(Circulation. 2001;103:1115.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Inducible Nitric Oxide Synthase Deficiency Does Not Affect the Susceptibility of Mice to Atherosclerosis but Increases Collagen Content in Lesions
From the Departments of Physiology (X.-L.N., X.Y., K.H., H.N.) and Microbiology (K.T., S.S., Y.K.), School of Medicine, Tokai University, Isehara, Kanagawa, Japan.
Correspondence to Prof H. Nakazawa, Department of Physiology, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193 Japan. E-mail: nakazawa{at}is.icc.u-tokai.ac.jp
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Abstract |
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Background—Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established.
Methods and Results—We compared the susceptibility of iNOS knockout (iNOS-/-) and wild-type (iNOS+/+) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS+/+ 285±73x103 µm2, iNOS-/- 293±82x103 µm2, n=10). iNOS mRNA was detected in the lesions of iNOS+/+ but not iNOS-/- mice through RT-PCR. Immunohistochemically, iNOS+/+ mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS-/- mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS+/+ mice showed a markedly decreased extracellular collagen content compared with those of iNOS-/- mice
Conclusions—iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.
Key Words: mice, inbred • atherosclerosis • nitric oxide synthase
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Introduction |
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Chronic inhibition of NO production accelerates atherosclerosis,1 2 whereas enhanced production of NO suppresses the development of atherosclerosis or even regresses the lesions.3 4 These effects of NO in large part depend on its ability to terminate LDL oxidation,5 to inhibit the expression of adhesion molecules and chemokines,6 7 and to attenuate smooth muscle cell migration8 and are principally due to NO produced by endothelial NO synthase (eNOS). Experiments with eNOS knockout mice9 10 and eNOS gene transfer11 confirmed that eNOS is an important antiatherogenic player. However, the direct influence of inducible NOS (iNOS) on the development of atherosclerosis is unknown, although the induction of iNOS consistently occurs in atherosclerotic vessels of humans12 13 and experimental animals.14 15 The induction of iNOS may be a defense mechanism against the disease. However, a high flux of NO from iNOS may favor formation of peroxynitrite, a powerful oxidant generated by the reaction of NO and superoxide,16 and produce nitrotyrosine, which was shown to be present in human atheroma plaque.17 Thus, it is important to determine the overall impact of iNOS on atherogenesis, because this may have implications for preventive and therapeutic strategy. To examine directly the influence of iNOS on the development of atherogenesis and on the characteristics of the lesion, we compared the susceptibility of iNOS-/- and iNOS+/+ mice to the development of atherosclerosis induced with an atherogenic diet and analyzed the composition of the atherosclerotic lesions in the 2 strains.
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Methods |
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Mice and Experimental Protocol
The iNOS-/- mice, with a mixed C57Bl/6Jx129 SvEv genetic background,18 were obtained from Merck & Co, Inc. The iNOS+/+ mice were obtained by crossing 129 SvEv mice with C57 Bl/6J mice twice. The iNOS-/- and iNOS+/+ strains have similar genetic backgrounds of
75% C57Bl/6J and 25% 129/SvEv. The animals were
maintained in a pathogen-free barrier facility with a 12-hour
light/dark cycle and had free access to food and water. Fifteen
age-matched (8-week-old) females of each strains were fed a diet that
contained 15% fat, 1% cholesterol, and 0.5% sodium
cholate19 for 15 weeks. Six
females of each strain (8 weeks old), fed normal chow for the same
period of time, served as controls. The study was approved by the
Animal Care Committee of Tokai University.
Measurement of Plasma Lipid Levels
Mice were deprived of food for 16 hours, and blood
was collected via the abdominal aorta into heparin-coated tubes with
the animals under pentobarbital sodium–induced anesthesia. Plasma was
obtained through centrifugation of the whole blood for 10 minutes at
12 000g at 4°C.
Concentrations of total cholesterol, HDL cholesterol, and triglycerides
were measured enzymatically with a Boehringer-Mannheim Hitachi 717
analyzer.
