(Circulation. 1999;99:2027-2033.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Increased NADH-Oxidase–Mediated Superoxide Production in the Early Stages of Atherosclerosis
Evidence for Involvement of the Renin-Angiotensin System
From the Abteilung für Kardiologie, Universitäts-Krankenhaus Eppendorf, University of Hamburg (A.W., E.S., R.M., J.H.B., M.S., T.H., T. Meinertz, T. Münzel); the Klinik III für Innere Medizin, University of Cologne (G.N., M.B.); and Bayer Leverkusen, Wuppertal (J.P.S.), Germany; and the Division of Cardiology, Emory University, Atlanta, Ga (K.K.G., D.G.H.).
Correspondence to Thomas Münzel, MD, Abteilung für Kardiologie, Universitäts-Krankenhaus Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany. E-mail muenzel{at}uke.uni-hamburg.de
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Abstract |
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Background—Angiotensin II activates NAD(P)H-dependent oxidases via AT1-receptor stimulation, the most important vascular source of superoxide (O2·-). The AT1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O2·- production and whether AT1-receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction.
Methods and Results—Vascular responses were determined by isometric tension studies, and relative rates of vascular O2·- production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT1-receptor mRNA was quantified by Northern analysis, and AT1-receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O2·- production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O2·- production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT1-receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O2·- and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT1 receptor mRNA was upregulated to 166±11%, accompanied by a comparable increase in AT1-receptor density.
Conclusions—Hypercholesterolemia is associated with AT1-receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O2·- production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT1-receptor antagonist suggests a crucial role of angiotensin II–mediated O2·- production in the early stage of atherosclerosis.
Key Words: superoxide • hypercholesterolemia • magnetic resonance spectroscopy • angiotensin
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Introduction |
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Clinical and experimental studies have identified a marked attenuation in endothelium-dependent vasodilatation as a characteristic of early stages of atherosclerosis.1 2 In some cases, this is related to enhanced inactivation of endothelium-derived nitric oxide (·NO) by superoxide (O2·-)3 rather than a consequence of decreased NO production.4 Recently, we found that incubation of cultured smooth muscle cells with LDL enhances AT1-receptor expression.5 Activation of the AT1 receptor activates membrane-associated NADH-dependent oxidase,6 7 the predominant source of O2·- in vascular cells.6 8 There is also increased expression of ACE in atherosclerotic lesions,9 which may serve as a source for local production of angiotensin II and ultimately increased stimulation of vascular O2·- production. Indeed, long-term treatment with the ACE inhibitor quinapril markedly improved endothelial vasomotor function in patients with coronary artery disease (Trial on Reversing Endothelial Dysfunction, TREND),10 possibly because of decreased O2·--mediated inactivation of NO.
On the basis of these considerations, the present study was designed to test whether membrane-bound NADH oxidase is activated in hypercholesterolemia and to examine mechanisms responsible for this phenomenon.
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Methods |
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Animals and Protocols
A total of 92 rabbits were studied. The study consisted of 3 protocols.
Protocol 1
Ten male New Zealand White rabbits and 10 male Watanabe rabbits
(hypercholesterolemia secondary to an
LDL-receptor defect; age, 2 to 3 months) were used to measure
endothelial function and
O2·- in intact vessels and NADH-oxidase
activity in vessel homogenates.
Protocol 2
Forty New Zealand White rabbits were fed either a normal diet or
a diet containing 0.5% cholesterol diet for 8 weeks. Half
of these animals received concomitant treatment with a recently
developed AT1-receptor antagonist,
Bay 10-6734.11 Bay 10-6734 was mixed with the diet to
achieve a daily dose of 25 mg · kg-1
· d-1.
Protocol 3
Thirty-two New Zealand White rabbits were fed either a control
or 0.5% cholesterol diet. Half of these received
amlodipine 5 mg · kg-1 ·
d-1 PO for 8 weeks, mixed with the diet, to test
whether a hypotensive agent other than an angiotensin II
receptor antagonist affects vascular
O2·- production and
endothelial function.
Vessel Preparation
On the day of the study, blood was drawn for determination of
total cholesterol levels. Thereafter, the animals were
given 1000 U heparin IV and sodium pentobarbital to produce death. The
chest was rapidly opened and the descending thoracic aorta removed.
Aortic rings were suspended in organ chambers as described
previously.12 Vasodilator responses were determined after
preconstriction with phenylephrine to 30% to 50% of
maximal (KCl-induced) tone.
