Cardiovascular Research 37 Ž1998. 738–747
Impairment of endothelium-dependent vasorelaxation in experimental
atherosclerosis is dependent on gender
a
Georg Kojda a,) , Bjorn
, Andreas Hacker a , Dorothea Perings a ,
¨ Husgen
¨
b
Ebbo Michael Schnaith , Eva Kottenberg a , Eike Noack a
a
Institut fur
Germany
¨ Pharmakologie, Heinrich-Heine-UniÕersitat,
¨ Moorenstr. 5, 40225 Dusseldorf,
¨
b
Praxis fur
¨ Laboratoriumsdiagnostik, Am Vechtufer 9, 48529 Nordhorn, Germany
Received 17 June 1997; accepted 25 September 1997
Abstract
Objective: Nitric oxide ŽNO. has been suggested to have antiatherosclerotic effects. It has also been demonstrated that there is a
greater basal release of endothelium derived relaxing factor ŽEDRF. in female as compared to male rabbit aorta, which also might have
beneficial effects in atherosclerosis. We thus sought to determine if gender influences the severity of atherosclerosis. Methods: We
studied 18 female and 18 male New Zealand White rabbits that were randomly divided in two groups of 9 animals each and fed either a
standard or a cholesterol diet Ž0.75%. for 15 weeks. Results: In cholesterol-fed rabbits the percentage of atherosclerotic lesions in the
aorta was identical in females and males and was inversely correlated with the maximal aortic relaxation to acetylcholine as assessed in
organ chamber experiments Žfemales: P - 0.0008, males: P - 0.0002.. Importantly, the cholesterol diet induced a significantly
Ž P - 0.025. more severe impairment of maximal vasorelaxation to acetylcholine in males from 78.4 " 1.2% to 29.4 " 10.2%. compared
to females Žfrom 84.4 " 1.2% to 60.7 " 8.5%.. Both male gender Ž P - 0.0001. and the extent of impairment of endothelium-dependent
relaxation Ž P - 0.0002. were associated with a reduced aortic sensitivity to S-nitroso-N-acetyl-D,L-penicillamine, which releases NO into
the organ bath. In contrast, the aortic sensitivity to the organic nitrates pentaerythritol tetranitrate and isosorbide 5-nitrate, which release
NO after enzymatic metabolization within the smooth muscle, was not reduced. Conclusions: These results suggest that the impairment
of endothelium-dependent vasorelaxation induced by atherosclerosis is dependent on gender. This may be due to a greater degradation of
extracellular NO in the vessel wall of males. q 1998 Elsevier Science B.V.
Keywords: Atherosclerosis; Endothelial dysfunction; Rabbit; Isosorbide mononitrate; Pentaerythritol tetranitrate; Nitric oxide; Gender; Superoxide
1. Introduction
The endothelium derived relaxing factor ŽEDRF. was
discovered in rabbit aorta w1x and identified as NO w2–4x or
a closely related compound w5x. It is synthesized in endothelial cells by a specific membrane bound enzyme w6x
converting L-arginine to NO and citrulline w7x. NO stimulates vascular soluble guanylate cyclase to produce cyclic
guanosine monophosphate, the intracellular second messenger promoting vasorelaxation w8,9x. In certain diseases
endothelium-dependent vasorelaxation is severely impaired
w10x. Atherosclerosis was the first disease shown to be
associated with impaired endothelium dependent vasorelaxation in rabbits, w11,12x primates w13x and also humans
w14x. In addition, impaired endothelium dependent vasorelaxation is an early pathological event in human atherosclerosis w15x that may occur even in the absence of
clinically detectable atherosclerotic lesions w16x.
An increasing body of evidence suggests that NO has
protective effects against development of atherosclerosis
w17x. Treatment of cholesterol-fed rabbits with L-arginine,
the precursor of NO biosynthesis, reduced the severity of
aortic and coronary atherosclerosis w18,19x, whereas longterm inhibition of endogenous NO synthesis had the oppo-
)
Corresponding author. Tel. Žq49-211. 811 2518; Fax Žq49-211.
811 4781.
Time for primary review 36 days.
0008-6363r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 7 . 0 0 2 6 8 - X
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
site effect w20x. In addition, organic nitrates reduce the
formation of atherosclerotic intima lesions and improve
endothelium-dependent vasorelaxation in cholesterol-fed
rabbits w21x. Thus, there is evidence that both endogenous
and exogenous NO may diminish atherosclerosis and
maintain endothelium-dependent vasorelaxation.