Tissue Preparation and Histological
Analysis
After the collection of blood, the heart and proximal
aorta were excised and embedded in OCT compound (Tissue-Tek), frozen on
dry ice, and stored at -70°C until sectioning for histological
analysis. The OCT-embedded heart with aorta was serially sectioned from
the middle of the ventricle to the root of the aorta into 5-µm
slices. For the quantitative evaluation of atherosclerotic
lesions,20 9 sections were
taken every 40 µm, stained with oil red O, and counterstained with
hematoxylin. Remaining sections were frozen at -70°C and used for
other stainings. The lesion size was quantified through microscopic
examination with a computer-assisted image analysis system (KS 300;
Carl Zeiss). The average size of lesions in the 9 sections was taken to
represent the lesion size for each animal. Cellular density in the
lesions was quantified by counting cell nuclei in the intima of the
aortic lesions at 3 levels (sections 2, 4, and 6) with sections stained
with oil red O and hematoxylin. Cellular density was expressed as the
number of cells per intimal area
(cells/mm2). The presence of collagen in the
lesions was examined in sections stained according to the standard Van
Gieson method. For evaluation of iNOS mRNA induction, the proximal
aorta was snap-frozen in liquid nitrogen and stored at -80°C until
use.
Immunohistochemistry
Serial cryostat sections were used to stain
macrophages, smooth muscle cells, iNOS, nitrotyrosine, and vascular
cell adhesion molecule (VCAM)-1. The primary antibodies that we used
were a polyclonal rabbit anti-mouse iNOS antibody (017-16001; Wako Pure
Chemical Industries; diluted 1:1000), a monoclonal rat anti-mouse CD11b
(Mac-1) antibody for macrophages (MAS 034p; Harlan Sera-Laboratory Ltd;
diluted 1:5), a polyclonal rabbit anti-nitrotyrosine antibody (06-248;
Upstate Biotechnology; diluted 1:100), a monoclonal rat anti-mouse
VCAM-1 antibody (RA 140070; Antigenix America Inc; diluted 1:50), and a
monoclonal mouse anti-human smooth muscle cell 
-actin antibody
(IgG2) (A 2547; Sigma Chemical Co;
diluted
1:400).21
Sections for immunohistochemical staining were fixed in acetone at -20°C for 5 minutes. All subsequent incubations were performed at room temperature, except for incubation with primary antibodies. The endogenous peroxidase activity was neutralized with H2O2 (0.3% vol/vol) for 10 minutes. An avidin-biotin blocking kit (Vector Laboratories) was used to block nonspecific endogenous biotin staining. After incubation with 4% (vol/vol) BSA (Sigma) diluted in PBS for 30 minutes, the sections were incubated with primary antibodies overnight at 4°C. Then biotinylated goat anti-rabbit IgG (for iNOS and nitrotyrosine; BA-1000, Vector Laboratories), biotinylated rabbit anti-rat IgG (for macrophages; BA-4001, Vector Laboratories), or biotinylated goat anti-mouse IgG2a antibody (for smooth muscle cells; GAM/IGG2A/BIO, Nordic Immunological Laboratories BV) was applied at 5 µg/mL and incubated for 45 minutes. Subsequently, the sections were incubated with streptavidin-peroxidase complex (Vector Laboratories) for 30 minutes. The staining was visualized with 9-amino-3-ethylene-carbazole (Vector Laboratories) or diaminobenzidine (Vector Laboratories). Nonimmune rabbit or rat serum was used in place of the primary antibody as a negative control.
Measurements of iNOS and mRNA
To determine iNOS mRNA levels, the frozen aorta was
homogenized in ISOGEN (Nippon Gene), total RNA was extracted, and
RT-PCR was applied as previously
described.22 Briefly, equal
amounts of RNA were reverse-transcribed into cDNA. The RT products were
amplified with the following primers for iNOS:
5'-CTTGCCCCTGGAAGTTTCTCTT-3 (upstream) and 3'-CTTTGTTGTCCTTGGATGGTCG-5'
(downstream), which afforded a 519-bp fragment. As an internal control,
ß2-microglobulin
(ß2M) mRNA was estimated under the same
cycling conditions with the primers 5'-TGACCGGCTTGTATGCTATC-3'
(upstream) and 5'-CAGTGTGAGCCAGGATATAG-3' (downstream), which gave a
223-bp fragment. The PCR products of iNOS and
ß2M were mixed and electrophoresed on a 1%
agarose Tris-acetate EDTA gel, which was stained with ethidium bromide.
