(Circulation. 1997;96:3048-3052.)
© 1997 American Heart Association, Inc.
Articles |
Prevention of Fatty Streak Formation of 17ß-Estradiol Is Not Mediated by the Production of Nitric Oxide in Apolipoprotein E– Deficient Mice
From INSERM U397 and Laboratoire de Physiologie, Institut L. Bugnard, Toulouse, France (R.E., F.B., J.-F.A.); Laboratoire de Pharmacologie, Faculté de Médecine, Rouen, France (V.R.); INSERM U325 et SERLIA, Institut Pasteur, Lille, France (C.F.); RPR-Gencell, Atherosclerosis Departement, Vitry sur Seine, France (N.D.); and Center for Molecular and Vascular Biology, Leuven, Belgium (P.H.).
Correspondence to J.F. Arnal, INSERM U397, Institut L. Bugnard, C.H.U. Rangueil, 1 ave Jean Poulhès, 31054 Toulouse Cedex, France. E-mail arnal{at}rangueil.inserm.fr
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Abstract |
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Background Estrogens have atheroprotective properties, the mechanisms of which remain obscure. Estrogens have recently been reported to increase endothelial NO synthase expression in castrated animals and to prevent the degradation of NO by decreasing superoxide anion production in cultured endothelial cells. In both cases, increased NO bioavailability would promote vasodilation, inhibit proliferation of the adjacent vascular smooth muscle, reduce platelet aggregation, and inhibit monocyte adhesion to the endothelium and the inflammatory reaction induced by cytokines, all key contributors in the development of atherosclerosis.
Methods and Results In the present work, the respective
roles of 17ß-estradiol and NO in the development of the
atherosclerotic process were investigated in castrated apolipoprotein
E–deficient (apo E KO) mice, which spontaneously develop fatty streak
lesions within 3 months.
N
-Nitro-L-arginine methyl ester
(L-NAME), an NO synthase inhibitor, 50 mg ·
kg-1 · d-1,
increased arterial blood pressure and decreased cerebellum
cGMP content, demonstrating the blockade of NO production, but
did not influence the atherogenic process in castrated apo E KO
mice.
Conclusions 17ß-Estradiol decreased the size of the aortic lesions approximately threefold, and the magnitude of this vasculoprotective effect was not altered by L-NAME. Moreover, L-NAME increased circulating malonyldialdehyde (MDA)-modified LDL, which was not altered by 17ß-estradiol, leading to a complete dissociation between circulating MDA-modified LDL and parietal lesions.
Key Words: atherosclerosis • apolipoproteins • lipoproteins • endothelium
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Introduction |
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The incidence of cardiovascular disease, the leading cause of mortality in western societies,1 is higher in men than in premenopausal women but increases in postmenopausal women. An abundance of epidemiological data supports a role for estrogens in this atheroprotective effect, prompting recommendations for their widespread use in postmenopausal estrogen replacement therapy.2 However, the mechanism(s) whereby this protection is mediated has remained obscure. It has traditionally been thought to be due to potentially favorable changes in blood lipids and lipoproteins,2 but a number of animal studies strongly suggest a direct effect on the vascular wall.3 4 5 6 7 8 It was recently demonstrated9 10 that E2 also prevents fatty streak formation in the arterial wall of apo E KO mice, confirming the direct action of E2 on cells of the vascular wall and showing that this animal model is well adapted to characterization of the molecular mechanisms that mediate E2 effects in this process.