Interestingly, female rabbits have a greater aortic basal
release of EDRF than males w22x, suggesting that endothelial function may exhibit gender differences. It is known
that the atherogenic process in humans is dependent on
gender w23x. Young women have a lower incidence of
coronary artery disease than men of the same age and this
difference is presumably a function of the relative differences in estrogen and androgenic hormones w24x. Furthermore, pre- and postmenopausal estrogen intake is associated with a reduced incidence of coronary heart disease
w25x.
We, therefore sought to determine if gender could
influence the severity of atherosclerosis and the degree of
impairment of endothelium-dependent vasorelaxation,
which develops during hypercholesterolemia. For this purpose, we directly compared the extent of atherosclerotic
lesions and the endothelium-dependent vasorelaxation in
the aorta of female and male rabbits fed a cholesterol-rich
diet. Furthermore, we wished to gain information on the
mechanism underlying the impairment of endothelium-dependent vasorelaxation that develops during hypercholesterolemia. For this purpose, we compared the sensitivity of
aortic rings to NO that was released either directly into the
organ bath or directly into the aortic smooth muscle cells
using S-nitroso-N-acetyl-D,L-penicillamine ŽSNAP. or pentaerythritol tetranitrate ŽPETN. and isosorbide 5-nitrate
ŽISMN., respectively.
2. Methods
2.1. Animal preparation
Eighteen female and 18 male New Zealand White rabbits, 10–12 weeks of age were used. The animals were
housed individually in stainless steel cages at a temperature of 18–208C, a humidity of 50–60% and a day-nightrhythm of 12 h and received water ad libitum. Both female
and male rabbits were randomly divided in two groups and
fed 40 grkg per day of a standard or a cholesterol-enriched Ž0.75%. rabbit chow for 15 weeks. During this time
the body weight of every animal was determined weekly
and in some rabbits, concentration of plasma lipids Žsee
below. were also monitored. A separate group of female
New Zealand White rabbits, 20–22 weeks of age, was
used for preliminary studies.
Permission for these studies was provided by the regional government Ž26.4203.1-24r92 and 23.4203.148r93. and the experiments were performed according to
the guidelines for the use of experimental animals as given
739
by ‘Deutsches Tierschutzgesetz’ and to the ‘Guide for the
care and use of laboratory animals’ of the US National
Institutes of Health.
2.2. Studies on isolated Õessel segments
After a 24-h fast rabbits were anesthetized by injection
of a mixture of xylazine Ž5 mgrkg. and ketamine Ž25
mgrkg. into the tibialis muscle. Blood samples for determination of plasma lipids and estradiol concentration were
obtained from the middle ear artery. The animals were
killed and the entire thoracic and abdominal aorta was
dissected free and rapidly immersed in cold oxygenated
Ž95% O 2 q 5% CO 2 . Krebs–Henseleit solution ŽpH 7.4. of
the following composition Žmmolrl.: Naq 143.07, Kq
5.87, Ca2q 1.6, Mg 2q 1.18, Cly 125.96, HCOy
3 25.00,
H 2 PO4y 1.18, SO42y 1.18 and glucose 5.05. Four ring
segments Ž4 mm width. of thoracic aorta were mounted
between stainless steel triangles in a water jacketed organ
bath Ž378C. for measurement of tension-development as
recently described w26x. Previous experiments with KCl
Ž10–60 mmolrl. revealed an optimal resting tension of 2 g
for development of contractile function in the vessels.
After equilibration Ž1 h., contractile function of aortic
segments was tested by application of KCl Ž60 mmolrl..
This was followed by a cumulative application of phenylephrine Ž0.01–10 mmolrl., which resulted in a maximal
tension of approximately 12 g Žsee Section 3.5.. Function
of endothelium was then examined by cumulative addition
of acetylcholine Ž0.01–10 mmolrl. following precontraction with a single dose of phenylephrine. A comparable
submaximal aortic tension within the different groups was
achieved by adding 0.5 mmolrl phenylephrine to rings
from female rabbits Žstandard diet: 5.9 " 0.6 g; cholesterol
diet: 5.1 " 0.5 g. and by adding 0.2 mmolrl of phenylephrine to aortic rings of male rabbits Žstandard diet
5.1 " 0.6 g; cholesterol diet 4.2 " 0.4 g.. Addition of 0.5
mmolrl phenylephrine to aortic rings from male rabbits
resulted in a significantly stronger tension Žstandard diet:
7.52 " 0.4 g; cholesterol diet: 7.28 " 0.4 g.. In aortic rings
of male rabbits the cumulative application of acetylcholine
was repeated in the presence of 10 mmolrl oxyhemoglobin, which scavenges extracellular NO Ž2 rings of each
animal., and 200 Urml superoxide dismutase Ž2 rings of
each animal.. Both, oxyhemoglobin and SOD were added
to the vessel segments 15 min before starting these experiments. Thereafter the aortic rings were divided in subgroups and the vasorelaxations to different type of NO
donors such as SNAP Ž1 nmolrl–10 mmolrl., ISMN Ž0.1
mmolrl–1 mmolrl. and PETN Ž1 nmolrl–100 mmolrl.
