuals,66 and one study of
endothelial dysfunction in young, otherwise healthy G6PD-
deficient individuals.67 G6PD deficiency is very common in
the black population, among whom it is found in 11.4% of
males and 2.5% of females (compared with ⬍0.1% of
whites). The B variant corresponds to the wild-type enzyme,
whereas the A⫹ variant (which has normal to mildly reduced
activity) is found in 20% of blacks and corresponds to an
Asn126Asp mutation. A⫺ variants comprise 3 additional
mutations on an A⫹ background, each leading to ⬇30% to
40% reductions in enzyme activity: Val68Met, Arg227Leu,
and Leu323Pro. Because blacks have an increased prevalence
of cardiovascular disease, these data suggest that G6PD
deficiency may contribute to endothelial dysfunction, vascu-
lar injury, and myocardial dysfunction.
Conclusions
Maintenance of redox balance in the cardiovascular system is
of paramount importance as uncompensated oxidant stress
contributes to endothelial dysfunction and vascular disease.
Risk factor-mediated injury, inflammation, and thrombosis
all promote oxidant stress and, thus, this intermediate molec-
ular phenotype appears to be an essential link among the
determinants of atherothrombotic disease. Many genetically
determined heritable differences in pro-oxidant and antioxi-
dant enzyme activities have been identified, and some have
been found to modulate atherothrombotic risk. How these
genetic variants interact with one another and with environ-
mental risk factors to modulate the resulting oxidative enzy-
mopathies are the subjects of ongoing studies of the mecha-
nisms of this complex disease process.
Acknowledgments
This work was supported by National Institutes of Health grants PO1
HL55993, RO1 HL58796, RO1 HL61795, and NO1 HV28178 (to
J.L.), and National Institutes of Health grant HL04399 and a
grant-in-aid from the American Heart Association (to J.A.L.). The
authors thank Stephanie Tribuna for expert secretarial support.
References
1. Stocker R, Keaney JF Jr. Role of oxidative modifications in atheroscle-
rosis. Physiol Rev. 2004;84:1381–1478.
2. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian
organs. Physiol Rev. 1979;59:527– 605.
3. Jarasch ED, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW.
Localization of xanthine oxidase in mammary-gland epithelium and cap-
illary endothelium. Cell. 1981;25:67– 82.
4. Romano M, Claria J. Cyclooxygenase-2 and 5-lipoxygenase converging
functions on cell proliferation and tumor angiogenesis: implications for
cancer therapy. FASEB J. 2003;17:1986 –1995.
5. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in
cardiovascular biology and disease. Circ Res. 2000;86:494 –501.
 Leopold and Loscalzo
6. Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS,
Karoui H, Tordo P, Pritchard KA Jr. Superoxide generation by endothe-
lial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci
U S A. 1998;95:9220 –9225.
7. Xia Y, Tsai AL, Berka V, Zweier JL. Superoxide generation from endo-
thelial nitric-oxide synthase. A Ca2⫹/calmodulin-dependent and tetrahy-
drobiopterin regulatory process. J Biol Chem. 1998;273:25804 –25808.
8. Stralin P, Karlsson K, Johansson BO, Marklund SL. The interstitium of
the human arterial wall contains very large amounts of extracellular
superoxide dismutase. Arterioscler Thromb Vasc Biol. 1995;15:
2032–2036.
9. Kirkman HN, Rolfo M, Ferraris AM, Gaetani GF. Mechanisms of pro-
tection of catalase by NADPH. Kinetics and stoichiometry. J Biol Chem.
1999;274:13908 –13914.
10. Gaetani GF, Ferraris AM, Sanna P, Kirkman HN. A novel NADPH:
(bound) NADP ⫹ reductase and NADH::(bound) NADP ⫹ transhydro-
genase function in bovine liver catalase. Biochem J. 2005;385:763–768.
11. Takahashi K, Avissar N, Whitin J, Cohen H. Purification and character-
ization of human plasma glutathione peroxidase: a selenoglycoprotein
distinct from the known cellular enzyme. Arch Biochem Biophys. 1987;
256:677– 686.
12. Bierl C, Voetsch B, Jin RC, Handy DE, Loscalzo J. Determinants of
human plasma glutathione peroxidase (GPx-3) expression. J Biol Chem.
2004;279:26839 –26845.
13. Yamawaki H, Haendeler J, Berk BC. Thioredoxin: a key regulator of
cardiovascular homeostasis. Circ Res. 2003;93:1029 –1033.