Different experimental7 8 11 and clinical
studies12 have suggested that the mechanism by which
E2 is active is the increased bioavailability of EDRF,
which is NO or a related nitrosocompound.13 14 Such an
increase could result from enhanced NO production, because
endothelial NO synthase gene expression was reported to
be increased by E2 in castrated guinea pigs and
rats.15 16 It could also result from a decreased breakdown
of NO, because we found that
O2-
production of
cultured bovine aortic endothelial cells was decreased
by estrogen treatment.17 In a poorly understood balance,
O2- interacts with NO to form
peroxynitrite, thereby destroying NO and diverting
O2- away from its dismutation
product, hydrogen peroxide.18 Besides regulation of
vascular tone and smooth muscle cell proliferation, NO might also play
an atheroprotective role19 20 by inhibiting leukocyte
adhesion to the endothelium,21 monocyte
chemotaxis,22 and inflammatory reaction induced by
cytokines.23 Indeed, inhibition of NO
production during the initial weeks of cholesterol
feeding in rabbits accelerates atherogenesis,24 25 and it
has been reported that NO production is decreased in
atherosclerotic vessels from both human26 27 and animal
models.28 29 Finally, NO probably protects against the
late events of atherosclerosis, such as thrombosis, by
inhibiting platelet adhesion and aggregation.19
To clarify the role of NO bioavailability in the vascular wall as a mediator of the prevention of fatty streak formation by estrogens, ie, the early stages of atherosclerosis, we sought to examine the effects of the inhibition of NO production in the development of lesions in castrated apo E KO mice treated or not treated with E2.
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Methods |
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Animals
Apo E KO mice were obtained by breeding three couples bought from the Jackson Laboratory (sixth generation of back-cross from 129/B6 F1 heterozygotes to C57BL/6). C57BL/6 mice were also obtained from the Jackson Laboratory. They were housed in stainless steel cages in groups of 5, kept in a temperature-controlled facility on a 12-hour light-dark cycle, and fed normal laboratory mouse chow containing 4.3% fat and 0.02% cholesterol. All experimental protocols were performed in accordance with the recommendations of the French Accreditation of Laboratory Animal Care.
Serum Hormone Concentrations
Radioimmunoassay kits for E2 were used according to
the manufacturer's instructions (Sorin Biomedica). Hormone levels were
assayed for each individual mouse in the same series of assays. The
intra-assay coefficient of variability was 4.5%. The assay
sensitivity, defined as 15% displacement of labeled tracer, was 0.5 pg
E2.
Lipid Analyses
Serum total cholesterol concentrations were measured
with Boehringer-Mannheim Biochemicals enzymatic assay kits. HDL
cholesterol content was determined after selective
precipitation of apo B–containing lipoproteins with phosphotungstic
acid/magnesium chloride (Boehringer-Mannheim) according to
Melhum et al.30 Comparative analysis of this
technique and an ultracentrifugation micromethod for
separation of serum lipoproteins31 showed a highly
significant linear correlation coefficient (n=60, r=.836,
P<.001). Serum apo A-1 concentrations were measured in
castrated mice and mice treated with 0.1 mg E2 by
immunonephelometry with mouse specific antibodies.30 Serum
levels of MDA-modified LDL were measured according to Holvoet et
al.32
Determination of the L-NAME Effective Dose
In preliminary experiments, groups of 5 C57BL/6 mice were given
L-NAME (Sigma), 0, 200, or 400 mg/L drinking water, ie, 0, 50,
or 100 mg · kg-1 ·
d-1, for 8 days. Mice were then
anesthetized with intraperitoneal ketalar.
After tracheotomy, they were intubated and ventilated with a
small-rodent respirator. Under a dissecting microscope, a PE10 catheter
was introduced into the left femoral artery. The PE10 catheter was
connected via a PE50 catheter to a Gould pressure transducer, and the
pressure signal was continuously recorded on a Gould recorder.
After a 10-minute stabilization period, three blood pressure tracings
were obtained (each 5 minutes apart), and blood pressure was averaged
from the three measurements.
Another series of C57BL/6 mice, given L-NAME 0 or 50 mg · kg-1 · d-1 (5 mice in each group) were killed with an overdose of ketalar and used for determination of cGMP (the second messenger of NO) content in the cerebellum. Cerebella were removed and frozen in liquid nitrogen, and the tissues were stored frozen (-80°C) until measurement of the cGMP level. Cerebella were homogenized with an all-glass homogenizer in 110 µL ice-cold 4N KOH solution, then 550 µL ice-cold 50 mmol/L sodium acetate buffer (pH 4.0) was added. cGMP levels were determined by enzyme immunoassay after acetylation according to the manufacturer's instructions (Cayman Chemical). All measurements of a single experiment were performed in duplicate and were made in the same enzyme immunoassay series, with an intra-assay coefficient of variability of 10%.