were studied following precontraction with phenylephrine
Ž3 mmolrl.. In rings of male rabbits the vasorelaxant
activity of SNAP was also evaluated in presence of superoxide dismutase Ž200 Urml.. SOD was added to the vessel
segments 15 min before starting these experiments. Each
drug was studied in at least one thoracic ring from each
animal.
740
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
Preliminary experiments revealed that the maximal
achieved concentration of dimethylsulfoxide Ž0.01%.,
which was necessary to dissolve SNAP and PETN, exhibited no influence on aortic contractile function. In another
set of preliminary experiments using rabbit aorta we confirmed that SNAP released NO largely extracellulary as
the p D 2 value of the dose-dependent relaxation caused by
SNAP showed a marked decrease in the presence of 1 mM
oxyhemoglobin Žfrom 7.24 " 0.08 to 6.23 " 0.09, respectively.. In contrast, this concentration of oxyhemoglobin
had no effect on vasorelaxations caused by the organic
nitrates PETN and ISMN.
2.3. Determination of plasma lipids and estradiol
Blood samples were collected in glass vials and centrifuged for 15 min at 4000 = g. The resulting supernatants
were directly used for enzymatic determination of cholesterol, high density lipoproteins ŽHDL. and triglycerides
according to published methods w27–29x. Frozen plasma
samples were thawed and used for determination of estradiol by gas chromatographyrmass spectrometry ŽGCrMS..
The analyte and the internal standard were extracted from
the plasma samples Ž1 ml. by liquidrliquid extraction and
derivatized to their pentaflourobenzoyl derivatives. These
were chromatographed on a DB-5 fused silica capillary
column ŽJ & W Scientific, Folcom, CA, USA. in a Carlo
Erba Mega series gas chromatograph ŽCarlo Erba, Italy..
Detection was by negative ion mass spectrometry ŽNermag
R 10-10, France. after chemical ionisation ŽNICI-MS.. As
reagent gas ammonia was used. Calibrated linear working
range was from 5 to 100 pg estradiol per ml plasma. Thus,
lower limit of quantification was 5 pg estradiol per ml
plasma.
2.4. Staining and determination of atherosclerotic lesions
After the tension experiments, all segments of throracic
aorta the aortic arch and the abdominal aortas of each
animal were fixed and stained as described previously w21x.
The tissues were fixed in formol solution Ž10 ml formaldehyde solution 37% and 90 ml distilled water containing
2.5 g Ca-acetate, pH 6.8. and stored after repeated changing of the fixative at 48C. Staining of the lesions was
achieved by pretreating the vessels for 2–3 min in 70%
ethanol and then placing them into a staining solution of
the following composition: Sudan IV 0.1 g, acetone 50 ml,
ethanol 70% 50 ml. The staining procedure was followed
by a repeated rinsing with 70% ethanol. The stained
preparations were stored in formol until examination.
The percentage of the stained area Žatherosclerotic area.
related to the whole intimal surface was determined in
each segment by use of a laser-scanning apparatus ŽMolecular Dynamics Densitometer, model 300 A, Sunnyvale,
CA 94086. and calculated by use of Image Quant, Version
3.15. The aortic segments were photographed under repro-
ducible conditions Žcamera: Mamiya RZ 67 professional,
objective: Mamiya Sekor 2.8r110 mm, film: Kodak TMX
6025, 6 = 7 cm negatives, black-and-white.. Both negatives and developed photographs were easily scanned and
yielded comparable results. The percentage values presented in Section 3.4 are based on scanned negatives.