Estimation of Vascular O2·-
Production and NADH/NADPH-Oxidase Activity
Vascular O2·- was estimated with
lucigenin-enhanced chemiluminescence (lucigenin concentration, 250
µmol/L) as previously described.12 Some vessels from
Watanabe rabbits were incubated with either diphenylene iodonium
10 µmol/L, oxypurinol 100 µmol/L,
NG-nitro-L-arginine
(L-NNA) 10 µmol/L, or rotenone 100 µmol/L to examine the
potential role of flavin-containing oxidases, xanthine oxidase, ·NO
synthase, and mitochondrial respiration enzymes, respectively. We also
examined the effect of the O2·- scavenger
Tiron 10 mmol/L on vascular O2·-. To
validate data obtained with high lucigenin concentrations,
chemiluminescence studies with low concentrations of lucigenin (5
µmol/L)13 and a cypridina luciferin analogue (CLA,
1 µmol/L),14 were performed. We also quantified
vascular O2·- with electron spin resonance
(ESR) studies using the O2·--specific spin
trap 1-hydroxy-3-carboxy-pyrrolidine.15 Intensities of ESR
signals were quantified by measurement of magnitudes of the low-field
component of triplet ESR signal of the carboxy-pyrrolidine
radical with a dual-probe resonator and an ESR standard probe (Bruker).
Settings of the ESR spectrometer (ECS 106, Bruker) were as recently
described.14
To measure NADH/NADPH-oxidase activity, thoracic aortas were homogenized as described.12 In some experiments, the endothelium of the vessel was mechanically removed before homogenization.
Morphometric Analysis
The macroscopic area detected by positive fat stain and
histological parameters of the plaque were
determined with a Zeiss/Kontron morphometry unit and KS 400 software.
The descending thoracic aorta was cut open and stained for 15 minutes
in "Fettrot" solution (Fettrot 7B, No. 1A727, Chroma). The vessels
were then washed in 50% ethanol for 10 minutes. The luminal surfaces
were photographed, scanned, and digitized. The red-stained area was
planimetered and expressed as a percentage of the total luminal
surface.
For histochemical analysis, vessels were fixed in 4% neutral buffered formaldehyde and mounted in paraffin blocks. Sections were stained with hematoxylin and eosin and Masson's trichrome stain. Immunohistochemical staining for macrophages was carried out with the monoclonal antibody RAM 11 (Dako) and the ABC technique with peroxidase/DAB detection by a Vecstatin standard kit (Vector Laboratories). Morphometric analysis of 8 aortic cross sections were performed at three 1-mm cross-sectional steps of each paraffin block and included the percent value of macrophage staining for plaques (% macrophage/stained area to total plaque cross-sectional area).
mRNA Isolation and Northern Analysis
Northern blots were performed as described
previously.16 Briefly, aortic segments were
homogenized and total RNA was isolated with
RNA-clean according to the manufacturer's protocol. Aliquots
(10 µg) were electrophoresed through agarose-formaldehyde gels and
transferred onto Hybond N membranes (Amersham). Northern blots were
prehybridized for 2 hours at 42°C and then hybridized for 15 hours at
42°C with a random-primed, [32P]dCTP-labeled
rat AT1-receptor cDNA probe.
Radioligand Binding Assays
Binding assays on homogenized aortas were performed
in 25 mmol/L Tris-HCl (pH 7.4), 5 mmol/L
MgCl2, and 100 mmol/L NaCl. Saturation
binding assays were conducted with increasing amounts of
125I-labeled angiotensin II or
[3H]prazosin (Amersham) as described
previously. Total and nonspecific binding points were measured in
duplicate. Nonspecific binding was determined in the presence of
10 µmol/L of the AT1-receptor
antagonist TCV-116 (Takeda). The samples were incubated for
90 minutes at 22°C, followed by rapid aspiration through Whatman GF/C
filters. Samples were counted in a gamma counter.
Materials
All chemicals were purchased from Sigma Chemical Co.
[32P]dCTP, Hybond N nylon membranes, and
125I-labeled angiotensin II were
obtained from Amersham. RNA-Clean was from AGS GmbH.
Statistical Analysis
Results are expressed as mean±SEM. The
ED50 value for each experiment was obtained by
logit transformation. To compare NADH- and NADPH-driven
O2·- production in normal and
hypercholesterolemic vessels, 1-way ANOVA was used.