14. Cappadoro M, Giribaldi G, O’Brien E, Turrini F, Mannu F, Ulliers D,
Simula G, Luzzatto L, Arese P. Early phagocytosis of glucose-6-
phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by
Plasmodium falciparum may explain malaria protection in G6PD defi-
ciency. Blood. 1998;92:2527–2534.
15. Leopold JA, Loscalzo J. Cyclic strain modulates resistance to oxidant
stress by increasing G6PDH expression in smooth muscle cells. Am J
Physiol Heart Circ Physiol. 2000;279:H2477–H2485.
16. Leopold JA, Cap A, Scribner AW, Stanton RC, Loscalzo J. Glucose-6-
phosphate dehydrogenase deficiency promotes endothelial oxidant stress
and decreases endothelial nitric oxide bioavailability. FASEB J. 2001;15:
Downloaded from http://ahajournals.org by on September 16, 2023
1771–1773.
17. Leopold JA, Zhang YY, Scribner AW, Stanton RC, Loscalzo J. Glucose-
6-phosphate dehydrogenase overexpression decreases endothelial cell
oxidant stress and increases bioavailable nitric oxide. Arterioscler
Thromb Vasc Biol. 2003;23:411– 417.
18. Mehrabian M, Allayee H, Wong J, Shi W, Wang XP, Shaposhnik Z, Funk
CD, Lusis AJ. Identification of 5-lipoxygenase as a major gene con-
tributing to atherosclerosis susceptibility in mice. Circ Res. 2002;91:
120 –126.
19. Kuhn H, Anton M, Gerth C, Habenicht A. Amino acid differences in the
deduced 5-lipoxygenase sequence of CAST atherosclerosis-resistance
mice confer impaired activity when introduced into the human ortholog.
Arterioscler Thromb Vasc Biol. 2003;23:1072–1076.
20. Spanbroek R, Grabner R, Lotzer K, Hildner M, Urbach A, Ruhling K,
Moos MP, Kaiser B, Cohnert TU, Wahlers T, Zieske A, Plenz G,
Robenek H, Salbach P, Kuhn H, Radmark O, Samuelsson B, Habenicht
AJ. Expanding expression of the 5-lipoxygenase pathway within the
arterial wall during human atherogenesis. Proc Natl Acad Sci U S A.
2003;100:1238 –1243.
21. Dwyer JH, Allayee H, Dwyer KM, Fan J, Wu H, Mar R, Lusis AJ,
Mehrabian M. Arachidonate 5-lipoxygenase promoter genotype, dietary
arachidonic acid, and atherosclerosis. N Engl J Med. 2004;350:29 –37.
22. Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S, Jonsdottir
H, Thorsteinsdottir U, Samani NJ, Gudmundsson G, Grant SF, Thor-
geirsson G, Sveinbjornsdottir S, Valdimarsson EM, Matthiasson SE,
Johannsson H, Gudmundsdottir O, Gurney ME, Sainz J, Thorhallsdottir
M, Andresdottir M, Frigge ML, Topol EJ, Kong A, Gudnason V,
Hakonarson H, Gulcher JR, Stefansson K. The gene encoding 5-lipoxy-
genase activating protein confers risk of myocardial infarction and stroke.
Nat Genet. 2004;36:233–239.
23. Papafili A, Hill MR, Brull DJ, McAnulty RJ, Marshall RP, Humphries
SE, Laurent GJ. Common promoter variant in cyclooxygenase-2
represses gene expression: evidence of role in acute-phase inflammatory
response. Arterioscler Thromb Vasc Biol. 2002;22:1631–1636.
24. Cipollone F, Toniato E, Martinotti S, Fazia M, Iezzi A, Cuccurullo C, Pini
B, Ursi S, Vitullo G, Averna M, Arca M, Montali A, Campagna F,
Ucchino S, Spigonardo F, Taddei S, Virdis A, Ciabattoni G, Notarbartolo
A, Cuccurullo F, Mezzetti A. A polymorphism in the cyclooxygenase 2
Oxidative Enzymopathies and Vascular Disease 1339
gene as an inherited protective factor against myocardial infarction and
stroke. JAMA. 2004;291:2221–2228.
25. Voetsch B, Loscalzo J. Genetic determinants of arterial thrombosis.
Arterioscler Thromb Vasc Biol. 2004;24:216 –229.