2.5. Substances and solutions
SNAP was synthesized in our laboratory according to
Field et al. w30x. The reaction product was recrystallized
once in methanol. Analysis of SNAP included a thin-layer
chromatography on Merck w RP18 plates with
methanolrwater Ž7:3, vrv. as a solvent. To determine free
sulfide groups spray detection was performed with 1.5 g of
sodium nitroprusside dissolved in a 10-ml aliquot of a
solution containing 5 ml 2 N hydrochloric acid, 95 ml
methanol and 10 ml ammonia Ž25%.. We found no detectable amount of free sulfides. Spray detection of the
nitroso group was achieved by two steps: Zn powder in
methanol and sulfanilic acidra-naphthylamine 250 mg
each in 30% acetic acid. This procedure resulted in a
concentration-dependent increase of the colored area. The
preparation decomposed at 148–1508C. In addition, the
NO liberating property of synthesized SNAP was determined using the oxyhemoglobin assay w31x. With 1 mmolrl
of SNAP, the rate of release of NO was 1.28 " 0.01
mmolrl per min Ž n s 3.. Furthermore, the spontaneous
release of NO from SNAP Žat 378C and pH 7.4. was
confirmed by direct measurement of NO using an NO
electrode ŽISO-NO, WPI-Instruments, Berlin, Germany.. A
concentration of 100 mmolrl SNAP produced a peak
concentration of 221 nmolrl NO.
Oxyhemoglobin was freshly prepared every day as described previously w31x ISMN and PETN were provided by
ISIS-Pharma, Zwickau, Germany; GTN was provided by
Schwarz Pharma, Monheim, Germany; Sudan VI, acetylcholine and phenylephrine were obtained from Sigma,
Deisenhofen, Germany; all other chemicals were obtained
from Merck, Darmstadt, Germany. GTN Ž4.404 mmolrl.
was available in isotonic glucose solution and used as
stock solution. Stock solutions Ž10 mmolrl. of acetylcholine, ISMN and phenylephrine in distilled water, those
of SNAP and PETN Ž100 mmolrl. in dimethylsulfoxide
were prepared daily, diluted with buffer as required and
kept on ice and protected from daylight until use. All
concentrations indicated in the text, figures and tables are
expressed as final bath concentrations.
2.6. Statistics
The concentrations of the half-maximal vasocontractile
effect of phenylephrine Žp D 2 values. were calculated from
the individual concentration-effect-curves as proposed by
Hafner et al. w32x and are related to the maximal achieved
vasoconstriction. Vasorelaxation due to treatment with the
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
drugs is expressed as percentage of the contractile response achieved with phenylephrine Ž0.2, 0.5 or 3 mmolrl.
at the beginning of the experiments. The concentrations of
acetylcholine or the nitrovasodilators for half-maximal inhibition of phenylephrine-induced preconstriction Žp D 2
values. were calculated as described above. All data were
analyzed by one-way analysis of variance ŽANOVA. with
subsequent Student–Newman Keuls test ŽSAS PC Software 6.04, PROC ANOVA, also used to calculate the
correlations and the p D 2 values. and are expressed as
mean values and standard error of the mean ŽS.E.M... The
p D 2 values, representing the negative logarithms of the
half-maximal effective concentrations, were taken to test
for significant differences and P - 0.05 was considered
significant.
3. Results
3.1. Body weight
At the beginning of the experiments the average body
weight of the rabbits was 1996 " 48 g Žfemales. and
2201 " 44 g Žmales.. Daily feeding with 40 grkg of the
different diets resulted in a weekly increase in body weight
of approximately 35–50 g Žstandard diet. and 50–65 g
Žcholesterol diet.. The chows were completely consumed
each day.
3.2. Plasma lipids
The cholesterol concentration measured at the end of
the feeding period was significantly different between
males Ž1358 " 111 mgrdl. and females Ž907 " 85.8
mgrdl, P - 0.0001.. The cholesterol diet did not affect
the concentration of HDL and triglycerides Ždata not
shown.. The cholesterol concentration in standard-fed rabbits showed no significant difference Žmales: 46 " 5.1
mgrdl; females: 69.8 " 10.0 mgrdl.. Control measurements of the plasma concentration of cholesterol performed at various times during the feeding period in a
subgroup of female rabbits revealed a rapid increase in
plasma cholesterol to a high level, which remained constant during at least 60 days of the feeding period.
