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1 Evans Department of Medicine, 2 Whitaker Cardiovascular Institute, Department of Pharmacology, and 3 Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cellular glutathione peroxidase (GPx-1)
is the most abundant intracellular isoform of the GPx antioxidant
enzyme family. In this study, we hypothesized that GPx-1 deficiency
directly induces an increase in vascular oxidant stress, with resulting
endothelial dysfunction. We studied vascular function in a murine model
of homozygous deficiency of GPx-1 (GPx-1
/). Mesenteric
arterioles of GPx-1/ mice demonstrated paradoxical
vasoconstriction to 
-methacholine and bradykinin, whereas wild-type
(WT) mice showed dose-dependent vasodilation in response to both
agonists. One week of treatment of GPx-1/ mice with
L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal
vascular reactivity in the mesenteric bed of GPx-1/
mice. We observed an increase of the isoprostane
iPF2
-III, a marker of oxidant stress, in the plasma and
aortas of GPx-1/ mice compared with WT mice, which
returned toward normal after OTC treatment. Aortic sections from
GPx-1/ mice showed increased binding of an
anti-3-nitrotyrosine antibody in the absence of frank vascular lesions.
These findings demonstrate that homozygous deficiency of GPx-1 leads to
impaired endothelium-dependent vasodilator function presumably due to a
decrease in bioavailable nitric oxide and to increased vascular oxidant
stress. These vascular abnormalities can be attenuated by increasing
bioavailable intracellular thiol pools.
nitric oxide; peroxynitrite; oxidant stress
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO) synthesized by the endothelium contributes to vascular tone (23), preserves endothelial integrity (13), inhibits smooth muscle cell migration and proliferation (16), and acts as an antioxidant (30). An increase in reactive oxygen species (ROS), leading to increased oxidant stress in the vasculature, promotes endothelial dysfunction (28) associated with NO insufficiency (15).
The enzyme glutathione peroxidase (GPx) is a selenocysteine-containing
protein that serves an important role in the cellular defense against
oxidant stress (24) by utilizing reduced glutathione (GSH)
to reduce hydrogen peroxide (H2O2) and lipid
peroxides to their corresponding alcohols (37). GPx exists
in several isoforms, and the most abundant intracellular isoform is
cellular GPx, or GPx-1. We (7, 36, 39) have previously
shown that elevated homocysteine concentrations suppress GPx-1
expression in endothelial cells in vitro and in mildly
hyperhomocysteinemic mice in vivo and suggested that this effect may
account, in part, for the vascular oxidant stress of
hyperhomocysteinemic states. Hydrogen peroxide forms the toxic oxygen
species hydroxyl radical (·OH), which is highly reactive and causes
lipid peroxidation, and hydroxide anion (OH
), which promotes alkaline
tissue damage, a process that is offset in part by catalase and
GPx-1-dependent reduction to H2O. Elevated levels of lipid
peroxides are accompanied by an increase in peroxyl radicals, which can
inactivate NO through the formation of lipid peroxynitrites (19,
30), although the precise molecular mechanism(s) by which these
peroxyl radicals form remains speculative (19). Thus a
deficiency of GPx-1 would theoretically lead to an increase in ROS and
a decrease in bioavailable NO.
Because GSH represents one of the most important intracellular antioxidants, primarily as a cosubstrate for GPx-1, we hypothesized that this antioxidant system plays a central role in protecting the vasculature in states of increased oxidant stress. To test the hypothesis, we studied the role of this system in modulating endothelial function by using a murine model of targeted gene disruption of the GPx-1 gene.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents and animals.
All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise
stated. Mice homozygous for disruption of the GPx-1 gene
(GPx-1/ mice) were kindly provided by Dr. Y. Ho (Wayne
State University; Detroit, MI) and subsequently bred at our
institution. GPx-1 was inactivated by insertion of a neomycin
resistance gene (NEO) cassette into an EcoRI site located in
exon 2 of the GPx-1 gene, which was then inserted into mouse embryonic
stem cells as previously described (11).
1 · day1, a dose
known to affect hepatic GSH levels. Actual ingestion was determined by
weighing the drinking bottles daily.
All mice were killed during full anesthesia with pentobarbital by
exsanguination during the collection of blood and tissue. All
procedures were approved by the Institutional Animal Care and Use
Committee at Boston University Medical Center.