Comparisons of vascular responses were performed by
multivariate ANOVA. A Scheffé post hoc test was
used to examine differences between groups when significance was
indicated. Probability values <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Plasma Lipid Profile
The total plasma cholesterol in Watanabe rabbits averaged 603±45 mg/dL and was therefore significantly higher than in controls (32±3 mg/dL). In cholesterol-fed rabbits (0.5% cholesterol for 8 weeks), the total cholesterol level was significantly increased (1362±92 versus 32±3 mg/dL).
Effects of Hyperlipidemia on Vasodilator Responses
to Acetylcholine and Nitroglycerin
Compared with control vessels, maximal relaxations to the
endothelium-dependent vasodilator acetylcholine were
significantly impaired in vessels from Watanabe rabbits, whereas the
sensitivity, as reflected by the ED50, was not
altered (Table 1
). The sensitivity
and maximal relaxations to the endothelium-independent
vasodilator nitroglycerin were comparable in both
groups (Table 1).
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Effects of Hyperlipidemia on Vasoconstriction to
Angiotensin II and Phenylephrine
Constrictions to angiotensin II were enhanced in
vessels from Watanabe rabbits compared with control vessels. In
contrast, vasoconstrictor responses to phenylephrine were
similar between control and Watanabe rabbits (Figure 1).
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O2·- Production by Aortas From
Watanabe and Control Rabbits
Rates of O2·- production,
estimated by lucigenin-enhanced chemiluminescence, were increased by
2-fold in aortic segments from Watanabe rabbits compared with
controls. This increase was abolished by denudation of the
endothelium (Figure 2A).
Diphenylene iodonium (DPI), the radical scavenger Tiron, and
oxypurinol, an inhibitor of xanthine oxidase, markedly
inhibited O2·- in tissue from
hyperlipidemic animals, whereas L-NNA and rotenone had
no effect (Figure 2B). The effect of DPI was significantly
greater than that of oxypurinol, suggesting that an enzyme other than
xanthine oxidase was contributing to the signal (Figure 2B).
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P<0.05 vs
Watanabe+endothelium. B, Superoxide-dependent,
lucigenin-enhanced chemiluminescence of intact aortic rings with
endothelium of Watanabe rabbits. Data are from 4 to 6
experiments. *P<0.05 vs Watanabe aortic rings without
preincubation,
Effect of Hyperlipidemia on Vascular NADH-Dependent
Oxidase Activity in Watanabe Rabbits
NADH oxidase activity, assessed by addition of NADH to the
vascular homogenates, was significantly increased in
Watanabe rabbits compared with controls. In contrast, NADPH-oxidase
activity was similar in the 2 groups (Figure 3A). Endothelial removal
decreased NADH-oxidase activity from 5.5±0.5 to 3.5±0.3 nmol
O2·- · mg-1
· min-1 (P<0.05) in
hypercholesterolemic vessels. NADH-dependent activity
in both control and hyperlipidemic animals was located
predominantly (>90%) in the particulate fraction (Figure 3B).
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Effects of AT1-Receptor Blockade on Plasma Lipid
Profile, Endothelial Function, and Vascular
O2·- in Cholesterol-Fed Animals
AT1-receptor blockade had no effect on
cholesterol levels in either control (32±3 versus 28±3
mg/dL) or hyperlipidemic (1362±92 versus 1312±121
mg/dL) animals. In cholesterol-fed rabbits,
endothelium-dependent vasodilation was reduced, whereas
endothelium-independent vasorelaxation was preserved
(Figure 4A and 4B). Furthermore, a
significant activation of NADH-dependent, membrane-associated oxidase
(Figure 5A and 5B) was observed.
AT1-receptor blockade (25 mg ·
kg-1 · d-1 PO) for
8 weeks inhibited sensitivity and potency of angiotensin II
with respect to control vessels and
hypercholesterolemic animals, compatible with a
sufficient blockade of the AT1 receptor (Table 2). In cholesterol-fed
animals, AT1-receptor blockade reduced
O2·- production in intact rings and
inhibited NADH-oxidase activity (Figure 5A and 5B). This
reduction in NADH-oxidase activity was associated with a marked
improvement in endothelium-dependent vasodilation in
cholesterol-fed animals, whereas responses to
nitroglycerin were comparable in all 4 groups (Figure 4A and 4B, Table 3). Short-term
incubation of aortas from hyperlipidemic animals with
the AT1-receptor antagonist Bay
10-6734 did not inhibit O2·-
production, indicating that the antagonist has no
intrinsic antioxidant properties (hyperlipidemic,
2.12±0.20 versus hyperlipidemic+Bay, 1.98±0.18
pmol · mg-1 ·
min-1).