3.3. Plasma leÕels of estradiol
The concentration of estradiol in the plasma of cholesterol-fed female and male rabbits, as determined by gas
chromatographyrmass spectrometry was always below the
detection limit of 5 pg estradiolrml plasma. In a few
samples extrapolation beyond the detection limit yielded
estradiol concentrations of 1.2–1.9 pgrml. In experiments
using plasma spiked with 30 pgrml estradiol the recovery
rate was ) 90%.
741
Table 1
Atherosclerotic lesion area in the intima of cholesterol fed rabbits
Aortic region
Aortic arch
Thoracic aorta
Abdominal aorta
Area of intima lesions w%x
females
males
67.5"2.8
44.6"3.4
47.5"4.3
57.8"6.4
40.6"4.7
40.3"5.5
Values are means"S.E.M. of determinations with aortas from 9 female
and 9 male rabbits fed a cholesterol diet. The area of intima lesions was
determined by a computerized laser-scanning approach after staining with
Sudan IV and is related to the whole surface of the intima Žno significant
differences between females and males..
3.4. Atherosclerotic lesions
Quantification of the severity of atherosclerotic lesions
in the aorta was performed by measuring the percentage of
the area of the Sudan IV-stained atherosclerotic lesions
related to the whole aortic inner surface using a computerized laser-scanner. The aortas of cholesterol-fed rabbits of
each group exhibited easily visible atherosclerotic lesions,
which were more pronounced in the aortic arch than in the
thoracic and the abdominal aortic segments ŽTable 1.. The
distribution of these aortic lesions was not homogenous
and the measured percentages of the involved area varied
within the thoracic ring segments used for the tension
studies. We observed no significant differences between
female and male rabbits.
3.5. Vascular smooth muscle contractile actiÕity
The contractile activity of thoracic ring segments was
evaluated by cumulative application of phenylephrine. The
maximal contractile response occurred at a concentration
of 10 mmolrl ŽFig. 1.. Preliminary experiments Ždata not
shown. and experiments with aorta from standard-fed rabbits ŽFig. 1. showed that rings from males respond significantly stronger to low concentrations of phenylephrine
than rings from females Žat 0.1 mmolrl 1.80 " 0.28 g vs.
1.0 " 0.2 g, respectively.. There was a significant difference Ž P - 0.0001. between the p D 2 values of phenylephrine evaluated in cholesterol-fed female Ž6.12 " 0.05.
and male rabbits Ž6.45 " 0.03., whereas in the standard fed
groups this value was comparable Žfemales: 6.25 " 0.02;
males 6.28 " 0.05.. The maximal tension induced by 10
mmolrl phenylephrine was identical in each group ŽFig.
1..
3.6. Function of aortic endothelium
Function of aortic endothelium was assessed by application of increasing doses of acetylcholine to ring segments
submaximally precontracted with phenylephrine to a similar tension Žsee Section 2.2.. Both, cholesterol diet Ž P 0.0001. and male gender Ž P - 0.025. significantly reduced
acetylcholine induced vasorelaxation.
742
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
tima-lesions ŽFig. 3, Table 2. demonstrating that in both
groups these events are directly related.
3.7. Effects of nitroÕasodilators
To determine whether an enhanced breakdown of EDRF
is involved in the impairment of endothelium-dependent
vasorelaxation that was observed in atherosclerotic rabbit
aorta, we compared dose–response curves of the NO-donor
SNAP, which releases NO into the organ bath, with those
of the organic nitrates ISMN and PETN, which release NO
after enzymatic metabolization within the smooth muscle.
In aortic segments of both female and male cholesterolfed rabbits SNAP was significantly less potent than in the
respective female and male controls ŽFig. 4, Table 3,
P - 0.0002.. In addition, we found in both cholesterol-fed
groups a significant direct correlation between the p D 2
values of SNAP evaluated in each animal and the corresponding maximal relaxation to acetylcholine ŽFig. 5, Table
2.. Thus, impairment of endothelium-dependent relaxation
induced by atherosclerosis is associated with an attenuated
vasorelaxant response to a NO donor, which releases NO
into the organ bath. The statistical analysis also showed
that SNAP was significantly more potent in aortic rings of
standard- and cholesterol-fed female rabbits Ž P - 0.0001;
p D 2 values see Table 3. as compared to male rabbits. In
contrast, the activity of the organic nitrates ISMN or PETN
was not reduced in atherosclerotic aorta ŽTable 3.: Neither
gender nor the feeding protocol showed a significant influence on the efficacy of PETN. ISMN induced a signifiFig. 1. The vasocontractile activity of phenylephrine in isolated thoracic
aorta of standard-fed Župper panel. and cholesterol-fed Žlower panel.
female and male New Zealand White rabbits. Each concentration–response curve was plotted by taking the respective mean values of nine
separate experiments ŽS.E.M. indicated by bars.. The respective p D 2
values of phenylephrine in aortic rings of cholesterol-fed rabbits are
significantly different Ž P - 0.05, data see Section 3.5..