Genotype determination.
DNA was obtained by extraction from mouse tail snips. DNA (1 µl) was
amplified in a 50-µl PCR reaction containing 10 mM Tris · HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.1 mM dNTP, and 0.18 µM each primer. To identify the WT GPx-1 gene, we
utilized the forward primer FinN (5'-GTTTCCCGTGCAATCAGTTCG-3') and the
reverse primer R3N (5'-TCGGACGTACCCTTGAGGGAAT-3') to detect the presence of a 293-bp fragment. To identify GPx-1/
mice, we used FinN and RpgkN (5'-CATTTGTCACGTCCTGCAC-3') as the reverse
primer to amplify a 509-bp fragment in the NEO insert. Reaction
products were analyzed by electrophoresis on a 1% agarose gel.
GPx-1/ mice were identified by an exclusive 509-bp
product, and WT mice were identified by an exclusive 293-bp product.
Hepatic GPx-1 activity.
Liver samples were snap-frozen in liquid nitrogen and stored at
80°C. Tissue (0.5 g) was homogenized in an ice-cold buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA (pH 8), and 1 mM
dithiothreitol (DTT). The homogenate was centrifuged at 10,000 g for 20 min at 4°C. GPx activity was then determined from
the supernatant by coupling the reduction of peroxides and the
oxidation of glutathione with the reduction of oxidized glutathione by
glutathione reductase using NADPH as a cofactor (9). The
reaction was carried out in a buffer containing 50 mM
Tris · HCl, 5 mM EDTA, 1 mM glutathione, 0.4 U/ml glutathione
reductase, and 0.2 mM NADPH (pH 7.6) and initiated by the addition of
tert-butyl-hydroperoxide (0.22 mM final concentration). GPx activity
was monitored by a decrease in light absorbance with oxidation of NADPH
at 340 nm. Enzyme activity was calculated using a molar extinction
coefficient for NADPH of 6,220 M1 · cm1 and normalized to protein concentration.
Mesenteric microcirculation studies.
Vascular reactivity in the mesenteric circulation in response to the
endothelium-dependent agonists -methacholine (BMC) and bradykinin
(BK) and the endothelium-independent vasodilator sodium nitroprusside
(SNP) was assessed in vivo using videomicroscopy as previously
described (7, 35).
cGMP levels.
cGMP content was measured in isolated thoracic aortas as previously
described (2) with slight modifications. In anesthetized mice, the aortas were gently and antegradely perfused with normal saline via puncture of the left ventricle, the thoracic aortas were
then excised, and loose connective tissue of the adventitia was
removed. The tissue was weighed, placed in Dulbecco's PBS containing
Ca2+ and Mg2+ (GIBCO-BRL Life Technologies,
distributed by Invitrogen; Carlsbad, CA), and then supplemented with
0.068 mM EDTA, 0.14 mM ascorbate, and 11.1 mM glucose. The vessels were
preincubated at 37°C, aerated with 95% O2-5%
CO2 for 15 min, stimulated with BK (105 M)
for 1 min, snap-frozen in liquid nitrogen, and then stored at 80°C
until analysis. The tissue was homogenized in ice-cold 50 mM phosphate
buffer containing 7.5% (vol/vol) trichloroacetic acid and centrifuged
at 2,000 g for 10 min at 4°C. Trichloroacetic acid was
removed from the supernatant by extraction with water-saturated diethyl
ether. cGMP content in the supernatant was then measured using a
commercially available immunoassay (Cayman Chemical; Ann Arbor, MI)
according to the manufacturer's instructions.
Aortic endothelial NO synthase expression. Expression of endothelial NO synthase (eNOS) in aortic tissue was assessed by immunoblot analysis. Thoracic aortas were homogenized in 50 mM Tris · HCl buffer (pH 7.5) in the presence of EDTA (2 mM) and protease inhibitors (1 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride) and adjusted to equal protein concentrations in the same buffer. Samples were reduced and denatured by adding DTT (0.05 M final concentration) and lithium dodecyl sulfate sample buffer (Invitrogen) and by boiling for 5 min. Samples of 10 µg protein each were electrophoresed through Bis-Tris · HCl-buffered (pH 6.4) 12% polyacrylamide gels (NuPAGE, Invitrogen) and blotted on nitrocellulose filters. Blots were blocked in 5% skimmed milk in PBS-Tween (PBS-T; 1× PBS + 0.05% Tween) for at least 15 min, followed by an overnight incubation with a monoclonal antibody to eNOS (1:1,000, Signal Transduction Laboratories; Lexington, KY). Blots were washed three times for at least 15 min each in PBS-T and then incubated with a peroxidase-conjugated second antibody for 1 h (1:2,500, Signal Transduction Laboratories). Immunoblots were developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech; Piscataway, NJ). Equal loading of protein was confirmed by staining parallel gels with Coomassie brilliant blue or the filters with Ponceau S.
