(Circulation. 2001;103:3099.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Genetic Deficiency of Inducible Nitric Oxide Synthase Reduces Atherosclerosis and Lowers Plasma Lipid Peroxides in Apolipoprotein E–Knockout Mice
From the Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, and Harvard Medical School, Boston, Mass.
Correspondence to Paul L. Huang, MD, PhD, Cardiovascular Research Center, Massachusetts General Hospital East, 149 E 13th St, Charlestown, MA 02129. E-mail huangp{at}helix.mgh.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Inducible nitric oxide synthase (iNOS) is expressed by leukocytes and smooth muscle cells in atherosclerotic lesions. To test whether NO produced by iNOS deficiency affects atherosclerosis, we studied apoE/iNOS–double knockout (dKO) and apoE-knockout (KO) control animals fed a "Western-type" diet.
Methods and Results—After 16 weeks of Western-type diet, the aortic lesion area in apoE/iNOS-dKO males and females was significantly reduced, by 22% and 21%, respectively, compared with apoE-KO males and females. This effect was more pronounced after 24 weeks of Western-type diet, after which lesion formation in male and female dKO mice was reduced by 38% and 40%, respectively. Plasma levels of lipoperoxides in apoE/iNOS-dKO mice (2.0±0.23 µmol/L) were significantly lower than in apoE-KO control animals (3.2±0.44 µmol/L; P=0.02). To test whether substrate deficiency plays a role in the proatherogenic actions of iNOS, we administered L-arginine to apoE-KO animals for 16 and 24 weeks. L-Arginine treatment did not affect lesion formation in apoE-KO animals fed a Western-type diet.
Conclusions—Genetic deficiency of iNOS decreases diet-induced atherosclerosis and lowers plasma levels of lipoperoxides, a marker for oxidative stress, in apoE-KO animals. Reduction in iNOS-mediated oxidative stress could partly explain protection from lesion formation in dKO animals. L-Arginine supplementation did not change lesion area in apoE-KO mice, indicating that substrate deficiency is not a likely cause for iNOS-mediated injury in this model of atherosclerosis.
Key Words: atherosclerosis • nitric oxide • nitric oxide synthase
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Although endothelial nitric oxide synthase (eNOS, NOS3) is constitutively expressed by the endothelium, the neuronal and inducible NOSs (nNOS [NOS1] and iNOS [NOS2]) are usually not detectable in undiseased vascular tissue. In human atherosclerotic lesions, however, all 3 isoforms are present.1 iNOS expression has been localized to vascular smooth muscle cells and mononuclear leukocytes in early and advanced atherosclerotic lesions.2
NOSs use L-arginine as substrate, converting a terminal guanidino nitrogen to NO. The activities of eNOS and nNOS, the constitutive isoforms, are regulated by intracellular Ca2+ concentration. In contrast, iNOS expression is induced by microbial endotoxins or cytokine stimulation, and its activity does not vary with intracellular Ca2+ concentration. Although strong evidence suggests that eNOS plays an important role in protection of the vessel wall from atherosclerosis, the role of iNOS in modulating the development of atherosclerosis is unclear.
iNOS may contribute to lesion formation by increasing oxidative stress in the vessel wall.3 Reactive intermediates derived from NO accelerate LDL oxidation under acidic conditions.4 NO can react with superoxide to form peroxynitrite, a strong oxidant, with resultant generation of hydroxyl radicals.5 6 Peroxynitrite also promotes LDL lipid oxidation in vitro6 7 and nitrosylates protein tyrosine residues in atherosclerotic lesions.8 Because superoxide reacts with NO to form peroxynitrite faster than superoxide can be scavenged by superoxide dismutase,9 the rate of superoxide formation in the vessel wall can profoundly influence the fate of NO.
To study the contribution of iNOS to lesion formation, we combined a genetic model of iNOS deficiency with a mouse model of atherosclerosis, the hyperlipidemic apolipoprotein E (apoE)–knockout (KO) mouse. ApoE-KO mice develop atherosclerotic lesions on a normal chow diet in a distribution closely resembling that in human disease.10 Disease progression can be substantially accelerated by feeding a "Western-type" atherogenic diet. We compared lesion formation in Western-type diet–fed apoE/iNOS–double knockout (dKO) mice and apoE-KO mice. This genetic approach overcomes problems with specificity and variability of NOS inhibitors. To avoid effects due to genetic background, we used highly inbred animals, all backcrossed 10 generations to the C57BL6 strain.