The gel was photographed under ultraviolet light.
Statistical Analysis
All data are expressed as mean±SEM. To analyze
differences in atherosclerotic lesion size between the 2 groups,
Student’s t test and
Mann-Whitney U analysis were
performed. To compare lipid measurements, 2-way ANOVA was performed.
Probability values of <0.05 were considered
significant.
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Results |
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Plasma Lipid Levels
Plasma cholesterol was markedly elevated with the atherogenic diet in both iNOS-/- and iNOS+/+ mice, but no significant difference was observed between the 2 groups (Table
).
Triglyceride levels were unaffected by the atherogenic diet, and there
were no significant intergroup differences. Basal levels of all lipids
were comparable in the 2 strains and remained in the same ranges after
15 weeks of normal chow (data not shown).
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iNOS Expression in Aorta
As shown in
Figure 1, iNOS mRNA was not observed in the aorta of control
mice of either strain. It also was not observed in
iNOS-/- mice fed the atherogenic diet, as
expected. However, iNOS mRNA expression was clearly demonstrated in the
aorta of iNOS+/+ mice fed the atherogenic
diet.
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Atherosclerotic Lesion Formation
All mice of both strains developed atherosclerotic
lesions along the proximal aortic wall and at the valve cusps after the
15-week atherogenic diet
(Figure 2). Neither strain developed aortic atherosclerotic
lesions when maintained on the normal chow for the same period of time.
Individual lesions varied from simple fatty streaks to complex fibrous
plaques, but there was no consistent difference in morphological
characteristics or sites of lesions between the 2 strains. The average
values of cumulative lesion size were similar in the 2 strains
(iNOS+/+
285±73x103
µm2, iNOS-/-
293±82x103
µm2 [mean±SEM], n=10)
(Figure 3).
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Collagen Content in Lesions
As shown in
Figure 4, extracellular collagen content was markedly
decreased in the lesions of iNOS+/+ mice
compared with that of comparable-sized lesions of
iNOS-/- mice. The cellular densities in
the lesions were equal in the 2 strains
(iNOS+/+ 3869±315
cells/mm2,
iNOS-/-: 3568±248
cells/mm2 [mean±SEM],
n=10).
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Immunohistochemistry
Immunocytochemical analyses showed that lesions
consisted primarily of macrophages
(Figures 5A1, 5B1, and 5B5) with minimal smooth muscle cell
involvement in both strains of mice (data not shown). iNOS staining was
extensive in the lesions of iNOS+/+ mice
(Figures 5B2 and 5B6) but was absent in those of
iNOS-/- mice
(Figure 5A2). Specifically, iNOS staining was seen in
macrophages (identified in adjacent sections by positive MAC-1
staining) distributed throughout the intimal lesions. Staining for iNOS
was also evident in medial smooth muscle cells beneath the intimal
lesions
(Figure 5B2). iNOS was not detected in the aorta of either of
these strains of mice maintained on normal chow (data not shown).
Nitrotyrosine staining in the lesions of the
iNOS+/+ mice showed a distribution similar
to that of iNOS stainings but was more marked in the intimal area of
lesions, where macrophages are the major component
(Figures 5B3 and 5B7). Nitrotyrosine staining was not present
in sections of aorta from iNOS-/- mice fed
the atherogenic diet or in such sections from both strains fed normal
chow (data not shown). VCAM-1 staining was seen in the lesions of both
strains, with no apparent difference in the intensity or distribution
between the 2 strains
(Figures 5A4 and 5B4).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This is the first study in which iNOS knockout mice have been used to directly examine whether iNOS-mediated pathways promote or protect against development of atherosclerosis in vivo. iNOS was induced in the atherosclerotic lesions only in iNOS+/+ mice, and the sizes of atherosclerotic lesions were similar in the iNOS-/- and iNOS+/+ mice.