Plasma and tissue isoprostane determination.
Mice were killed and plasma was obtained by centrifugation of blood
collected in tubes containing a final concentration of 0.1 M EDTA,
snap-frozen in liquid nitrogen, and stored at 80°C. Tissue samples
were weighed as described previously (7), snap-frozen in
liquid nitrogen, and stored at 80°C.
-III was purified from plasma
samples. Plasma was diluted 1:15 in ultrapure water, and samples were applied to a reverse-phase C-18 column (Alltech; Deerfield, IL) at pH 3 and eluted with 1:1 (vol/vol) ethyl acetate-heptane. The eluant was
then further purified on a Silica column (Alltech) and eluted with 1:1
(vol/vol) ethyl acetate-methanol. iPF2-III was measured
from the eluant using a commercially available immunoassay (Cayman Chemical).
Tissue samples were minced, and iPF2-III was extracted
overnight using 2:1 (vol/vol) chloroform-methanol with 0.0005% butylated hydroxytoluene. Samples were filtered through glass wool, and
the organic phase was evaporated at 40°C under nitrogen. The dried
sample was resuspended in methanol, saponified using 15% KOH, and then
incubated for 60 min at 40°C. The sample was then adjusted to pH 2 and applied to the C-18 column as above. iPF2-III was
assayed from the eluant of the C-18 column as described above.
Tissue phospholipid hydroperoxide content. Measurement of phospholipid hydroperoxide levels in hepatic tissue was performed by HPLC using a LC-Si column as previously described (32).
Histological assessment.
Tissue, including the heart, aorta, and mesentery, was excised from
GPx-1/ and WT mice. The tissues were fixed in 4%
formalin, embedded in paraffin, and stained with hematoxylin and eosin
as well as Masson trichrome.
Immunohistochemistry for 3-nitrotyrosine. The production of reactive nitrogen species was assessed in aortic tissue by the presence of the stable endproduct of their interaction with cellular tyrosine residues, 3-nitrotyrosine, as previously described (7, 10).
Data analysis. Continuous data are expressed as means ± SE. The Kruskal-Wallis test was used for multigroup comparisons of continuous variables, followed by Mann-Whitney U-tests to compare differences between two groups. Differences in the dose response to agonists between groups were tested using two-way repeated-measures ANOVA with post hoc analysis performed using Scheffé's F-test and Bonferroni-Dunn methods. Statistical significance was defined as a P value <0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hepatic GPx-1 activity.
Hepatic cellular GPx activity was undetectable in
GPx-1/ mice compared with WT mice (1.5 ± 0.9 vs. 579.4 ± 6.1 mU/mg protein, P < 0.0001).
Mesenteric vascular reactivity.
Superfusion with BMC and BK produced dose-dependent vasodilation
of mesenteric arterioles in WT mice, with a maximal vascular response
(MVR) of 10.0 ± 0.7% (BMC) and 12.2 ± 1.3% (BK),
respectively, at concentrations of 104 M and
105 M, respectively. However, superfusion of mesentery
from GPx-1/ mice with either endothelium-dependent
agonist resulted in paradoxical arteriolar vasoconstriction, with a MVR
of 14.0 ± 0.7% (BMC) and 10.1 ± 1.2% (BK),
respectively (Fig. 1,
A and B), that was statistically different from the response of WT mice (P < 0.0001). Superfusion of the mesentery with SNP resulted in
dose-dependent arteriolar vasodilation that was similar in the two
groups of animals (Fig. 1C). These results indicate that
GPx-1/ mice have impaired endothelium-dependent
vasodilation and intact endothelium-independent vasodilation in
mesenteric arterioles, suggesting a depletion of bioavailable NO.
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Aortic cGMP levels and eNOS expression.