We show that genetic deficiency of the inducible NOS protects from atherosclerosis in apoE/iNOS-dKO mice. Furthermore, apoE/iNOS-dKO mice had significantly lower plasma levels of lipid peroxides, suggesting that the reduction in lesion formation may be due to a decrease in lipid peroxidation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Mice
iNOS-KO and apoE-KO animals were obtained from Jackson Laboratories.11 12 All mice were backcrossed for 10 generations to a C57BL/6J genetic background. iNOS-KO and apoE-KO animals were crossed to generate double-heterozygous mice, which were intercrossed and the offspring genotyped for iNOS and apoE by polymerase chain reaction. ApoE-KO animals that were wild-type or knockout for iNOS were weaned at 21 days and fed a Western-type diet (42% of total calories from fat; 0.15% cholesterol; Harlan Teklad) for 16 or 24 weeks. L-Arginine was given to apoE-KO mice by supplementing the drinking water with 2.5% L-arginine, a method previously shown to reduce lesion formation in hypercholesterolemic rabbits and LDL receptor–knockout mice.13 14 All procedures were approved by the institutional animal use and care committee and conform with NIH guidelines.
Lesion Assessment
The aorta was dissected and analyzed as
previously described.15
Animals were euthanized with pentobarbital (80 µg/kg IP), and the
aorta was perfused with PBS (pH 7.4), dissected from the aortic valve
to the iliac bifurcation, and fixed in 4%
paraformaldehyde overnight. Adventitial tissue was
removed, and the aorta was opened longitudinally and pinned onto a
black wax surface with micro needles (Fine Science Tools). Serial
images of the submerged vessels were captured with a video camera and a
still 35-mm camera. Lipid-rich intraluminal lesions were stained with
Sudan IV.15 Image
analysis was performed with Image Pro Plus (version 3.0.1;
Media Cybernetics). The amount of aortic lesion formation in each
animal was measured as percent lesion area per total area of the
aorta.
Tissue Preparation and Histology
Animals were euthanized with pentobarbital (80
µg/kg IP) and perfused with 0.9% NaCl followed by 10%
phosphate-buffered formalin through a catheter placed in the left
ventricle. The heart was fixed in 10% phosphate-buffered formalin for
48 hours and embedded in paraffin. Serial 5-µm sections were taken
from the aortic valve area. Sections were stained with
hematoxylin-eosin, Masson’s trichrome, Weigert’s method for elastic
fibers, and von Kossa stain for mineral salts.
Immunohistochemistry
Immunohistochemistry was carried out on
paraffin-embedded sections with a goat anti-mouse IgG1 antibody raised
against iNOS (clone 2, N52920, Transduction
Laboratories). A Vectastain ABC kit
with DAB as the substrate was used for
visualization.
Lipids and Lipoprotein Characterization
Animals were fasted for 4 hours, and blood was drawn
from the left ventricle of anesthetized animals. Plasma total
cholesterol was measured with Sigma
kit 352. Lipoprotein cholesterol distribution of plasma
samples was evaluated after fractionation by fast protein liquid
chromatography (FPLC) gel filtration. Plasma (200 µL)
was fractionated on a Superose 6 column with an ÄKTA-FPLC
(Amersham Pharmacia Biotech) system.
Cholesterol concentrations were measured with a microtiter
plate assay (Sigma kit 352; Spectra MAX 250; Molecular
Devices).
Plasma Lipoperoxide Measurement
Malondialdehyde–thiobarbituric acid (MDA-TBA) adduct
was measured by high-performance liquid
chromatography (HPLC) as
described.16 Blood was drawn
from the left ventricle of animals under pentobarbital
anesthesia (40 to 60 mg/kg IP) and collected in tubes
containing EDTA. Plasma was separated, and fractionation of the
protein-free extract was done with a C18 column (Micro Bondapak,
Waters) in an ÄKTApurifier HPLC system
(Amersham Pharmacia Biotech). A standard curve was constructed with
tetraethoxypropane standards.
Statistical Analysis
Statistical analysis was performed with
StatView 4.51 (Abacus Concepts, Inc). Two-way
ANOVA was used for repeated measures, followed by Scheffé’s F test.
A probability value of P<0.05
was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Inspection of the longitudinally opened Sudan IV–stained aorta showed that apoE/iNOS-dKO and apoE-KO animals developed lesions at typical sites of predilection, namely, at branch vessels and curvature (Figure 1
). After 16 weeks of Western-type diet,
analysis of hematoxylin and eosin–stained and Masson’s
trichrome–stained sections through the aortic valve showed advanced
lesions with fibrous cap and necrotic core in apoE/iNOS-dKO and apoE-KO
control animals
(Figure 2). von Kossa–stained sections showed calcification
of the aortic valve leaflets in apoE/iNOS-dKO and apoE-KO animals.
Sections stained with Weigert’s method for elastic fibers revealed
thinning of the vessel wall in apoE-KO that was not seen in
apoE/iNOS-dKO animals. Atherosclerotic lesions of apoE-KO animals
stained strongly for iNOS immunoreactivity, but lesions from
apoE/iNOS-dKO animals did not
(Figure 2).
|
|
The body weight, total area of the aorta, and total
cholesterol did not differ between apoE-KO mice and
apoE/iNOS-dKO mice within each sex
(Table).