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Effects of AT1-Receptor Blockade on Macrophage
Infiltration and Plaque Area in Cholesterol-Fed Animals
The macroscopic fat-stained area was reduced in
cholesterol-fed animals treated with the
AT1-receptor blocker compared with
cholesterol-fed animals without
AT1-receptor blockade (5.3±1.4% versus
28.6±7.5%, P<0.05, Figure 6). Histologically, in
animals treated with Bay 10-6734, macrophage infiltration
averaged 1±0.2% compared with 58.8±15% in plaques from
cholesterol-fed animals without receptor blockade.
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Effects of Therapy With Amlodipine on Endothelial
Function and Vascular O2·-
Production in Cholesterol-Fed Animals
In marked contrast to the effect of the angiotensin II
receptor blockade, treatment of control rabbits with amlodipine for 8
weeks significantly increased NADH-induced
O2·- production (from 4.3±0.3 to
8.2±0.2 nmol · mg-1 ·
min-1) but did not alter NADH-oxidase activity
in aortas from cholesterol-fed animals (7.2±0.2 versus
7.1±0.8 nmol · mg-1 ·
min-1; P<0.05 for all). This
increase in O2·- did not alter responses to
nitroglycerin but did decrease responses to
acetylcholine (Table 4).
Endothelium-dependent vasodilation was not modified by
amlodipine in hypercholesterolemic animals.
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Lucigenin Validation Experiments
It has recently been shown that high concentrations of lucigenin
can produce a redox cycle with flavin-containing enzymes,
leading to artifactual increases in estimates of superoxide
production.17 18 This has not been found to occur
with low concentrations of lucigenin (5 µmol/L).13
Experiments with 5 µmol/L lucigenin revealed a significant
increase in vessels from hypercholesterolemic Watanabe
rabbits (980±107 counts · mg-1 ·
min-1, n=5) and cholesterol-fed New
Zealand White rabbits (1058±90 counts ·
mg-1 · min-1, n=5)
compared with control (524±39 counts ·
mg-1 · min-1,
n=5). Differences of similar magnitude were observed with the
chemiluminescence probe CLA (control, 9529±489 counts ·
mg-1 · min-1;
Watanabe, 27 405±3285 counts ·
mg-1 · min-1;
cholesterol-fed animals, 21 159±2225 counts ·
mg-1 · min-1;
P<0.05). Likewise, ESR studies also indicated an increase
in vascular superoxide production in
hypercholesterolemic vessels (Figure 7). In summary, with several independent
methods, we found a 2- to 4-fold increase in vascular
O2·- production in vessels from
hypercholesterolemic animals compared with vessels from
controls.
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Effect of Hyperlipidemia on Vascular
AT1-Receptor Density and Expression
Saturation binding assays on vessel membranes from Watanabe and
New Zealand White rabbits using 125I-labeled
angiotensin II revealed a significant increase in
AT1-receptor density without changes in binding
affinity (Figure 8). Likewise,
AT1-receptor mRNA levels were markedly enhanced
in hypercholesterolemic Watanabe rabbit vessels
compared with control animals. GAPDH mRNA levels were similarly
expressed in both normocholesterolemic and
hypercholesterolemic rabbits (Figures 9 and 10).
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Discussion |
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In this study, we demonstrate that hypercholesterolemia is associated with AT1-receptor upregulation and increased vascular O2·- production secondary to an activation of the vascular NADH oxidase. AT1-receptor blockade normalized the activity of the oxidase, reduced plaque area and macrophage infiltration, and in parallel improved endothelial dysfunction. These findings suggest a pathogenic role for the renin-angiotensin system in early stages of atherosclerosis.
Although numerous epidemiological studies have shown that elevated levels of LDL are associated with the onset of hypertension and atherosclerosis (for review, see Reference 1919 ), the underlying mechanisms remain unclear. ACE inhibition has been shown to promote regression and even prevention of atherosclerosis, suggesting a link between atherosclerosis and the renin-angiotensin system.20
Endothelial Dysfunction and
O2·- Production in
Hypercholesterolemia
Our present findings are in accord with previous
observations3 showing that
hypercholesterolemia is associated with
endothelial dysfunction and increased vascular
O2·- production. In
hyperlipidemic Watanabe rabbits, vascular
O2·- was increased 2-fold compared with
controls. Incubation of vessels from Watanabe rabbits with diphenylene
iodonium and oxypurinol inhibited O2·-
production, whereas L-NNA and rotenone were ineffective,
suggesting that the flavoprotein containing xanthine oxidase rather
than ·NO synthase or mitochondrial NADH dehydrogenase is a
significant source of O2·- in these vessels.