Aortic tissue from GPx-1/ mice tended to accumulate
less cGMP upon stimulation with bradykinin for 1 min compared with
aortic tissue from WT mice (0.38 ± 0.07 vs. 0.80 ± 0.12 pmol/ml tissue, P < 0.02; Fig.
2). This effect was independent of aortic
eNOS expression because immunoblot analysis of eNOS protein did not show any appreciable difference between GPx-1/ and WT
mice (data not shown).
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Isoprostanes/hepatic phospholipid hydroperoxides.
iPF2-III levels were elevated in the plasma and aortas
of 16- to 20-wk-old GPx-1/ mice compared with WT mice
[plasma: 168.4 ± 25.4 vs. 84.6 ± 7.6 pg/ml,
n = 6 mice/group, P < 0.005 (Fig.
3A); and aortas: 13.3 ± 2.8 vs. 8.4 ± 1.1 pg/mg tissue, n = 6 mice/group,
P < 0.01 (Fig. 3B)]. By 40 wk of age,
plasma iPF2-III increased further to 214.0 ± 18.0 pg/ml in GPx-1/ and 100.5 ± 5.4 pg/ml in WT mice
(n = 6 mice/group, P < 0.001). Aortic
iPF2-III levels did not increase further (14.9 ± 1.8 vs. 8.2 ± 0.5 pg/mg tissue, respectively, for
GPx-1/ and WT mice, n = 6 mice/group,
P < 0.01). Cardiac iPF2-III also
increased in GPx-1/ mice compared with WT mice
(17.2 ± 0.4 vs. 5.6 ± 1.1 pg/mg tissue, n = 6 mice/group, P < 0.001), as did hepatic
iPF2-III levels (1.8 ± 0.2 pg/mg tissue for
GPx-1/ mice vs. 0.6 ± 0.2 pg/mg tissue for WT
mice, n = 6 mice/group, P < 0.01).
Hepatic phospholipid hydroperoxides were also elevated in
GPx-1/ mice compared with WT mice almost twofold
(199 ± 31% vs. 100 ± 9%, n = 6 mice/group, P < 0.05).
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Effect of OTC treatment.
Treatment of mice with OTC resulted in an increase in hepatic GPx-1
activity in WT mice (679.4 ± 17.6 vs. 579.4 ± 6.1 mU/mg protein for untreated, P < 0.01) but did not affect
enzyme activity in GPx-1/ mice (1.7 ± 2.5 vs.
1.5 ± 0.9 mU/mg protein for untreated).
/ mice restored normal
dose-dependent arteriolar vasodilation to BMC (MVR = 14.8 ± 3.0%) and BK (MVR = 13.1 ± 1.9%), which was similar to the
response observed in WT mice treated with OTC (MVR = 11.8 ± 9% for BMC and 11.2 ± 0.9% for BK; Fig. 1, A and
B). There was no significant difference in the vascular
reactivity with superfusion of SNP in either group of mice (Fig.
1C).
GPx-1/ mice tended to accumulate less cGMP in aortic
tissue compared with WT mice. cGMP levels in aortic tissue from
OTC-treated GPx-1/ mice (0.082 ± 0.08 pmol/mg
tissue), however, were not different from the levels in WT mice and in
OTC-treated WT mice (0.79 ± 0.07 pmol/mg tissue; Fig. 2). OTC
treatment did not affect aortic eNOS expression, which was similar in
WT and GPx-1/ mice without and with OTC treatment (data
not shown).
OTC treatment also resulted in a significant reduction of plasma
iPF2-III (96.7 ± 16.0 pg/ml, P < 0.05 compared with untreated GPx-1/ mice; Fig.
3A) and a trend to reduction in aortic
iPF2-III (8.7 ± 3.4 pg/mg tissue for OTC-treated
GPx-1/ mice vs. 13.3 ± 2.8 pg/mg tissue for
untreated GPx-1/ mice, P = 0.074; Fig.
3B).
Histological assessment and 3-nitrotyrosine staining.