Effects of L-NAME and of E2 on Fatty Streak
Formation
In a second series of experiments, 40 female apo E KO mice were
anesthetized with intraperitoneal ketalar
and ovariectomized at 4 weeks of age. These mice were given either
60-day time-release E2 pellets (0.1 mg 17ß-estradiol,
Innovative Research of America) or placebo-containing pellets,
implanted subcutaneously into the back of the animals with a sterile
trochar and forceps and randomized into four groups: group 1 (n=10) was
provided with placebo-containing pellets, group 2 (n=10) was
provided with E2-containing pellets, group 3 (n=10) was
provided with placebo-containing pellets and given L-NAME 50 mg
· kg-1 · d-1
in the drinking water, and group 4 (n=10) was provided with
E2-containing pellets and given L-NAME 50 mg ·
kg-1 · d-1 in
the drinking water.
The body weight was determined 8 weeks later, after a 16-hour fast period. Blood was collected by retro-orbital bleeding in two ways: either without any chemicals (serum) or in 10% (vol/vol) of a buffer citrate containing 1 mmol/L EDTA, 20 µmol/L vitamin E, 10 µmol/L butylated hydroxytoluene, 20 µmol/L dipyridamole, and 15 µmol/L theophylline (plasma). These antioxidants and inhibitors were used to prevent in vitro LDL oxidation and platelet activation, respectively. Plasma and serum were separated by centrifugation for 10 minutes at 12 000g at 4°C. Mice were killed with an overdose of ketalar, and the cerebellum (for determination of cGMP content), the heart together with the ascending aorta (for fatty streak lesion analysis), and the uterus were quickly removed.
The lesions were estimated according to Paigen et al.33 Briefly, the heart and ascending aorta were washed in PBS and fixed with phosphate-buffered paraformaldehyde (4%, pH 7.4) for 24 hours. Each heart was frozen on a cryostat mount with OCT compound (Tissue-Tek) and stored at -70°C. One hundred sections 10 µm thick were prepared from the top of the left ventricle, where the aortic valves were first visible, up to a position in the aorta at which the valve cusps were just disappearing from the field. After drying for 2 hours, the sections were stained with oil red O and counterstained with Mayer's hematoxylin. Ten of the 100 sections, each separated by 90 µm, were used for morphometric evaluation with a computerized Biocom morphometry system. The mean lesion size, expressed in square micrometers, in these 10 sections was used to evaluate the lesion size of each animal.
Statistics
Results are expressed as mean±SEM. To test the respective role
of E2 treatment and of NO blockade (L-NAME) on different
parameters, a two-factor ANOVA was performed (comparison of
the four groups). When the F test allowed rejection of the null
hypothesis of no difference between groups, paired comparisons were
performed with the Scheffé procedure. A value of
P<.05 was considered statistically significant.
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Results |
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Determination of the L-NAME Effective Dose
In a preliminary experiment, the effective dose of L-NAME was determined by two approaches guided by data previously obtained in Wistar rats.34 We first measured blood pressure in anesthetized C57BL/6 mice given L-NAME 0, 50, or 100 mg · kg-1 · d-1 for 8 days. Compared with the mean arterial pressure of the controls (72±2 mm Hg), mice receiving L-NAME 50 or 100 mg · kg-1 · d-1 were similarly hypertensive (104±4 and 100±8 mm Hg, respectively, each P<.01 versus control). A dose of 50 mg · kg-1 · d-1 L-NAME was thus considered to maximally inhibit endothelial NO synthase in mice. Furthermore, L-NAME 50 mg/kg IV bolus did not induce any change in blood pressure in mice receiving L-NAME long-term, demonstrating the maximal blockade of NO synthase in L-NAME–treated mice (not shown).
The effect of L-NAME was then assessed in a tissue very rich in neuronal NO synthase and easy to dissect, ie, the cerebellum. The cGMP content in the cerebellum of C57BL/6 mice treated with L-NAME 50 mg · kg-1 · d-1 for 8 days demonstrated a 4.5-fold drop in the second messenger of NO and was not further influenced by a dose of 100 mg · kg-1 · d-1.
These two approaches demonstrated that a dose of 50 mg · kg-1 · d-1 L-NAME maximally inhibited the NO synthases (endothelial and neuronal) in mice.