This result is consistent with recent observations suggesting
that vascular and/or circulating xanthine oxidase is a significant
source of O2·- in
cholesterol-fed rabbits.3 21 Because the
effects of DPI on steady-state O2·-
production were significantly greater than that of oxypurinol,
an additional flavin-containing oxidase (such as NADH/NADPH oxidase)
probably also serves as a source of O2·- in
hypercholesterolemic vessels. NADH/NADPH oxidase has
recently been demonstrated to be the predominant
O2·- source in both
endothelial and smooth muscle cells.6 8
Direct measurement of NADH/NADPH-oxidase activity in
homogenates of aortas from controls,
hyperlipidemic Watanabe rabbits, and
cholesterol-fed rabbits confirmed this observation. In both
models of hypercholesterolemia, NADH-oxidase
activity was increased compared with controls.
Lucigenin Validation Experiments
The majority of data regarding
O2·- formation in vessels and vessel
homogenates are based on measurements of the intensity of
lucigenin-enhanced chemiluminescence.7 12 The validity of
these data was recently questioned because lucigenin itself increases
formation of O2·- in the presence of enzymes
that are capable of providing a 1-electron reduction of
lucigenin2+, such as ·NO synthase and
xanthine/xanthine oxidase.17 18 We therefore performed
experiments using low concentrations of lucigenin (5 µmol/L),
which have recently been shown not to produce additional
O2·-.13 Measurements were also
made using CLA-enhanced chemiluminescence14 and ESR
spectroscopy with a spin trap recently shown to detect
O2·- with a high sensitivity and
specificity.15 All 3 approaches demonstrated a 2- to
4-fold increase in vascular O2·- in vessels
from hypercholesterolemic animals compared with vessels
from controls. These data therefore confirm the initial observation
from our group that in the setting of
hypercholesterolemia, vascular
O2·- levels are increased3 and
validate our lucigenin measurements.
Hypercholesterolemia and
Angiotensin II Receptor Expression
To gain insights into how
hypercholesterolemia increases oxidase
activity, AT1-receptor gene expression was
assessed. Recent studies have shown that incubation of vascular smooth
muscle cells with LDL increases expression of the
angiotensin II subtype
AT1.22 Similar results were obtained
in cholesterol-fed animals.16 The present
investigation illustrates that an upregulation of vascular
AT1-receptor expression also occurs in Watanabe
rabbit aortas. It is likely that increased smooth muscle levels of the
AT1 receptor underlie the increase in constrictor
responses to angiotensin II found in these vessels.
AT1-receptor activation has been shown to increase the activity of NADH/NADPH oxidase in smooth muscle6 and endothelial cells.23 There is a growing body of evidence that in hypercholesterolemia, the vascular renin-angiotensin system is activated. Uehara et al24 reported a significant correlation between chymase-like activity (a major non–ACE-dependent angiotensin II generation pathway) in internal mammary artery and serum total cholesterol levels. Moreover, it was demonstrated that O2·- activates vascular ACE.25 Of note, atherosclerotic lesions contain large amounts of angiotensin I–converting enzyme,9 and activated macrophages in atherosclerotic plaques can produce angiotensin II.26 In aggregate, together with our present studies, these data strongly suggest a key role of the renin-angiotensin system in atherosclerosis.
Mechanisms of ACE Inhibitor–Induced Improvement in
Endothelial Dysfunction
Endothelial dysfunction in
hypercholesterolemic animals has been shown to be
improved by ACE inhibitors.20 Some of this
benefit was diminished by a bradykinin antagonist. It was
therefore hypothesized that inhibition of bradykinin breakdown (and
subsequent high concentrations of ·NO and
endothelium-derived hyperpolarizing factor) rather than
inhibition of angiotensin II formation
itself27 was important in this protective effect. Our
present studies indicate that improvement in
endothelium-dependent vasodilation and vascular
O2·- production can be achieved
independently of bradykinin preservation.
Recently, Mancini et al10 showed that treatment of patients with coronary artery disease with an ACE inhibitor markedly improved coronary vasomotor function. The TREND study demonstrated that quinapril 40 mg/d given for 6 months markedly improved acetylcholine-provoked vasoconstriction. The authors speculated that one of the basic mechanisms responsible for this improvement may be inhibition of angiotensin II–sensitive, NAD(P)H-dependent O2·--producing enzymes, resulting in a reduction of ·NO inactivation. Our results support this concept and provide possible molecular mechanisms involved in this phenomenon. Indeed, in the present study, AT1-receptor blockade inhibited NADH-oxidase activity and in parallel improved endothelial dysfunction in cholesterol-fed animals.