Female mice, however, were smaller than male mice of the same
genotype. The lipoprotein profile, as assessed by FPLC, also
did not differ between apoE-KO mice and apoE/iNOS-dKO mice (data not
shown).
|
After 16 weeks of Western-type diet, the aortic lesion areas
in apoE/iNOS-dKO males and females were reduced by 22% and 21%,
respectively (males: mean area 11.4±0.7%, n=9; females: mean area
14.2±0.7%, n=7), compared with apoE-KO males and females (mean area
14.6±0.8%, n=7, P=0.035, and
18.0±0.6%, n=8, P=0.005)
(Figure 3, left). After 24 weeks of Western-type diet, lesion
formation in male and female dKO mice was reduced by 38% and 40%,
respectively (males: mean area 19.5±1.5%, n=9; females: mean area
19.2±1.4%, n=8), compared with apoE-KO control animals (males: mean
area 31.3±1.6%, n=11,
P=0.0006; females: mean area
32.1±3.3%, n=6, P=0.0017)
(Figure 3, right).
|
L-Arginine–supplemented
drinking water was given to male and female apoE-KO mice fed the
Western-type diet. After 16 weeks of Western-type diet and
L-arginine supplementation,
lesion formation in male and female apoE-KO mice (males: mean area
16.2±1.1%, n=5, P
0.6;
females: mean area 18.7±1.0%, n=5,
P0.9) did not differ from
that in apoE-KO mice fed the Western-type diet alone
(Figure 4, left). This result did not change when we examined
the 24-week time point (males: mean area 30.7±2.1%, n=8,
P0.9; females: mean area
27.7±1.0%, n=8, P0.5) with
apoE-KO animals fed the Western-type diet alone for the same amount of
time
(Figure 4, right).
|
Plasma levels of lipoperoxides, assayed as MDA-TBA adducts
by HPLC, were significantly reduced in apoE/iNOS-dKO mice (2.01±0.23
µmol/L, n=12) compared with apoE-KO control animals (3.20±0.44
µmol/L, n=7, P=0.018) at the
4-month time point, as seen in
Figure 5. In good agreement with the basal lipoperoxide
levels measured in rats (1.4 µmol/L) by Wong et
al,16 the basal plasma
lipoperoxide level in wild-type C57BL6 mice on a normal chow diet
averaged 0.99±0.11 µmol/L (n=8), which was significantly lower than
the values measured in apoE/iNOS-dKO mice
(P=0.037) and apoE-KO animals
(P
0.0001).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The development of atherosclerosis is a dynamic process that results from excessive inflammatory and fibroproliferative responses.17 NO may normally prevent atherosclerosis by inhibiting smooth muscle cell proliferation, inhibiting platelet aggregation and adhesion, and inhibiting leukocyte activation and adhesion.18 19 20 21 Although eNOS is an important regulator of normal vascular function, the inducible isoform and the neuronal isoform are usually not detectable in undiseased vessels.1 In human atherosclerotic lesions, however, all 3 isoforms of NOS—iNOS, nNOS, and eNOS—are present.1 In contrast to nNOS and eNOS, iNOS is fully active at basal Ca2+ levels. Once activated, iNOS is capable of generating higher NO concentrations, in the micromolar range, with higher substrate demands and with less regulation than the constitutive isoforms.
The role of iNOS in vascular pathology is variable and poorly defined in atherosclerosis. Transplant atherosclerosis is exacerbated in a model of chronic cardiac rejection in iNOS-KO mice, suggesting a protective role for iNOS.22 The same authors reported, however, that iNOS deficiency reduced inflammatory infiltrates and lowered rejection scores in a model of acute cardiac allograft rejection, suggesting a pathological role for iNOS.23 Recently, Knowles et al24 reported that absence of iNOS had no effect on atherosclerosis development in apoE/iNOS-dKO mice, but details were not provided. The potential effects of iNOS in atherosclerosis have clinical implications beyond a molecular mechanism of atherogenesis as well. Gene transfer of iNOS to injured vascular segments reduces neointima formation in normocholesterolemic animals,25 providing impetus for studies involving gene transfer of iNOS in patients. Thus, a possible involvement of iNOS in the development of atherosclerotic lesions has important clinical implications.
In the present study, we show that genetic deficiency of iNOS decreases atherosclerosis in apoE/iNOS-dKO mice. Male and female apoE/iNOS-dKO animals showed significant 22% and 21% reductions in aortic lesion area after 16 weeks and 38% and 40% reductions after 24 weeks of Western-type diet, respectively. Thus, protection from lesion formation in genetic iNOS deficiency increases over time, suggesting that iNOS accelerates lesion progression from an early time point and that the amount of iNOS-mediated injury increases over time.