Effects of L-NAME and of E2 on Fatty Streak
Formation
As shown in the Table
, L-NAME
treatment (50 mg · kg-1 ·
d-1) did not influence body weight, serum
total and HDL cholesterol, and apo A-I. It again decreased
the cGMP content in the cerebellum (4.4-fold drop, P<.0001)
and increased plasma MDA-modified LDL concentrations (Fig 1, P<.01).
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Under E2 treatment, serum E2 concentrations and
uterine weight were not significantly different whether associated with
L-NAME or not. E2 treatment did not influence body weight
and total cholesterol but decreased serum HDL
cholesterol and apo A-I concentrations; it increased the
cGMP content in the cerebellum in control animals, as previously
observed by Weiner et al in guinea pigs.15 E2
treatment did not influence plasma MDA-modified LDL concentrations (Fig 1
).
As shown in Fig 2, inhibition of NO
synthesis by L-NAME did not influence the constitution of fatty streak
in castrated apo E KO mice. In contrast, E2 highly
significantly protected the mice from this process. E2 was
as effective in L-NAME–treated as in untreated animals.
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Discussion |
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The lesions analyzed in 3-month-old apo E KO mice correspond to the constitution of fatty streak in terms of infiltration of the initial area by macrophages, a common step in the development of atherosclerosis in all species.35 36 In previous works, we10 and others9 observed that chronic E2 treatment of apo E KO mice decreased fatty streak formation and macrophage accumulation in a dose-dependent fashion. This atheroprotective effect occurred despite a major atherogenic lipid profile (ie, decreased serum HDL cholesterol and apo A-I concentrations), strongly suggesting a direct action of E2 on cells of the vascular wall. The dose-response curve analysis also revealed that a maximal effect was reached at 0.83 µg/d in females. Therefore, 60-day time-release E2 pellets containing twice this amount were used in the present study.
Arginine antagonists are useful tools to inhibit NO production in vitro and in vivo. Because NO is a short-lived free radical, it is difficult to measure its production in vivo, and endothelium-derived NO activity was indirectly evaluated by two approaches guided by data previously obtained in Wistar rats.34 It was initially planned to measure the aortic wall content of cGMP, but in agreement with data obtained by Rupin et al37 in hypercholesterolemic rabbits, great variability was observed. Changes in arterial blood pressure were then used to define the effective dose of L-NAME in C57BL/6 mice. We found that L-NAME 50 mg · kg-1 · d-1 maximally inhibited neuronal NO synthase activity and induced a level of hypertension that was not further increased by 100 mg · kg-1 · d-1, showing that this dose maximally inhibited both endothelial and neuronal isoforms of NO synthase in mice.
The L-NAME treatment was as effective in apo E KO mice as in C57BL/6
mice, as shown by inhibition of the neuronal NO synthase activity.
However, this treatment did not alter the aortic intimal lesion area
compared with castrated apo E KO mice. To the best of our knowledge,
the effect of inhibition of NO production has never been
reported in a mouse model of atherosclerosis. This
question has been previously addressed in a model of
hypercholesterolemia-induced
atherosclerosis in rabbit. In this model, Cayatte et
al25 and Naruse et al24 reported an
acceleration of the atherosclerotic process in the aorta with L-NAME,
but Böger et al38 did not find any effect on lesions
of the carotid artery. Our results agree with this latter study.
Although the effect of L-NAME on atherosclerotic lesions could be
dependent on the anatomic site and on the species, these observations
could have been expected from previously published data.
Endothelium-derived NO bioactivity is already depressed
during the atherosclerotic process, as demonstrated in both advanced
and early atherosclerosis in humans26 39
and animal models.28 29 Several mechanisms probably
contribute to the depressed EDRF activity. Atherosclerotic
arterial wall of hypercholesterolemic
animals is characterized by increased generation of
O2- ,38 40 41
which may result in accelerated degradation of NO and contribute to the
endothelial dysfunction.42 Several
mechanisms probably contribute to the depressed EDRF activity. In
particular, atherosclerotic arterial wall of
hypercholesterolemic animals is characterized by
increased generation of O2-
,38 40 41 which may result in accelerated degradation of
NO and contribute to the endothelial
dysfunction.41 42 Conversely, the protective effect of NO
against O2- -dependent
oxidative stress has been clearly demonstrated in vivo.18
Although the site(s) and mechanism(s) of oxidation leading to
circulating levels of MDA-modified LDL are unknown,32 the
present study demonstrates that the plasma levels of the modified
LDL are very significantly increased by L-NAME treatment, suggesting
that NO blockade increases oxidative stress in the compartment (ie,
liver, parietal wall, or any other organ) in which the modification is
taking place.