There were no appreciable differences in aortic morphology by
hematoxylin and eosin or Masson trichrome staining in the
GPx-1/ mice compared with WT mice. However,
immunostaining of aortic tissue with an 3-nitrotyrosine antibody showed
greater staining for 3-nitrotyrosine in GPx-1/ mice
compared with WT mice (Fig. 4). This
increase in staining was primarily on the endothelial surface and
adventitia, supporting increased reactive nitrogen species formation at
these sites.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We found that homozygous deficiency of the GPx-1 gene produces
vascular dysfunction as well as oxidative and nitrosative stress. The
vascular dysfunction we observed was detected in response to mesenteric
superfusion with BMC and BK, not SNP, indicating that the aberrant
microvascular function is endothelium dependent. The trend to lower
cGMP accumulation in aortic tissue from GPx-1/ mice
after stimulation with BK indicates a decrease in bioavailable endothelium-derived NO, which is not caused by decreased expression of
eNOS protein, as indicated by immunoblot analysis.
NO plays a critical role in the endothelial response to muscarinic agonists (20). In normal arteries, muscarinic agonists stimulate NO release, which attenuates the muscarinic agonist-induced vasoconstrictor effect and causes vascular relaxation. However, with a depletion of bioavailable NO in the presence of increased oxidant stress, vasoconstriction occurs because there is unopposed muscarinic stimulation of vascular smooth muscle cells. Abnormal endothelial responses to muscarinic agonists have been shown to be a marker for the development of atherothrombosis (18) as well as a risk factor for a worse prognosis in the presence of atherosclerotic vascular disease (18, 31, 34). Because endothelial response to a direct vasodilator, SNP, was similar between both groups, we can infer that the effect of endothelial dysfunction from homozygous GPx-1 deficiency is not a consequence of impaired vascular smooth muscular function. Therefore, a depletion of bioavailable NO likely plays a role in the endothelial dysfunction that occurs in GPx-1 deficiency. Importantly, this dysfunction occurs in the absence of frank structural vascular lesions, suggesting that the endothelial dysfunction may precede the development of vascular lesions.
Because GPx-1 plays a central role in defending the cell from ROS, its
deficiency should lead to increased oxidant stress in the cell. GPx-1
has a much higher Michaelis-Menten constant for
H2O2 than catalase (12), the other
important cellular enzyme involved in the detoxification of
H2O2. In endothelial cells, 70% of
H2O2 generated by activated polymorphonuclear
leukocytes is detoxified by GPx-1 (6); importantly, there
is no change in catalase activity in GPx-1/ mice
(11). Chemical inhibition of GPx-1 activity in macrophages results in increased cellular peroxide formation and increased superoxide (O
/ mice have decreased survival compared with WT
mice after a lethal injection of the xenobiotic paraquat
(3), which induces formation of O
Increased ROS in the presence of GPx-1 deficiency induces lipid
peroxidation, which we evaluated by measuring the isoprostane iPF2-III and hepatic phospholipid hydroperoxides.
Isoprostanes are formed largely (but not exclusively) from the
non-cyclooxygenase (COX)-dependent peroxidation of arachidonic acid
(14, 22) and serve as an index of oxidant stress. The
isoprostane iPF2-III has been shown to be elevated in
several conditions associated with endothelial dysfunction, such as
diabetes mellitus (5) and hypercholesterolemia (4,
26). Isoprostanes may contribute to vascular pathology by
serving as mitogens (14) and vasoconstrictors (22). There is some capacity for iPF2-III
to form in a COX-dependent fashion; however, urinary
iPF2-III excretion in diabetics (5) was
unaffected by the administration of a reversible or an irreversible COX
inhibitor, whereas urinary 11-dehydro-thromboxane B2 (a
thromboxane metabolite that depends on COX for its formation) excretion
was decreased. Riley and colleagues (25) have demonstrated similar results using an irreversible COX inhibitor in cigarette smokers (25), as has McAdam and co-workers
(17) in normal volunteers given endotoxin. Thus the
enzymatic formation of isoprostanes may be of little relevance in vivo.
GPx-1/ mice had significantly increased levels of
iPF2-III compared with WT mice in aortic tissue and
plasma. GPx-1/ mice also had increased phospholipid
hydroperoxide levels in the liver compared with WT mice, consistent
with a previous report (8) of GPx-1 deficiency that was
generated in a different fashion than this model. These two indexes of
lipid peroxidation reflect the increased oxidant stress occurring
throughout the lifetime of these animals and may contribute to the
aforementioned endothelial dysfunction.