26. Casas JP, Bautista LE, Humphries SE, Hingorani AD. Endothelial nitric
oxide synthase genotype and ischemic heart disease: meta-analysis of 26
studies involving 23028 subjects. Circulation. 2004;109:1359 –1365.
27. Tsukada T, Yokoyama K, Arai T, Takemoto F, Hara S, Yamada A,
Kawaguchi Y, Hosoya T, Igari J. Evidence of association of the ecNOS
gene polymorphism with plasma NO metabolite levels in humans.
Biochem Biophys Res Commun. 1998;245:190 –193.
28. San Jose G, Moreno MU, Olivan S, Beloqui O, Fortuno A, Diez J, Zalba
G. Functional effect of the p22phox -930A/G polymorphism on p22phox
expression and NADPH oxidase activity in hypertension. Hypertension.
2004;44:163–169.
29. Moreno MU, San Jose G, Orbe J, Paramo JA, Beloqui O, Diez J, Zalba
G. Preliminary characterisation of the promoter of the human p22(phox)
gene: identification of a new polymorphism associated with hypertension.
FEBS Lett. 2003;542:27–31.
30. Ito D, Murata M, Watanabe K, Yoshida T, Saito I, Tanahashi N, Fukuuchi
Y. C242T polymorphism of NADPH oxidase p22 PHOX gene and is-
chemic cerebrovascular disease in the Japanese population. Stroke. 2000;
31:936 –939.
31. Cahilly C, Ballantyne CM, Lim DS, Gotto A, Marian AJ. A variant of
p22(phox), involved in generation of reactive oxygen species in the vessel
wall, is associated with progression of coronary atherosclerosis. Circ Res.
2000;86:391–395.
32. Guzik TJ, West NE, Black E, McDonald D, Ratnatunga C, Pillai R,
Channon KM. Functional effect of the C242T polymorphism in the
NAD(P)H oxidase p22phox gene on vascular superoxide production in
atherosclerosis. Circulation. 2000;102:1744 –1747.
33. Wyche KE, Wang SS, Griendling KK, Dikalov SI, Austin H, Rao S, Fink
B, Harrison DG, Zafari AM. C242T CYBA polymorphism of the
NADPH oxidase is associated with reduced respiratory burst in human
neutrophils. Hypertension. 2004;43:1246 –1251.
34. Li A, Prasad A, Mincemoyer R, Satorius C, Epstein N, Finkel T,
Quyyumi AA. Relationship of the C242T p22phox gene polymorphism to
angiographic coronary artery disease and endothelial function. Am J Med
Genet. 1999;86:57– 61.
35. Goth L. A new type of inherited catalase deficiencies: its characterization
and comparison to the Japanese and Swiss type of acatalasemia. Blood
Cells Mol Dis. 2001;27:512–517.
36. Hirono A, Sasaya-Hamada F, Kanno H, Fujii H, Yoshida T, Miwa S. A
novel human catalase mutation (358 T–⬎del) causing Japanese-type
acatalasemia. Blood Cells Mol Dis. 1995;21:232–234.
37. Goth L, Vitai M. The effects of hydrogen peroxide promoted by homo-
cysteine and inherited catalase deficiency on human hypocatalasemic
patients. Free Radic Biol Med. 2003;35:882– 888.
38. Forsberg L, Lyrenas L, de Faire U, Morgenstern R. A common functional
C-T substitution polymorphism in the promoter region of the human
catalase gene influences transcription factor binding, reporter gene tran-
scription and is correlated to blood catalase levels. Free Radic Biol Med.
2001;30:500 –505.
39. Jiang Z, Akey JM, Shi J, Xiong M, Wang Y, Shen Y, Xu X, Chen H, Wu
H, Xiao J, Lu D, Huang W, Jin L. A polymorphism in the promoter region
of catalase is associated with blood pressure levels. Hum Genet. 2001;
109:95–98.
40. Sentman ML, Brannstrom T, Westerlund S, Laukkanen MO, Yla-
Herttuala S, Basu S, Marklund SL. Extracellular superoxide dismutase
deficiency and atherosclerosis in mice. Arterioscler Thromb Vasc Biol.
2001;21:1477–1482.
41. Kakko S, Paivansalo M, Koistinen P, Kesaniemi YA, Kinnula VL,
Savolainen MJ. The signal sequence polymorphism of the MnSOD gene
is associated with the degree of carotid atherosclerosis. Atherosclerosis.