Reduction in lesion formation was associated with a decrease in plasma lipoperoxide concentrations. Plasma levels of MDA, a highly reactive compound generated during lipid peroxidation by phagocytic cells, were significantly higher in apoE-KO animals than in apoE/iNOS-dKO animals and normocholesterolemic C57BL6 mice. In addition, MDA levels in apoE/iNOS-dKO animals were significantly higher than in C57BL6 animals. This demonstrates that iNOS is not the sole contributor to the elevated MDA levels seen in Western-type diet–fed apoE-KO mice. The decrease in atherosclerotic lesion area and lowered plasma lipoperoxide levels are not due to secondary changes in plasma total cholesterol, lipoprotein distribution, or body weight, because none of these parameters differed between apoE-KO and apoE/iNOS-dKO animals.
NO has both oxidant and antioxidant effects. NO may inhibit lipid peroxidation by scavenging lipid radicals and terminating the peroxidative chain reaction.26 Conversely, NO reacts with superoxide to form peroxynitrite. Peroxynitrite can oxidize LDL, nitrosylate tyrosine residues within proteins, and generate hydroxyl radicals. Hydroxyl radicals, in turn, can attack unsaturated fatty acids to yield lipid hydroperoxides.6 MDA generated during lipid oxidation can itself modify LDL so that it is recognized by the macrophage scavenger receptor, an important step in foam cell formation.27 28
It has been suggested that the balance between local concentrations of NO and O2- may determine whether peroxynitrite forms and lipid peroxidation occurs.29 Conditions that favor peroxynitrite formation would favor lipid peroxidation, whereas excess NO without O2- may block lipid peroxidation by scavenging peroxyl radicals. The role of iNOS in this balance is complex, because iNOS can generate not only NO but also O2-, particularly under conditions of decreased L-arginine availability.30 31 L-Arginine supplementation reduces atherosclerosis development in hyperlipidemic rabbits and in LDL receptor–knockout mice.13 14
To test whether L-arginine supplementation modulates atherosclerosis in apoE-KO animals, we supplemented the drinking water of Western-type diet–fed animals with 2.5% L-arginine. After 16 and 24 weeks of L-arginine supplementation, the lesion area in apoE-KO mice did not differ from animals that were fed the Western-type diet alone. There are several potential explanations for this finding. First, in vitro studies show that O2- production by iNOS is inhibited only by high L-arginine concentrations. Whereas O2- production by nNOS is completely blocked by 100 µmol/L L-arginine, iNOS-mediated O2- formation was essentially unaltered. Even in the presence of 1 mmol/L L-arginine, O2- production was only partially blocked. Physiological cytosolic L-arginine concentrations (200 to 800 µmol/L) may thus not be able to block O2- generation from iNOS. Second, if there are additional sources of O2- that are independent of iNOS, reducing the contribution of iNOS to O2- formation may not affect the net amount of O2- present. In fact, by providing more NO, L-arginine supplementation may increase the amount of OONO- formed under these conditions. Third, the effects of L-arginine on atherosclerosis may depend on the relative amounts of the 3 NOS isoforms and their contribution to NO and O2- production in the various animal models of atherosclerosis. For example, L-arginine may result in increased eNOS production of NO, accounting for protection in some models independent of iNOS. Our results suggest that L-arginine may not be important in reducing iNOS-mediated atherogenesis in Western-type diet–fed apoE-KO animals. Additional cofactor deficiency, low local L-arginine concentration despite supplementation, or the fact that iNOS is not substrate deficient in first place cannot be excluded, however. Our results differ from those in LDL receptor–KO mice,14 in which L-arginine supplementation reduced atherosclerosis. In addition to the above-mentioned factors, the 2 animal models differ in several important ways. Our apoE-KO mice were fed a Western-type diet containing 0.15% cholesterol, whereas the LDL receptor–KO mice were fed a high-cholesterol diet containing 1.25% cholesterol. Lesion areas are much greater in the apoE-KO model than the LDL receptor–KO model.32
While our manuscript was in preparation, Knowles et al24 reported that iNOS deficiency has no effect on atherosclerosis development in apoE/iNOS-dKO mice. The difference in phenotype seen in the 2 studies may be due to the mixed genetic background of the animals, their use of normal chow diet rather than our use of a Western-type diet, or our study of later time points and the different assay for atherosclerosis development used. A more recent report confirms our results that iNOS deficiency reduces atherosclerosis in apoE-KO mice.33 Our study differs, however, because we assayed en face lesion area as opposed to aortic root lesions, we used animals that were all backcrossed to the C57BL6 strain, and we provide data about levels of plasma MDA-TBA adducts.
In summary, genetic deficiency of iNOS decreases atherosclerosis in Western-type diet–fed apoE-KO animals. The reduction in atherosclerosis in dKO animals was associated with decreased plasma levels of lipoperoxides, suggesting that reduction in iNOS-mediated oxidative stress may explain the protection from lesion formation in dKO animals. L-Arginine supplementation did not change lesion area in apoE-KO mice after 16 and 24 weeks of Western-type diet.
|
Acknowledgments |
|---|
This work was supported by NINDS grant NS-33335 and NHLBI grant HL-52818 to Dr Huang. Dr Huang is an Established Investigator of the American Heart Association.