The involvement of iNOS in atherosclerosis has been shown
through a variety of observations, including the detection of iNOS
protein or mRNA in the atherosclerotic lesions of both
humans12 13 and
experimental animals.14 Our
results confirm and extend previous reports of the induction of iNOS in
atherosclerotic vessels. iNOS may have paradoxical effects on the
development of atherosclerosis due to the antiatherogenic character of
NO5 6 7 8
and the proatherogenic property of NO-derived
peroxynitrite.15 23 24
Our study provides direct evidence that the overall impact of iNOS on
the progression of atherosclerosis is remarkably little. We know of
only 1 previous study in which iNOS knockout mice were used to examine
the influence of iNOS on vascular
lesions.25 Using a
transplant arteriosclerosis model in which an allogenic heart was
heterotropically transplanted into an
iNOS-/- or
iNOS+/+ recipient, those authors concluded
that iNOS has an antiarteriosclerotic effect, based on the
marked inhibition of intimal hyperplasia of coronary arteries in hearts
transplanted into iNOS+/+ mice. Experiments
with adenovirus-mediated iNOS gene transfer also demonstrated that iNOS
inhibited intimal hyperplasia in allograft
arteriosclerosis26 and
balloon-injured arteries.27
These findings are not in conflict with our result, because the
pathological processes that underlie intimal hyperplasia are not the
same as those in hyperlipidemic atherosclerosis. In intimal
hyperplasia, immunological reaction is the initial trigger and
neointimal smooth muscle cell migration and proliferation are the major
characteristics,25 whereas
modified LDL accumulation and monocyte/macrophage recruitment play
important roles in hyperlipidemic
atherosclerosis.28 As shown
in
Figures 5A
1, 5B, and 5B5, macrophages are the predominant
cells in the lesion, and the VCAM-1 induction, which is 1 of the
earliest events in the development of
atherosclerosis,28 was
comparable in iNOS-/- and
iNOS+/+ mice. Thus, induced iNOS appears not
to have affected the lesion size or adhesion molecule expression.
However, it may increase plaque instability, because a marked decrease
in extracellular collagen content was observed in the lesions of
iNOS+/+ mice compared with those of
iNOS-/- mice. The collagen content in the
lesion determines the biomechanical strength of the atherosclerotic
lesion and its vulnerability to
disruption.28 29 30
NO-induced apoptosis or inhibition of cell proliferation is unlikely to
account for the decreased collagen content, because lesion cellularity
was similar in both strains. Other possible mechanisms are NO-induced
inhibition of collagen
formation31 and the
activation of collagen-degrading
metalloproteinase.3 29 30 32 33
We have recently shown that peroxynitrite is a novel activator of
procollagenase,34 so the
finding that iNOS was colocalized with nitrotyrosine, which is formed
by peroxynitrite-induced
nitration,17 in the lesions
of iNOS+/+ mice suggests that iNOS-catalyzed
NO production may have enhanced peroxynitrite formation and activated
MMP. It remains to be examined whether MMP induction or inhibition of
collagen formation contributed to the reduced collagen content in our
experiment. As a next step, it would be useful to confirm our findings
by using mice crossed iNOS-/- and either
apoE or LDL receptor-/- mice, which
develop advanced atherosclerotic lesions that are more relevant to
human disease than the current
model.35
In conclusion, iNOS appeared to have neither a proatherogenic nor an antiatherogenic influence in hyperlipidemia-induced atherosclerosis under our in vivo conditions, but it does decrease the collagen content of the lesion, which may increase plaque instability. This finding may offer a novel therapeutic approach to the prevention of acute coronary events.
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Acknowledgments |
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This work was supported by a grant from Tokyo Biochemical Research Foundation of Japan and a Grant-in-Aid for Scientific Research (C) (11838018) from the Ministry of Education, Science, Sports and Culture.
Received July 6, 2000; revision received August 29, 2000; accepted September 12, 2000.
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