2003;168:147–152.
42. Forgione MA, Weiss N, Heydrick S, Cap A, Klings ES, Bierl C,
Eberhardt RT, Farber HW, Loscalzo J. Cellular glutathione peroxidase
deficiency and endothelial dysfunction. Am J Physiol Heart Circ Physiol.
2002;282:H1255–H1261.
43. Dayal S, Brown KL, Weydert CJ, Oberley LW, Arning E, Bottiglieri T,
Faraci FM, Lentz SR. Deficiency of glutathione peroxidase-1 sensitizes
hyperhomocysteinemic mice to endothelial dysfunction. Arterioscler
Thromb Vasc Biol. 2002;22:1996 –2002.
44. Shiomi T, Tsutsui H, Matsusaka H, Murakami K, Hayashidani S, Ikeuchi
M, Wen J, Kubota T, Utsumi H, Takeshita A. Overexpression of gluta-
 1340 Arterioscler Thromb Vasc Biol. July 2005
thione peroxidase prevents left ventricular remodeling and failure after
myocardial infarction in mice. Circulation. 2004;109:544 –549.
45. Forgione MA, Cap A, Liao R, Moldovan NI, Eberhardt RT, Lim CC,
Jones J, Goldschmidt-Clermont PJ, Loscalzo J. Heterozygous cellular
glutathione peroxidase deficiency in the mouse: abnormalities in vascular
and cardiac function and structure. Circulation. 2002;106:1154 –1158.
46. Weiss N, Zhang YY, Heydrick S, Bierl C, Loscalzo J. Overexpression of
cellular glutathione peroxidase rescues homocyst(e)ine-induced endothe-
lial dysfunction. Proc Natl Acad Sci U S A. 2001;98:12503–12508.
47. Blankenberg S, Rupprecht HJ, Bickel C, Torzewski M, Hafner G, Tiret L,
Smieja M, Cambien F, Meyer J, Lackner KJ. Glutathione peroxidase 1
activity and cardiovascular events in patients with coronary artery
disease. N Engl J Med. 2003;349:1605–1613.
48. Winter JP, Gong Y, Grant PJ, Wild CP. Glutathione peroxidase 1 geno-
type is associated with an increased risk of coronary artery disease. Coron
Artery Dis. 2003;14:149 –153.
49. Hamanishi T, Furuta H, Kato H, Doi A, Tamai M, Shimomura H,
Sakagashira S, Nishi M, Sasaki H, Sanke T, Nanjo K. Functional variants
in the glutathione peroxidase-1 (GPx-1) gene are associated with
increased intima-media thickness of carotid arteries and risk of macro-
vascular diseases in japanese type 2 diabetic patients. Diabetes. 2004;53:
2455–2460.
50. Freedman JE, Loscalzo J, Benoit SE, Valeri CR, Barnard MR, Michelson
AD. Decreased platelet inhibition by nitric oxide in two brothers with a
history of arterial thrombosis. J Clin Invest. 1996;97:979 –987.
51. Kenet G, Freedman J, Shenkman B, Regina E, Brok-Simoni F, Holzman
F, Vavva F, Brand N, Michelson A, Trolliet M, Loscalzo J, Inbal A.
Plasma glutathione peroxidase deficiency and platelet insensitivity to
nitric oxide in children with familial stroke. Arterioscler Thromb Vasc
Biol. 1999;19:2017–2023.
52. Voetsch B, Bierl C, Handy DE, Loscalzo J. Adverse functional conse-
quences of promoter polymorphisms in the plasma glutathione peroxidase
gene. Circulation. 2003;108:II-230.
53. Jin RC, Voetsch B, Handy DE, Loscalzo J. Promoter polymorphisms in
the plasma glutathione peroxidase (GPx-3) gene affect transcriptional
activation by hypoxia. FASEB J. 2004;18:C92.
Downloaded from http://ahajournals.org by on September 16, 2023
54. Voetsch B, Bierl C, Damasceno BP, Annichino-Bizzacchi JM, Arruda
VR, Loscalzo J. A novel promoter polymorphism in the plasma gluta-
thione peroxidase gene associated with arterial ischemic stroke in the
young. Circulation. 2000;102:II-704.
55. Voetsch B, Bierl C, Camargo ECS, Deus-Silva L, Ozelo MC, Massaro
AR, Annichino-Bizzacchi JM, Loscalzo J. Promoter polymorphisms in
the plasma glutathione peroxidase gene: a novel risk factor for cerebral
venous thrombosis among young patients. Neurology. 2003;60.