Received December 12, 2000; revision received February 14, 2001; accepted February 19, 2001.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997;17:2479–2488.
2. Esaki T, Hayashi T, Muto E, et al. Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits. Atherosclerosis. 1997;128:39–46.[Medline] [Order article via Infotrieve]
3. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med. 1996;20:707–727.[Medline] [Order article via Infotrieve]
4.
Chang GJ, Woo P,
Honda HM, et al. Oxidation of LDL to a biologically active form by
derivatives of nitric oxide and nitrite in the absence of superoxide:
dependence on pH and oxygen. Arterioscler
Thromb. 1994;14:1808–1814.
5.
Beckman JS, Beckman
TW, Chen J, et al. Apparent hydroxyl radical production by
peroxynitrite: implications for endothelial injury from
nitric oxide and superoxide. Proc Natl
Acad Sci
U S A. 1990;87:1620–1624.
6. Radi R, Beckman JS, Bush KM, et al. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys. 1991;288:481–487.[Medline] [Order article via Infotrieve]
7. Graham A, Hogg N, Kalyanaraman B, et al. Peroxynitrite modification of low-density lipoprotein leads to recognition by the macrophage scavenger receptor. FEBS Lett. 1993;330:181–185.[Medline] [Order article via Infotrieve]
8. Ischiropoulos H, Zhu L, Chen J, et al. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys. 1992;298:431–437.[Medline] [Order article via Infotrieve]
9.
Beckman JS,
Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the
bad, and the ugly. Am J
Physiol. 1996;271:C1424–C1437.
10.
Nakashima Y,
Plump AS, Raines EW, et al. ApoE-deficient mice develop lesions of all
phases of atherosclerosis throughout the
arterial tree. Arterioscler
Thromb. 1994;14:133–140.
11.
Laubach VE,
Shesely EG, Smithies O, et al. Mice lacking inducible nitric oxide
synthase are not resistant to
lipopolysaccharide-induced death.
Proc Natl Acad Sci
U S A. 1995;92:10688–10692.
12.
Piedrahita JA,
Zhang SH, Hagaman JR, et al. Generation of mice carrying a mutant
apolipoprotein E gene inactivated by gene targeting in
embryonic stem cells. Proc Natl Acad Sci
U S A. 1992;89:4471–4475.
13. Boger RH, Bode-Boger SM, Mugge A, et al. Supplementation of hypercholesterolaemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis. 1995;117:273–284.[Medline] [Order article via Infotrieve]
14.
Aji W, Ravalli S,
Szabolcs M, et al.
L-Arginine prevents
xanthoma development and inhibits atherosclerosis in
LDL receptor knockout mice [see comments].
Circulation. 1997;95:430–437.
15.
Palinski W, Ord
VA, Plump AS, et al. ApoE-deficient mice are a model of lipoprotein
oxidation in atherogenesis: demonstration of oxidation-specific
epitopes in lesions and high titers of autoantibodies to
malondialdehyde-lysine in serum.
Arterioscler Thromb. 1994;14:605–616.
16.
Wong SH, Knight
JA, Hopfer SM, et al. Lipoperoxides in plasma as measured by
liquid-chromatographic separation of
malondialdehyde-thiobarbituric acid adduct.
Clin Chem. 1987;33:214–220.
17. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
18. Radomski MW, Palmer RM, Moncada S. Modulation of platelet aggregation by an L-arginine-nitric oxide pathway. Trends Pharmacol Sci. 1991;12:87–88.[Medline] [Order article via Infotrieve]
19. Mooradian DL, Hutsell TC, Keefer LK. Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J Cardiovasc Pharmacol. 1995;25:674–678.[Medline] [Order article via Infotrieve]
20. Lefer AM, Ma XL, Weyrich A, et al. Endothelial dysfunction and neutrophil adherence as critical events in the development of reperfusion injury. Agents Actions Suppl. 1993;41:127–135.[Medline] [Order article via Infotrieve]
21. Bath PM. The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cGMP concentrations in vitro. Eur J Clin Pharmacol. 1993;45:53–58.[Medline] [Order article via Infotrieve]
22.
Koglin J,
Glysing-Jensen T, Mudgett JS, et al. Exacerbated transplant
arteriosclerosis in inducible nitric
oxide–deficient mice.
Circulation. 1998;97:2059–2065.
23.
Koglin J,
Glysing-Jensen T, Mudgett JS, et al. NOS2 mediates opposing effects in
models of acute and chronic cardiac rejection: insights from
NOS2-knockout mice. Am J
Pathol. 1998;153:1371–1376.
24. Knowles JW, Reddick RL, Jennette JC, et al. Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)Apoe(-/-) mice are ameliorated by enalapril treatment. J Clin Invest. 2000;105:451–458.[Medline] [Order article via Infotrieve]
25. Shears LL, Kawaharada N, Tzeng E, et al. Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis. J Clin Invest. 1997;100:2035–2042.[Medline] [Order article via Infotrieve]
26.