These findings cannot be attributed to cholesterol-lowering effects, because treatment with the AT1-receptor blocker had no effect on this parameter. It is also unlikely that the AT1-receptor antagonist had direct radical scavenging effects, because short-term incubation of intact segments with Bay 10-6734 did not reduce the lucigenin signal. The inhibitory effects of Bay 10-6734 on vascular O2·- production were also not secondary to nonspecific vasodilator effects,28 because the use of the vasodilator amlodipine activated, rather than inhibiting, NADH-mediated O2·- production in vascular tissue. We cannot exclude, however, that changes in endothelial function and/or reduction in NADH-oxidase activity is mediated in part by ongoing activation of alternative receptors such as the AT IV or AT2·- subtype, which is particularly manifest in the context of AT1 blockade.29
The reduction in vascular O2·- production caused by AT-receptor blockade was associated with attenuation of plaque formation and macrophage infiltration in cholesterol-fed animals. These findings are in line with recent observations demonstrating that lipid peroxidation and plaque formation are significantly reduced in apolipoprotein E–deficient mice treated with losartan.30
A major mechanism for reduction in atherosclerosis caused by AT1-receptor blockade relates to the observed reduction in vessel macrophages. It is unlikely, however, that macrophages are the source of the increased vascular O2·-, because the increase in oxidase activity in vessels from hypercholesterolemic animals was NADH-dependent, whereas macrophages utilize NADPH as a substrate to produce O2·-.
Clinical Implications
The present study provides novel information concerning
O2·- sources in the early stages of
atherosclerosis. It is evident that in
hyperlipidemic animals, in addition to vascular and/or
circulating xanthine oxidase, NADH oxidase represents a major
vascular source of O2·-. Because
hypercholesterolemia is associated with
increased expression of the AT1 receptor and
AT1-receptor blockade improves
endothelial dysfunction, it is tempting to speculate
that the observed increased activity of NADH oxidase in
hypercholesterolemia is at least in part
secondary to increased local angiotensin II generation.
Together with previous data, our findings suggest that the
renin-angiotensin system plays an important role in both
the initiation and acceleration of the atherosclerotic process and that
inhibition of the renin-angiotensin system may have benefit
in treatment of this disease.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (Mu 1079/2-1, Ni 397/2-1) and in part by a vascular biology grant from the William Harvey Institute, London.
Received September 12, 1998; revision received November 19, 1998; accepted December 17, 1998.
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 T. Inoguchi, T. Sonta, H. Tsubouchi, T. Etoh, M. Kakimoto, N. Sonoda, N. Sato, N. Sekiguchi, K. Kobayashi, H. Sumimoto, et al. Protein Kinase C-Dependent Increase in Reactive Oxygen Species (ROS) Production in Vascular Tissues of Diabetes: Role of Vascular NAD(P)H Oxidase J. Am. Soc. Nephrol., August 1, 2003; 14(90003): S227 - 232. [Abstract] [Full Text] [PDF]
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A. Warnholtz and T. Munzel The failing human heart: Another battlefield for the NAD(P)H oxidase? J. Am. Coll. Cardiol., June 18, 2003; 41(12): 2172 - 2174. [Full Text] [PDF]
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B. M. Singh and J. L. Mehta Interactions Between the Renin-Angiotensin System and Dyslipidemia: Relevance in the Therapy of Hypertension and Coronary Heart Disease Arch Intern Med, June 9, 2003; 163(11): 1296 - 1304. [Abstract] [Full Text] [PDF]
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D. G Harrison, Hua Cai, U. Landmesser, and K. K Griendling The Pickering Lecture British Hypertension Society, 10th September 2002: Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease Journal of Renin-Angiotensin-Aldosterone System, June 1, 2003; 4(2): 51 - 61. [Abstract] [PDF]
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J. Galle, A. Mameghani, S.-S. Bolz, S. Gambaryan, M. Gorg, T. Quaschning, U. Raff, H. Barth, S. Seibold, C. Wanner, et al. Oxidized LDL and its Compound Lysophosphatidylcholine Potentiate AngII-Induced Vasoconstriction by Stimulation of RhoA J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1471 - 1479. [Abstract] [Full Text] [PDF]