In vessel segments of all standard-fed rabbits, acetylcholine Ž1 nmolrl–0.5 mmolrl. produced vasorelaxations,
which were similar between males and females ŽFig. 2.. In
accordance, the p D 2 values of acetylcholine in rings of
female Ž6.91 " 0.03. and male rabbits Ž6.99 " 0.10. were
identical.
Likewise, the percentage of aortic vasorelaxation was
similar at all concentrations of acetylcholine, except the
highest, where acetylcholine produced constriction in the
males and further vasodilation in the females.
In vessel segments from cholesterol-fed rabbits the
relaxant response to acetylcholine was significantly impaired ŽFig. 2, P - 0.0001.. Importantly, this effect was
much more pronounced in rings of male compared to rings
of female rabbits ŽFig. 2.. In both cholesterol-fed groups,
the extent of endothelium-dependent vasorelaxation was
inversely correlated with the degree of atherosclerotic in-
Fig. 2. The vasorelaxant activity of acetylcholine in isolated thoracic
aorta segments of female and male New Zealand White rabbits fed either
a standard or a cholesterol-rich diet. The vasodilatory response is expressed as percentage of precontraction induced by phenylephrine. Each
concentration–response curve was plotted by taking the respective mean
values of nine separate experiments ŽS.E.M. indicated by bars.. Maximal
vasorelaxation of atherosclerotic aorta is significantly greater in females
as compared to males.
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
Fig. 3. Correlation between the percentage of maximal vasodilatation
induced by acetylcholine in segments of isolated thoracic aorta of female
ŽA. and male ŽB. New Zealand White rabbits Žfour rings per animal, nine
animals per gender. precontracted with phenylephrine and the percentage
of Sudan IV-stained atherosclerotic lesions detected at the luminal surface
of the same vessel segments. The levels of significance were P - 0.0008
Žfemales. and P - 0.0002 Žmales.. Mean values of maximal vasorelaxation are plotted in Fig. 2. Mean values of the area of luminal atherosclerotic lesions are shown in Table 1 Ž asslope, bs interception,
r s correlation coefficient..
743
Fig. 4. The vasorelaxing activity of S-nitroso-N-acetyl-D,L-penicillamine
ŽSNAP. in isolated segments of thoracic aorta of female ŽA. and male ŽB.
New Zealand White rabbits fed either a standard diet or a cholesterol diet.
The vasodilatory response is expressed as percentage of precontraction
induced by phenylephrine. Each dose–response curve was plotted by
taking the respective mean value of nine separate experiments ŽS.E.M.
indicated by bars..
Table 2
Correlation coefficients for the linear relationships
Gender
x-axis
y-axis
a
Male
plasma cholesterol
intimal lesion area
relaxation to acetylcholine
intimal lesion area
relaxation to acetylcholine
y0.01
0.02
0.00
0.01
52.84
8.31
45.72
52.3
Male
Female
intimal lesion area
relaxation to acetylcholine
relaxation to acetylcholine
y1.65
y2.21
102.28
155.89
y0.936
y0.950
Male
Female
relaxation to acetylcholine
p D 2 value for SNAP
p D 2 value for SNAP
0.01
0.01
5.77
6.15
0.727
0.897
Female
b
r
P
0.0201
0.253
0.012
0.118
0.61
0.51
0.98
0.76
0.0002
0.0008
0.03
0.001
Linear correlations of parameters given in the x-axis and y-axis columns. The data of these parameters were evaluated in ring segments of isolated thoracic
aorta of female and male New Zealand White rabbits Ž1–4 rings per animal, 9 animals per group. fed a cholesterol-rich diet. Only data deriving from
experiments within the same ring segments were correlated. Mean results are given in Table 1 Žintimal lesion area. and in Section 3.2 Žplasma cholesterol.
and Sections 3.6 and 3.7 Žmaximal relaxation to acetylcholine.. Plots of the significant relationships are given in Fig. 3 and 5 Ž as interception, bsslope,
r s correlation coefficient, P s level of significance..