Peroxynitrite (ONOO) and peroxynitrous acid are potent
oxidants and reactive nitrogen species that are formed from the
reaction of NO with O/ mice compared with WT mice. GSH has been shown
in vitro to be dependent on GPx to defend against peroxynitrite
toxicity (peroxynitrite reductase activity) (33); our
observations suggest the same in vivo, because these immunostaining
experiments show that there is increased nitrosative stress in
GPx-1/ mice.
The administration of OTC increases hepatic GSH (41), cysteine, and glutathione levels in vascular tissue, plasma (1), and lymphocytes (21) as well as total blood cysteine concentration (38). OTC is transported into cells and converted to cysteine by the enzyme 5-oxoprolinase (40); thus OTC represents an effective way to increase intracellular thiol pools.
OTC treatment reversed the vasoconstrictor response to superfusion with
BMC and BK observed in the mesenteric bed of GPx-1/
mice and the decrease in aortic cGMP accumulation after stimulation with BK, indicating an increase in bioavailable NO. Vita and colleagues (38) have reported an improvement in NO-dependent brachial
artery responses to shear stress as measured by ultrasound after OTC treatment in patients with coronary artery disease. OTC treatment also
decreased plasma iPF2-III and demonstrated a trend
toward a decrease of aortic iPF2-III in
GPx-1/ mice. Preliminary data show that OTC treatment
significantly increased total low-molecular-weight thiols in cardiac
tissue extracts by 36% from GPx-1/ mice
(P < 0.01 compared with untreated
GPx-1/ controls). This indicates that increasing
intracellular thiol pools (principally glutathione) decreases the
amount of oxidant stress present with GPx-1/ deficiency
and may contribute to the normalization of endothelial function seen
after OTC treatment by minimizing oxidative inactivation of NO.
In summary, mice with homozygous deficiency of GPx-1 have impaired endothelium-dependent vascular reactivity in resistance vessels in the presence of increased oxidative and nitrosative stress. Increasing intracellular thiol pools attenuates the increased oxidant stress and reverses the endothelial dysfunction in resistance vessels induced by homozygous deficiency of GPx-1. We conclude that GPx-1 contributes to the maintenance of a normal endothelial function as well as protects against oxidative and nitrosative stress in the blood vessel.
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ACKNOWLEDGEMENTS |
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We thank Stephanie Tribuna and Katherine Seropian for assistance in the preparation of this manuscript and Anne Ward Scribner for technical assistance.
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FOOTNOTES |
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* M. A. Forgione and N. Weiss contributed equally to this work.
First published December 6, 2001;10.1152/ajpheart.00598.2001
This work was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-55993, HL-58976, and HL-61795. M. A. Forgione was supported by NHLBI Cardiovascular Training Grant HL-07224-24. N. Weiss was supported by Deutsche Forschungsgemeinschaft Grant WE 1984/2-1.
These results were presented in part at the American Heart Association 72nd Annual Scientific Sessions, November 1999, in Atlanta, GA, and Experimental Biology '99 meeting, April 1999, in Washington, DC.
Present address of N. Weiss: Klinikum der Universität München, Medizinische Poliklinik-Innenstadt, Munich, Germany D-80336.
Address for reprint requests and other correspondence: J. Loscalzo, Boston Univ. School of Medicine, 715 Albany St., W507, Boston, MA 02118 (E-mail: jloscalz{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2001; accepted in final form 19 November 2001.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Boesgaard, S,
Aldershvile J,
Poulsen HE,
Loft S,
Anderson ME,
and
Meister A.
Nitrate tolerance in vivo is not associated with depletion of arterial or venous thiol levels.
Circ Res
74:
115-120,
1994.
2.
Bossaller, C,
Habib GB,
Yamamoto H,
Williams C,
Wells S,
and
Henry PD.
Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta.
J Clin Invest
79:
170-174,
1987.
3.
Cheng, WH,
Ho YS,
Valentine BA,
Ross DA,
Combs GF, Jr,
and
Lei XG.
Cellular glutathione peroxidase is the mediator of body selenium to protect against paraquat lethality in transgenic mice.
J Nutr
128:
1070-1076,
1998.
4.
Davi, G,
Alessandrini P,
Mezzetti A,
Minotti G,
Bucciarelli T,
Costantini F,
Cipollone F,
Bon GB,
Ciabattoni G,
and
Patrono C.
In vivo formation of 8-epi-prostaglandin F2 alpha is increased in hypercholesterolemia.