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T. Matsumoto, K. Minai, H. Horie, N. Ohira, H. Takashima, Y. Tarutani, Y. o Yasuda, T. Ozawa, S. Matsuo, M. Kinoshita, et al. Angiotensin-converting enzyme inhibition but not angiotensin II type 1 receptor antagonism augments coronary release of tissue plasminogen activator in hypertensive patients J. Am. Coll. Cardiol., April 16, 2003; 41(8): 1373 - 1379. [Abstract] [Full Text] [PDF]
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S. Spiekermann, U. Landmesser, S. Dikalov, M. Bredt, G. Gamez, H. Tatge, N. Reepschlager, B. Hornig, H. Drexler, and D. G. Harrison Electron Spin Resonance Characterization of Vascular Xanthine and NAD(P)H Oxidase Activity in Patients With Coronary Artery Disease: Relation to Endothelium-Dependent Vasodilation Circulation, March 18, 2003; 107(10): 1383 - 1389. [Abstract] [Full Text] [PDF]
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P. A. Fischer, G. N. Dominguez, L. A. Cuniberti, V. Martinez, J. P. Werba, A. J. Ramirez, and L. D. Masnatta Hyperhomocysteinemia Induces Renal Hemodynamic Dysfunction: Is Nitric Oxide Involved? J. Am. Soc. Nephrol., March 1, 2003; 14(3): 653 - 660. [Abstract] [Full Text] [PDF]
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H.-Y. Sohn, F. Krotz, S. Zahler, T. Gloe, M. Keller, K. Theisen, T. M Schiele, V. Klauss, and U. Pohl Crucial role of local peroxynitrite formation in neutrophil-induced endothelial cell activation Cardiovasc Res, March 1, 2003; 57(3): 804 - 815. [Abstract] [Full Text] [PDF]
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T. J. Anderson, J. Hubacek, D. G. Wyse, and M. L. Knudtson Effect of chelation therapy on endothelial function in patients with coronary artery disease: PATCH substudy J. Am. Coll. Cardiol., February 5, 2003; 41(3): 420 - 425. [Abstract] [Full Text] [PDF]
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 U. Landmesser and H. Drexler Oxidative stress, the renin-angiotensin system, and atherosclerosis Eur. Heart J. Suppl., January 1, 2003; 5(suppl_A): A3 - A7. [Abstract] [PDF]
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 H. Cai, Z. Li, S. Dikalov, S. M. Holland, J. Hwang, H. Jo, S. C. Dudley Jr., and D. G. Harrison NAD(P)H Oxidase-derived Hydrogen Peroxide Mediates Endothelial Nitric Oxide Production in Response to Angiotensin II J. Biol. Chem., December 6, 2002; 277(50): 48311 - 48317. [Abstract] [Full Text] [PDF]
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P. T. Schumacker Angiotensin II Signaling in the Brain: Compartmentalization of Redox Signaling? Circ. Res., November 29, 2002; 91(11): 982 - 984. [Full Text] [PDF]
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 J. S. Forrester Prevention of Plaque Rupture: A New Paradigm of Therapy Ann Intern Med, November 19, 2002; 137(10): 823 - 833. [Abstract] [Full Text] [PDF]
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S. Klahr and J. Morrissey Obstructive nephropathy and renal fibrosis Am J Physiol Renal Physiol, November 1, 2002; 283(5): F861 - F875. [Abstract] [Full Text] [PDF]
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U. Rueckschloss, M. T. Quinn, J. Holtz, and H. Morawietz Dose-Dependent Regulation of NAD(P)H Oxidase Expression by Angiotensin II in Human Endothelial Cells: Protective Effect of Angiotensin II Type 1 Receptor Blockade in Patients With Coronary Artery Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2002; 22(11): 1845 - 1851. [Abstract] [Full Text] [PDF]
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C. A. Hamilton, M. J. Brosnan, S. Al-Benna, G. Berg, and A. F. Dominiczak NAD(P)H Oxidase Inhibition Improves Endothelial Function in Rat and Human Blood Vessels Hypertension, November 1, 2002; 40(5): 755 - 762. [Abstract] [Full Text] [PDF]
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T. Gori and J. D. Parker The Puzzle of Nitrate Tolerance: Pieces Smaller Than We Thought? Circulation, October 29, 2002; 106(18): 2404 - 2408. [Full Text] [PDF]