The atheroprotective effect of E2 demonstrated in rabbits
and primates3 4 6 was also observed in apo E KO
mice.9 10 This animal model provided an appropriate model
for investigating the effect of estrogens, because E2
decreased the fatty streak area approximately threefold in a
dose-dependent fashion. The atheroprotective effect occurred despite a
more atherogenic lipid profile (ie, decreased serum HDL
cholesterol and apo A-I concentrations, analyzed
and discussed in Reference 1010 ), which was maintained under L-NAME
treatment, strongly suggesting a direct action of E2 on
cells of the vascular wall in both conditions. The most important
finding of the present studies was that the atheroprotective effect
of E2 was not impaired by L-NAME treatment. In the same
animals, E2 was also unable to decrease plasma levels of
MDA-modified LDL, suggesting that in apo E KO mice, E2
could not counteract the oxidative stress responsible for the LDL
modification. These results excluded NO bioactivity, produced by either
an upregulation of the endothelial NO synthase
expression or a decreased O2-
production, as an important mediator of the protective effect
of estrogens against fatty streak formation in the conditions of our
studies.
Conclusions
The present data show that inhibition of NO production
does not accelerate the atherosclerotic process in apo E KO mice and
clearly demonstrate that in these animals, the atheroprotective effect
of E2 is not mediated by NO. They also show that
measurements of circulating MDA-modified LDL do not appear as a marker
of atherogenic risk because they may be completely dissociated from
parietal lesions, as evidenced in the present study. Our recent
observations that E2 prevents fatty streak formation and
monocyte/macrophage accumulation in the arterial
wall of apo E–deficient mice in parallel suggest that an influence of
E2 on the local inflammatory and immune components of early
atherosclerotic lesions, involving recruitment, migration, and
differentiation of the mononuclear cells, should now be considered.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by INSERM, the Ministère de la Recherche et de la Technologie, the Groupe de Recherche et de Réflexion Cardio-Vasculaire, and the Conseil Régional Midi-Pyrénées. We thank Jean-Paul Henry and Didier Ludovic for excellent technical assistance.
Received February 18, 1997; revision received June 6, 1997; accepted June 19, 1997.
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M. Noris, M. Todeschini, S. Zappella, S. Bonazzola, C. Zoja, D. Corna, F. Gaspari, F. Marchetti, S. Aiello, and G. Remuzzi 17beta -Estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases Am J Physiol Renal Physiol, October 1, 2000; 279(4): F626 - F635. [Abstract] [Full Text] [PDF]
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K. Kauser, V. da Cunha, R. Fitch, C. Mallari, and G. M. Rubanyi Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1679 - H1685. [Abstract] [Full Text] [PDF]
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L. Zhang, M. C. Fishman, and P. L. Huang Estrogen Mediates the Protective Effects of Pregnancy and Chorionic Gonadotropin in a Mouse Model of Vascular Injury Arterioscler Thromb Vasc Biol, September 1, 1999; 19(9): 2059 - 2065. [Abstract] [Full Text] [PDF]
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C. A. D. Nascimento, K. Kauser, and G. M. Rubanyi Effect of 17beta -estradiol in hypercholesterolemic rabbits with severe endothelial dysfunction Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1788 - H1794. [Abstract] [Full Text] [PDF]
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B. Darblade, C. Pendaries, A. Krust, S. Dupont, M.-J. Fouque, J. Rami, P. Chambon, F. Bayard, and J.-F. Arnal Estradiol Alters Nitric Oxide Production in the Mouse Aorta Through the {alpha}-, but not {beta}-, Estrogen Receptor Circ. Res., March 8, 2002; 90(4): 413 - 419. [Abstract] [Full Text] [PDF]
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