Arterioscler Thromb Vasc Biol
17:
3230-3235,
1997.
5.
Davi, G,
Ciabattoni G,
Consoli A,
Mezzetti A,
Falco A,
Santarone S,
Pennese E,
Vitacolonna E,
Bucciarelli T,
Costantini F,
Capani F,
and
Patrono C.
In vivo formation of 8-iso-prostaglandin F2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation.
Circulation
99:
224-229,
1999.
6.
Dobrina, A,
and
Patriarca P.
Neutrophil-endothelial cell interaction. Evidence for and mechanisms of the self-protection of bovine microvascular endothelial cells from hydrogen peroxide-induced oxidative stress.
J Clin Invest
78:
462-471,
1986.
7.
Eberhardt, RT,
Forgione MA,
Cap A,
Leopold JA,
Rudd MA,
Trolliet M,
Heydrick S,
Stark R,
Klings ES,
Moldovan NI,
Yaghoubi M,
Goldschmidt-Clermont PJ,
Farber HW,
Cohen R,
and
Loscalzo J.
Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.
J Clin Invest
106:
483-491,
2000.
8.
Esposito, LA,
Kokoszka JE,
Waymire KG,
Cottrell B,
MacGregor GR,
and
Wallace DC.
Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene.
Free Radic Biol Med
28:
754-766,
2000.
9.
Flohé, L,
and
Günzler WA.
Assays of glutathione peroxidase.
Methods Enzymol
105:
114-121,
1984.
10.
Hammerman, SI,
Klings ES,
Hendra KP,
Upchurch GR, Jr,
Rishikof DC,
Loscalzo J,
and
Farber HW.
Endothelial cell nitric oxide production in acute chest syndrome.
Am J Physiol Heart Circ Physiol
277:
H1579-H1592,
1999.
11.
Ho, YS,
Magnenat JL,
Bronson RT,
Cao J,
Gargano M,
Sugawara M,
and
Funk CD.
Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia.
J Biol Chem
272:
16644-16651,
1997.
12.
Jones, DP,
Eklow L,
Thor H,
and
Orrenius S.
Metabolism of hydrogen peroxide in isolated hepatocytes: relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H2O2.
Arch Biochem Biophys
210:
505-516,
1981.
13.
Kurose, I,
Kubes P,
Wolf R,
Anderson DC,
Paulson J,
Miyasaka M,
and
Granger DN.
Inhibition of nitric oxide production: mechanisms of vascular albumin leakage.
Circ Res
73:
164-171,
1993.
14.
Lawson, JA,
Rokach J,
and
Fitzgerald GA.
Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo.
J Biol Chem
274:
24441-24444,
1999.
15.
Levine, GN,
Keaney JF, Jr,
and
Vita JA.
Cholesterol reduction in cardiovascular disease. Clinical benefits and possible mechanisms.
N Engl J Med
332:
512-521,
1995.
16.
Marks, DS,
Vita JA,
Folts JD,
Keaney JF, Jr,
Welch GN,
and
Loscalzo J.
Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide.
J Clin Invest
96:
2630-2638,
1995.
17.
McAdam, BF,
Mardini IA,
Habib A,
Burke A,
Lawson JA,
Kapoor S,
and
Fitzgerald GA.
Effect of regulated expression of human cyclooxygenase isoforms on eicosanoid and isoeicosanoid production in inflammation.
J Clin Invest
105:
1473-1482,
2000.
18.
McLenachan, JM,
Williams JK,
Fish RD,
Ganz P,
and
Selwyn AP.
Loss of flow-mediated endothelium-dependent dilation occurs early in the development of atherosclerosis.
Circulation
84:
1273-1278,
1991.
19.
O'Donnell, VB,
and
Freeman BA.
Interactions between nitric oxide and lipid oxidation pathways.
Circ Res
88:
12-21,
2000.
20.
Palmer, RM,
Ferrige AG,
and
Moncada S.
Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
Nature
327:
524-526,
1987.
21.
Porta, P,
Aebi S,
Summer K,
and
Lauterburg BH.
L-2-Oxothiazolidine-4-carboxylic acid, a cysteine prodrug: pharmacokinetics and effects on thiols in plasma and lymphocytes in human.
J Pharmacol Exp Ther
257:
331-334,
1991.
22.