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A. Warnholtz, H. Mollnau, T. Heitzer, A. Kontush, T. Moller-Bertram, D. Lavall, A. Giaid, U. Beisiegel, S. L. Marklund, U. Walter, et al. Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits J. Am. Coll. Cardiol., October 2, 2002; 40(7): 1356 - 1363. [Abstract] [Full Text] [PDF]
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A. Virdis, M. F. Neves, F. Amiri, E. Viel, R. M. Touyz, and E. L. Schiffrin Spironolactone Improves Angiotensin-Induced Vascular Changes and Oxidative Stress Hypertension, October 1, 2002; 40(4): 504 - 510. [Abstract] [Full Text] [PDF]
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U. Landmesser, H. Cai, S. Dikalov, L. McCann, J. Hwang, H. Jo, S. M. Holland, and D. G. Harrison Role of p47phox in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II Hypertension, October 1, 2002; 40(4): 511 - 515. [Abstract] [Full Text] [PDF]
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A. R. Brasier, A. Recinos III, and M. S. Eledrisi Vascular Inflammation and the Renin-Angiotensin System Arterioscler. Thromb. Vasc. Biol., August 1, 2002; 22(8): 1257 - 1266. [Abstract] [Full Text] [PDF]
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T. M. Paravicini, L. M. Gulluyan, G. J. Dusting, and G. R. Drummond Increased NADPH Oxidase Activity, gp91phox Expression, and Endothelium-Dependent Vasorelaxation During Neointima Formation in Rabbits Circ. Res., July 12, 2002; 91(1): 54 - 61. [Abstract] [Full Text] [PDF]
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S. Wassmann, S. Hilgers, U. Laufs, M. Bohm, and G. Nickenig Angiotensin II Type 1 Receptor Antagonism Improves Hypercholesterolemia-Associated Endothelial Dysfunction Arterioscler. Thromb. Vasc. Biol., July 1, 2002; 22(7): 1208 - 1212. [Abstract] [Full Text] [PDF]
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A. H Chester Mast cells feel the strain Cardiovasc Res, July 1, 2002; 55(1): 13 - 15. [Full Text] [PDF]
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S. Itoh, S. Umemoto, M. Hiromoto, Y. Toma, Y. Tomochika, S. Aoyagi, M. Tanaka, T. Fujii, and M. Matsuzaki Importance of NAD(P)H Oxidase-Mediated Oxidative Stress and Contractile Type Smooth Muscle Myosin Heavy Chain SM2 at the Early Stage of Atherosclerosis Circulation, May 14, 2002; 105(19): 2288 - 2295. [Abstract] [Full Text] [PDF]
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J. Herrmann, P. J. Best, E. L. Ritman, D. R. Holmes Jr, L. O. Lerman, and A. Lerman Chronic endothelin receptor antagonism prevents coronary vasa vasorum neovascularization in experimental hypercholesterolemia J. Am. Coll. Cardiol., May 1, 2002; 39(9): 1555 - 1561. [Abstract] [Full Text] [PDF]
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S. Keidar, R. Heinrich, M. Kaplan, and M. Aviram Oxidative stress increases the expression of the angiotensin-II receptor type 1 in mouse peritoneal macrophages Journal of Renin-Angiotensin-Aldosterone System, March 1, 2002; 3(1): 24 - 30. [Abstract] [PDF]
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B.-q. Zhu, R. E Sievers, A. E. Browne, R. T Hillman, K. Chair, R. J Lee, K. Chatterjee, S. A Glantz, and W. W Parmley The renin-angiotensin system does not contribute to the endothelial dysfunction and increased infarct size in rats exposed to second hand smoke Journal of Renin-Angiotensin-Aldosterone System, March 1, 2002; 3(1): 54 - 60. [Abstract] [PDF]
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A. Shihabi, W.-G. Li, F. J. Miller Jr., and N. L. Weintraub Antioxidant therapy for atherosclerotic vascular disease: the promise and the pitfalls Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H797 - H802. [Full Text] [PDF]
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J.-M. Li, A. M. Mullen, S. Yun, F. Wientjes, G. Y. Brouns, A. J. Thrasher, and A. M. Shah Essential Role of the NADPH Oxidase Subunit p47phox in Endothelial Cell Superoxide Production in Response to Phorbol Ester and Tumor Necrosis Factor-{alpha} Circ. Res., February 8, 2002; 90(2): 143 - 150. [Abstract] [Full Text] [PDF]
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G. Nickenig and D. G. Harrison The AT1-Type Angiotensin Receptor in Oxidative Stress and Atherogenesis: Part II: AT1 Receptor Regulation Circulation, January 29, 2002; 105(4): 530 - 536. [Full Text] [PDF]
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