Goss SP, Hogg N,
Kalyanaraman B. The effect of nitric oxide release rates on the
oxidation of human low density lipoprotein.
J Biol Chem. 1997;272:21647–21653.
27.
Fogelman AM,
Shechter I, Seager J, et al. Malondialdehyde alteration of low density
lipoproteins leads to cholesteryl ester accumulation in human
monocyte-macrophages. Proc Natl
Acad Sci
U S A. 1980;77:2214–2218.
28. Gerrity RG. The role of the monocyte in atherogenesis, II: migration of foam cells from atherosclerotic lesions. Am J Pathol. 1981;103:191–200.[Abstract]
29. Darley-Usmar V, Wiseman H, Halliwell B. Nitric oxide and oxygen radicals: a question of balance. FEBS Lett. 1995;369:131–135.[Medline] [Order article via Infotrieve]
30.
Xia Y, Roman LJ,
Masters BS, et al. Inducible nitric-oxide synthase generates superoxide
from the reductase domain. J Biol
Chem. 1998;273:22635–22639.
31.
Xia Y, Zweier JL.
Superoxide and peroxynitrite generation from inducible nitric oxide
synthase in macrophages. Proc Natl
Acad Sci
U S A. 1997;94:6954–6958.
32. Smith JD. Mouse models of atherosclerosis. Lab Anim Sci. 1998;48:573–579.[Medline] [Order article via Infotrieve]
33.
Detmers PA,
Hernandez M, Mudgett J, et al. Deficiency in inducible nitric oxide
synthase results in reduced atherosclerosis in
apolipoprotein E-deficient mice. J
Immunol. 2000;165:3430–3435.
This article has been cited by other articles:
 |
|
 |
|
  P. Ponnuswamy, E. Ostermeier, A. Schrottle, J. Chen, P. L. Huang, G. Ertl, B. Nieswandt, and P. J. Kuhlencordt Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase Am. J. Pathol., June 1, 2009; 174(6): 2400 - 2410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Lukowski, P. Weinmeister, D. Bernhard, S. Feil, M. Gotthardt, J. Herz, S. Massberg, A. Zernecke, C. Weber, F. Hofmann, et al. Role of Smooth Muscle cGMP/cGKI Signaling in Murine Vascular Restenosis Arterioscler. Thromb. Vasc. Biol., July 1, 2008; 28(7): 1244 - 1250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. von Lukowicz, P. O. Hassa, C. Lohmann, J. Boren, V. Braunersreuther, F. Mach, B. Odermatt, M. Gersbach, G. G. Camici, B. E. Stahli, et al. PARP1 is required for adhesion molecule expression in atherogenesis Cardiovasc Res, April 1, 2008; 78(1): 158 - 166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. W.T. Liu and P. L. Huang Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice Cardiovasc Res, January 1, 2008; 77(1): 19 - 29. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Upmacis, M. J. Crabtree, R. S. Deeb, H. Shen, P. B. Lane, L. E. S. Benguigui, N. Maeda, D. P. Hajjar, and S. S. Gross Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2878 - H2887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Romeo, K. S. Moulton, and A. Kazlauskas Attenuated Expression of Profilin-1 Confers Protection From Atherosclerosis in the LDL Receptor Null Mouse Circ. Res., August 17, 2007; 101(4): 357 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ahluwalia, A. Y. Lin, A. M. Tager, I. E. Pruitt, T. J. T. Anderson, F. Kristo, D. Shen, A. R. Cruz, M. Aikawa, A. D. Luster, et al. Inhibited Aortic Aneurysm Formation in BLT1-Deficient Mice J. Immunol., July 1, 2007; 179(1): 691 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Moens and D. A. Kass Tetrahydrobiopterin and Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2439 - 2444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Lowenstein Beneficial effects of neuronal nitric oxide synthase in atherosclerosis. Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1417 - 1418. [Full Text] [PDF]
|
|
|
|
|
|
P. J. Kuhlencordt, S. Hotten, J. Schodel, S. Rutzel, K. Hu, J. Widder, A. Marx, P. L. Huang, and G. Ertl Atheroprotective Effects of Neuronal Nitric Oxide Synthase in Apolipoprotein E Knockout Mice Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1539 - 1544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. V. d'Uscio and Z. S. Katusic Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2466 - H2471. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Heller, E. Liu, A. M. Tager, Q. Yuan, A. Y. Lin, N. Ahluwalia, K. Jones, S. L. Koehn, V. M. Lok, E. Aikawa, et al. Chemokine CXCL10 Promotes Atherogenesis by Modulating the Local Balance of Effector and Regulatory T Cells Circulation, May 16, 2006; 113(19): 2301 - 2312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Matsui, S. Xu, K. A. Maitland, R. Mastroianni, J. A. Leopold, D. E. Handy, J. Loscalzo, and R. A. Cohen Glucose-6-Phosphate Dehydrogenase Deficiency Decreases Vascular Superoxide and Atherosclerotic Lesions in Apolipoprotein E-/- Mice Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 910 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Mayr, Y. Zou, Z. Zhang, H. Dietrich, Y. Hu, and Q. Xu Accelerated Arteriosclerosis of Vein Grafts in Inducible NO Synthase-/- Mice Is Related to Decreased Endothelial Progenitor Cell Repair Circ. Res., February 17, 2006; 98(3): 412 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Hofmann, R. Feil, T. Kleppisch, and J. Schlossmann Function of cGMP-Dependent Protein Kinases as Revealed by Gene Deletion Physiol Rev, January 1, 2006; 86(1): 1 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Deeb, H. Shen, C. Gamss, T. Gavrilova, B. D. Summers, R. Kraemer, G. Hao, S. S. Gross, M. Laine, N. Maeda, et al. Inducible Nitric Oxide Synthase Mediates Prostaglandin H2 Synthase Nitration and Suppresses Eicosanoid Production Am. J. Pathol., January 1, 2006; 168(1): 349 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakata, M. Tsutsui, H. Shimokawa, M. Tamura, H. Tasaki, T. Morishita, O. Suda, S. Ueno, Y. Toyohira, Y. Nakashima, et al. Vascular Neuronal NO Synthase Is Selectively Upregulated by Platelet-Derived Growth Factor: Involvement of the MEK/ERK Pathway Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2502 - 2508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Feinberg, Z. Cao, A. K. Wara, M. A. Lebedeva, S. SenBanerjee, and M. K. Jain Kruppel-like Factor 4 Is a Mediator of Proinflammatory Signaling in Macrophages J. Biol. Chem., November 18, 2005; 280(46): 38247 - 38258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Phan, O. Ivanovski, T. Nguyen-Khoa, N. Mothu, J. Angulo, R. Westenfeld, M. Ketteler, N. Meert, J. Maizel, I. G. Nikolov, et al. Sevelamer Prevents Uremia-Enhanced Atherosclerosis Progression in Apolipoprotein E-Deficient Mice Circulation, November 1, 2005; 112(18): 2875 - 2882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. C. Mehra, V. S. Ramgolam, and J. R. Bender Cytokines and cardiovascular disease J. Leukoc. Biol., October 1, 2005; 78(4): 805 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. W. Q. Moore and D. Y. Hui Apolipoprotein E inhibition of vascular hyperplasia and neointima formation requires inducible nitric oxide synthase J. Lipid Res., October 1, 2005; 46(10): 2083 - 2090. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-i. Kodama, Y. Nishio, O. Sekine, Y. Sato, K. Egawa, H. Maegawa, and A. Kashiwagi Bidirectional regulation of monocyte chemoattractant protein-1 gene at distinct sites of its promoter by nitric oxide in vascular smooth muscle cells Am J Physiol Cell Physiol, September 1, 2005; 289(3): C582 - C590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Heller, E. Liu, A. M. Tager, S. Sinha, J. D. Roberts, S. L. Koehn, P. Libby, E. R. Aikawa, J. Q. Chen, P. Huang, et al. Inhibition of Atherogenesis in BLT1-Deficient Mice Reveals a Role for LTB4 and BLT1 in Smooth Muscle Cell Recruitment Circulation, July 26, 2005; 112(4): 578 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Barbato, B. S. Zuckerbraun, M. Overhaus, K. G. Raman, and E. Tzeng Nitric oxide modulates vascular inflammation and intimal hyperplasia in insulin resistance and the metabolic syndrome Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H228 - H236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. F. Bodary, Y. Shen, F. B. Vargas, X. Bi, K. A. Ostenso, S. Gu, J. A. Shayman, and D. T. Eitzman {alpha}-Galactosidase A Deficiency Accelerates Atherosclerosis in Mice With Apolipoprotein E Deficiency Circulation, February 8, 2005; 111(5): 629 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-A. Devynck, A. Simon, M.-G. Pernollet, G. Chironi, J. Gariepy, F. Rendu, and J. Levenson Plasma cGMP and Large Artery Remodeling in Asymptomatic Men Hypertension, December 1, 2004; 44(6): 919 - 923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Loscalzo L-Arginine and Atherothrombosis J. Nutr., October 1, 2004; 134(10): 2798S - 2800S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. V. Smirnova, M. Kajstura, T. Sawamura, and M. S. Goligorsky Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H782 - H790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Forbes, L. T. L. Yee, V. Thallas, M. Lassila, R. Candido, K. A. Jandeleit-Dahm, M. C. Thomas, W. C. Burns, E. K. Deemer, S. R. Thorpe, et al. Advanced Glycation End Product Interventions Reduce Diabetes-Accelerated Atherosclerosis Diabetes, July 1, 2004; 53(7): 1813 - 1823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Meir and E. Leitersdorf Atherosclerosis in the Apolipoprotein E-Deficient Mouse: A Decade of Progress Arterioscler. Thromb. Vasc. Biol., June 1, 2004; 24(6): 1006 - 1014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Feinberg, M. Watanabe, M. A. Lebedeva, A. S. Depina, J.-i. Hanai, T. Mammoto, J. P. Frederick, X.-F. Wang, V. P. Sukhatme, and M. K. Jain Transforming Growth Factor-{beta}1 Inhibition of Vascular Smooth Muscle Cell Activation Is Mediated via Smad3 J. Biol. Chem., April 16, 2004; 279(16): 16388 - 16393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Alp, M. A. McAteer, J. Khoo, R. P. Choudhury, and K. M. Channon Increased Endothelial Tetrahydrobiopterin Synthesis by Targeted Transgenic GTP-Cyclohydrolase I Overexpression Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 445 - 450. [Abstract] [Full Text]