Pratico, D.
F(2)-isoprostanes: sensitive and specific non-invasive indices of lipid peroxidation in vivo.
Atherosclerosis
147:
1-10,
1999.
23.
Quyyumi, AA,
Dakak N,
Anrews NP,
Husain S,
Arora S,
Gilligan DM,
Panza JA,
and
Cannon RO, III.
Nitric oxide activity in the human coronary circulation. Impact of risk factors for coronary atherosclerosis.
J Clin Invest
95:
1747-1755,
1995.
24.
Raes, M,
Michiels C,
and
Remacle J.
Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase.
Free Radic Biol Med
3:
3-7,
1987.
25.
Reilly, M,
Delanty N,
Lawson JA,
and
Fitzgerald GA.
Modulation of oxidant stress in vivo in chronic cigarette smokers.
Circulation
94:
19-25,
1996.
26.
Reilly, MP,
Pratico D,
Delanty N,
DiMinno G,
Tremoli E,
Rader D,
Kapoor S,
Rokach J,
Lawson J,
and
Fitzgerald GA.
Increased formation of distinct F2 isoprostanes in hypercholesterolemia.
Circulation
98:
2822-2828,
1998.
27.
Rosenblat, M,
and
Aviram M.
Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDL: in vitro and in vivo studies.
Free Radic Biol Med
24:
305-317,
1998.
28.
Ross, R.
Atherosclerosis-an inflammatory disease.
N Engl J Med
340:
115-126,
1999.
29.
Rubbo, H,
and
Freeman BA.
Nitric oxide regulation of lipid oxidation reactions: formation and analysis of nitrogen-containing oxidized lipid derivatives.
Methods Enzymol
269:
385-394,
1996.
30.
Rubbo, H,
Radi R,
Trujillo M,
Telleri R,
Kalyanaraman B,
Barnes S,
Kirk M,
and
Freeman BA.
Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.
J Biol Chem
269:
26066-26075,
1994.
31.
Schachinger, V,
Britten MB,
and
Zeiher AM.
Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease.
Circulation
101:
1899-1906,
2000.
32.
Shwaery, GT,
Samii JM,
Frei B,
and
Keaney JF, Jr.
Determination of phospholipid oxidation in cultured cells.
Methods Enzymol
300:
51-57,
1999.
33.
Sies, H,
Sharov VS,
Klotz LO,
and
Briviba K.
Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase.
J Biol Chem
272:
27812-27817,
1997.
34.
Suwaidi, JA,
Hamasaki S,
Higano ST,
Nishimura RA,
Holmes DR, Jr,
and
Lerman A.
Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction.
Circulation
101:
948-954,
2000.
35.
Suzuki, H,
Zweifach BW,
and
Schmid-Schonbein GW.
Vasodilator response of mesenteric arterioles to histamine in spontaneously hypertensive rats.
Hypertension
26:
397-400,
1995.
36.
Upchurch, GR, Jr,
Welch GN,
Fabian AJ,
Freedman JE,
Johnson JL,
Keaney JF, Jr,
and
Loscalzo J.
Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.
J Biol Chem
272:
17012-17017,
1997.
37.
Ursini, F,
Maiorino M,
Brigelius-Flohe R,
Aumann KD,
Roveri A,
Schomburg D,
and
Flohe L.
Diversity of glutathione peroxidases.
Methods Enzymol
252:
38-53,
1995.
38.
Vita, JA,
Frei B,
Holbrook M,
Gokce N,
Leaf C,
and
Keaney JF, Jr.
L-2-Oxothiazolidine-4-carboxylic acid reverses endothelial dysfunction in patients with coronary artery disease.
J Clin Invest
101:
1408-1414,
1998.
39.
Weiss, N,
Zhang Y,
and
Loscalzo J.
Homocyst(e)ine impairs cellular glutathione peroxidase expression and promotes endothelial dysfunction in an animal model of hyperhomocyst(e)inemia (Abstract).
Circulation
102:
II-238,
2000.
40.
Williamson, JM,
Boettcher B,
and
Meister A.
Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis.
Proc Natl Acad Sci USA
79:
6246-6249,
1982.
41.
Williamson, JM,
and
Meister A.
Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate.
Proc Natl Acad Sci USA
78:
936-939,
1981.
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P < 0.01 compared with WT mice at each specific
concentration of agonist.