56. Miller EA, Pankow JS, Millikan RC, Bray MS, Ballantyne CM, Bell DA,
Heiss G, Li R. Glutathione-S-transferase genotypes, smoking, and their
association with markers of inflammation, hemostasis, and endothelial
function: the atherosclerosis risk in communities (ARIC) study. Athero-
sclerosis. 2003;171:265–272.
57. Izzotti A, Cartiglia C, Lewtas J, De Flora S. Increased DNA alterations in
atherosclerotic lesions of individuals lacking the GSTM1 genotype.
Faseb J. 2001;15:752–757.
58. Habdous M, Siest G, Herbeth B, Vincent-Viry M, Visvikis S. [Glutathione
S-transferases genetic polymorphisms and human diseases: overview of epi-
demiological studies]. Ann Biol Clin (Paris). 2004;62:15–24.
59. Ono K, Goto Y, Takagi S, Baba S, Tago N, Nonogi H, Iwai N. A promoter
variant of the heme oxygenase-1 gene may reduce the incidence of ischemic
heart disease in Japanese. Atherosclerosis. 2004;173:315–319.
60. Schillinger M, Exner M, Minar E, Mlekusch W, Mullner M, Mannhalter
C, Bach FH, Wagner O. Heme oxygenase-1 genotype and restenosis after
balloon angioplasty: a novel vascular protective factor. J Am Coll
Cardiol. 2004;43:950 –957.
61. Chen YH, Chau LY, Lin MW, Chen LC, Yo MH, Chen JW, Lin SJ. Heme
oxygenase-1 gene promotor microsatellite polymorphism is associated
with angiographic restenosis after coronary stenting. Eur Heart J. 2004;
25:39 – 47.
62. Ono K, Mannami T, Iwai N. Association of a promoter variant of the
haeme oxygenase-1 gene with hypertension in women. J Hypertens.
2003;21:1497–1503.
63. Naylor CE, Rowland P, Basak AK, Gover S, Mason PJ, Bautista JM,
Vulliamy TJ, Luzzatto L, Adams MJ. Glucose 6-phosphate dehydro-
genase mutations causing enzyme deficiency in a model of the tertiary
structure of the human enzyme. Blood. 1996;87:2974 –2982.
64. Leopold JA, Walker J, Scribner AW, Voetsch B, Zhang YY, Loscalzo AJ,
Stanton RC, Loscalzo J. Glucose-6-phosphate dehydrogenase modulates
vascular endothelial growth factor-mediated angiogenesis. J Biol Chem.
2003;278:32100 –32106.
65. Jain M, Cui L, Brenner DA, Wang B, Handy DE, Leopold JA, Loscalzo
J, Apstein CS, Liao R. Increased myocardial dysfunction after ischemia-
reperfusion in mice lacking glucose-6-phosphate dehydrogenase. Circu-
lation. 2004;109:898 –903.
66. Gaskin RS, Estwick D, Peddi R. G6PD deficiency: its role in the high
prevalence of hypertension and diabetes mellitus. Ethn Dis. 2001;11:
749 –754.
67. Forgione MA, Loscalzo J, Holbrook M, Scribner AW, Palmisano J,
Maxwell C, Baldwin C, Vita J. The A326G (A⫹) variant of the glucose-
6-phosphate dehydrogenase gene is associated with endothelial dys-
function in African Americans. J Am Coll Cardiol. 2003;41:249A.
68. Holmgren A. Antioxidant function of thioredoxin and glutaredoxin
systems. Antioxid Redox Signal. 2000;2:811– 820.
69. Tamer L, Ercan B, Camsari A, Yildirim H, Cicek D, Sucu N, Ates NA,
Atik U. Glutathione S-transferase gene polymorphism as a susceptibility
factor in smoking-related coronary artery disease. Basic Res Cardiol.
2004;99:223–229.
70. Hoekstra KA, Godin DV, Cheng KM. Protective role of heme oxygenase
in the blood vessel wall during atherogenesis. Biochem Cell Biol. 2004;
82:351–359.
71. Stadtman ER, Moskovitz J, Berlett BS, Levine RL. Cyclic oxidation and
reduction of protein methionine residues is an important antioxidant
mechanism. Mol Cell Biochem. 2002;234 –235:3–9.