|
|
|
|
|
|
J. H. von der Thusen, M. L. Fekkes, R. Passier, A.J. van Zonneveld, V. Mainfroid, T. J.C. van Berkel, and E. A.L. Biessen Adenoviral Transfer of Endothelial Nitric Oxide Synthase Attenuates Lesion Formation in a Novel Murine Model of Postangioplasty Restenosis Arterioscler. Thromb. Vasc. Biol., February 1, 2004; 24(2): 357 - 362. [Abstract] [Full Text]
|
|
|
|
|
|
R. Feil, S. M. Lohmann, H. de Jonge, U. Walter, and F. Hofmann Cyclic GMP-Dependent Protein Kinases and the Cardiovascular System: Insights From Genetically Modified Mice Circ. Res., November 14, 2003; 93(10): 907 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wolfsgruber, S. Feil, S. Brummer, O. Kuppinger, F. Hofmann, and R. Feil A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells PNAS, November 11, 2003; 100(23): 13519 - 13524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. S. Katusic, N. M. Caplice, and K. A. Nath Nitric Oxide Synthase Gene Transfer as a Tool to Study Biology of Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2003; 23(11): 1990 - 1994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B Chesebro, E. Blessing, C.-C. Kuo, M. E Rosenfeld, M. Puolakkainen, and L. A. Campbell Nitric oxide synthase plays a role in Chlamydia pneumoniae-induced atherosclerosis Cardiovasc Res, October 15, 2003; 60(1): 170 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A.C. 't Hoen, C. A.C. Van der Lans, M. Van Eck, M. K. Bijsterbosch, T. J.C. Van Berkel, and J. Twisk Aorta of ApoE-Deficient Mice Responds to Atherogenic Stimuli by a Prelesional Increase and Subsequent Decrease in the Expression of Antioxidant Enzymes Circ. Res., August 8, 2003; 93(3): 262 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Cooke, K. Sydow, J. Chen, and P. Huang A Peculiar Result and a Fanciful Hypothesis Regarding L-Arginine * Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 1128 - 1128. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zanetti, L. V. d'Uscio, I. Kovesdi, Z. S. Katusic, and T. O'Brien In Vivo Gene Transfer of Inducible Nitric Oxide Synthase to Carotid Arteries From Hypercholesterolemic Rabbits Stroke, May 1, 2003; 34(5): 1293 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Attia, R. Goldschmeding, M. A. Attia, P. Boer, H. A. Koomans, and J. A. Joles Male gender increases sensitivity to renal injury in response to cholesterol loading Am J Physiol Renal Physiol, April 1, 2003; 284(4): F718 - F726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. V. d'Uscio, S. Milstien, D. Richardson, L. Smith, and Z. S. Katusic Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity Circ. Res., January 10, 2003; 92(1): 88 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Loscalzo L-Arginine in Atherosclerosis: Consequences of Methylation Stress in a Complex Catabolism? Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 3 - 5. [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, P. Kuhlencordt, F. Urano, H. Ichinose, J. Astern, and P. L. Huang L-Arginine on Atherosclerosis in ApoE Knockout and ApoE/Inducible NO Synthase Double-Knockout Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 97 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Eguchi, L. V. d'Uscio, C. Wambi, D. Weiler, I. Kovesdi, T. O'Brien, and Z. S. Katusic Inhibitory effect of recombinant iNOS gene expression on vasomotor function of canine basilar artery Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2560 - H2566. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Matucci Cerinic and M. B. Kahaleh Beauty and the Beast. The nitric oxide paradox in systemic sclerosis Rheumatology, August 1, 2002; 41(8): 843 - 847. [Full Text] [PDF]
|
|
|
|
 |
|
 M. W Manning, L. A Cassis, J. Huang, S. J Szilvassy, and A. Daugherty Abdominal aortic aneurysms: fresh insights from a novel animal model of the disease Vascular Medicine, February 1, 2002; 7(1): 45 - 54. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||