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
744
Table 3
The vasorelaxant efficacy of ISMN, PETN and SNAP in isolated segments of thoracic aorta from female and male rabbits fed different diets
Gender
Diet
ISMN Žylog molrl.
PETN Žylog molrl.
SNAP Žylog molrl.
Female
standard diet
cholesterol diet
standard diet
cholesterol diet
4.17"0.15
4.67"0.09 )
4.16"0.05
4.05"0.21
7.18"0.14
7.39"0.09
7.46"0.10
7.11"0.26
7.34"0.09
6.91"0.03 )
6.76"0.06
6.22"0.15 )
Male
Concentrations of the nitrovasodilators Žmean"S.E.M., ns9. that produced a half-maximal inhibition of the vessel tension elicited by 3 mmolrl
phenylephrine as calculated from the individual concentration–response curves Ž ) P - 0.02, cholesterol diet vs. standard diet..
cantly more potent vasorelaxation in aortic rings of cholesterol-fed female rabbits ŽTable 3, P - 0.02..
3.8. Effects of superoxide dismutase
Superoxide dismutase Ž200 Urml. significantly improved the dose-dependent relaxation of aortic rings of
standard-fed male rabbits elicited by SNAP as indicated by
the increase of the p D 2 value from 6.76 " 0.1 to 7.06 " 0.1
Ž n s 9.. In aortic rings of cholesterol-fed animals superoxide dismutase Ž200 Urml. did not affect endothelium-dependent relaxation. The maximal relaxation to acetylcholine in aortas of cholesterol-fed male rabbits was 29.1
" 7.2% and 29.4 " 10.2% in the absence and presence of
SOD, respectively. Aortic relaxation induced by SNAP
was not changed after application of SOD, as indicated by
similar p D 2 values in the absence Ž6.22 " 0.15, see Table
3. and presence of this enzyme Ž6.25 " 0.2..
4. Discussion
Fig. 5. Correlation between the efficacy Žp D 2 values. of the spontaneous
NO-donor S-nitroso-N-acetyl-D,L-penicillamine ŽSNAP. in segments of
isolated thoracic aorta of female ŽA. and male ŽB. New Zealand White
rabbits Žfour rings per animal, nine animals per gender. precontracted
with phenylephrine and the percentage of maximal vasodilatation induced
by acetylcholine in the same segments. The levels of significance were
P - 0.001 Žfemales. and P - 0.03 Žmales.. The mean p D 2 values of
SNAP are given in Table 3 and the corresponding mean dose–response
curves a depicted in Fig. 4. The mean values of vasorelaxation induced
by acetylcholine are plotted in Fig. 2 and given in Results Ž asslope,
bs interception, r s correlation coefficient..
The new findings of the present study are that male
gender significantly affects the severity of endothelium-dependent vascular relaxations to acetylcholine and endothelium-independent relaxations to the NO-donor SNAP in
cholesterol-fed rabbits. In contrast, cholesterol-feeding had
little effect on relaxations to the organic nitrates PETN or
ISMN in either male or female animals.
In this study, the severity of atherosclerosis was not
different between male and female rabbits. In each group
about one-half of the intimal aortic surface was covered by
atherosclerotic lesions ŽTable 1.. The relationship between
the extent of atherosclerosis and the impairment of endothelium-dependent vascular relaxation, however, was
quite different between male and female animals, as
demonstrated in Fig. 4. Examining this relationship, it is
apparent that for a percent lesion area of 50%, the maximal
relaxation to acetylcholine averaged approximately 20% in
males and 40% in females. Overall the maximal relaxations to acetylcholine were more than twice as great in
cholesterol-fed females as compared to males ŽFig. 3.. Our
results are in accord with previous studies in primates and
rabbits in which exogenous estrogens have been shown to
improve endothelium-dependent vascular relaxations in
cholesterol-fed animals subjected to previous oopherectomy w33x. The new finding in this study is that there is an
intrinsic difference between female and male animals in
their susceptability for development of abnormal endothelium-dependent vascular relaxations during cholesterolfeeding. This would indicate that endogenous hormone
status in addition to exogenous hormone replacement may
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
modulate this difference in response to hypercholesterolemia.
Our findings may provide some insight into the mechanisms of altered endothelium-dependent vascular relaxation in cholesterol-fed rabbits. To address this issue, we
examined responses to two forms of exogenous nitrovasodilators. We used SNAP as a method of generating NO
extracellularly. Although there is some debate as to the
rate and method of release of NO from various nitrosothiols, there is general agreement that these are capable of
release of NO in solutions w34–36x. In preliminary studies
Žsee Section 2.2., we were able to confirm previous studies
showing that SNAP spontaneously released NO at a rate of
1.28 " 0.01 mmolrl per min in our buffer solutions. NO
released in this form is exposed to the interstitial space and
must traverse the vascular smooth muscle cell membrane
to elicit vascular relaxation. We also examined the effect
of two organic nitrates, PETN and ISMN. Again, there is
debate regarding the mechanisms responsible for release of
NO from these drugs, but it is generally agreed that they
undergo biotransformation to NO in the vascular smooth
muscle cells w37x. Thus, NO released in this compartment
may not be subjected to the same degradation pathways as
NO released in the extracellular space. Our findings that
relaxations to NO generated outside of the vascular smooth
muscle cells were abnormal in atherosclerotic vessels,
while those to NO generated intracellularly were preserved
suggest that extracellular NO may be inactivated in atherosclerotic vessels. Altered endothelium-dependent vascular
relaxations are similar to relaxations to SNAP, in that the
endothelium-derived NO must also transverse the extracellular space. These findings are in keeping with recent
observations that vessels from cholesterol-fed rabbits produce excess superoxide anions and that treatment with
membrane targeted forms of superoxide dismutase improve endothelium-dependent vascular relaxations w38,39x.
Taken with these previous studies, our findings would
suggest that the site of NO degradation in these studies
may be outside of the vascular smooth muscle. It is of note
that altered vasodilations to sodium nitroprusside, which
also releases NO outside the smooth muscle cells, have
been found to be abnormal in young hypercholesterolemic
individuals w40x.
We found that the relaxations to acetylcholine and
SNAP were relatively preserved in cholesterol-fed female
as compared to male animals. This would suggest that the
rate of degradation of NO released from SNAP and by the
endothelium is less in female compared to males. The
mechanisms responsible for this remain unclear, however,
changes in expression of either superoxide-generation systems or superoxide dismutase may be involved. In preliminary experiments, we sought to correct abnormalities of
endothelium-dependent vascular relaxation and relaxations
to SNAP by acutely treating vessels with CurZn SOD
Ž200 Urml.. While this had a small effect in normal male
animals, there was no effect in the cholesterol-fed rabbits.
745
This does not exclude a role for superoxide in this process.
Because of its hydrophilicity, CurZn SOD is sterically and
electrostatically repelled from surface of vascular cells and
it is not surprising that conventional superoxide dismutase
was ineffective in our experimental setting w38,41x.
Previously, it has been suggested that vessels from
female animals produce more NO under basal conditions
than do vessels from male animals w22x. Others have
reported that estradiol decreases superoxide production in
endothelial cells w42x. Our findings are particularly compatible with these latter studies, in that a decrease in vascular
superoxide production would enhance relaxations to exogenous and endogenous NO. It is interesting to speculate
that during cholesterol-feeding and the development of
atherosclerosis, the factors leading to increases in the
oxidative state of the vessel may be modified by endogenous estrogens present in the female animals. Over the
long term, one would expect that such changes would
decrease the severity of atherosclerosis. This was not
observed in the present study, as the extent of atherosclerotic involvement was similar between the genders. We
attempted to correlate our experimental findings with endogenous estradiol levels. Unfortunately, the levels of
estradiol in these animals were consistently below the limit
of detection by our assay Ž5 pgrml.. Others have reported
similarly low levels of estradiol in rabbits w43x.
Cholesterol feeding resulted in slightly higher cholesterol levels in the male compared to the female animals. It
is unlikely that this contributed to the differences in endothelium-dependent vascular relaxations in this study. We
found that there was no correlation between cholesterol
levels and acetylcholine responses in either groups of
rabbits ŽTable 2.. Thus, acetylcholine responses were often
quite abnormal in male rabbits in whom cholesterol levels
were only modestly increased. Further, there was a wide
range of cholesterol levels in both groups of animals. To
ascertain if the data in the male animals were skewed by
spuriously high levels in a minority of male rabbits, we
performed another analysis of acetylcholine-induced relaxations in which data from the two animals with the highest
cholesterol levels were eliminated. In this analysis, the
average acetylcholine responses continued to be different
in male and female cholesterol-fed animals.
In these studies, we attempted to attain similar levels of
preconstriction to study relaxations to acetylcholine. This
required slightly higher concentrations of phenylephrine in
female as compared to male rabbits in both control and
cholesterol-fed animals. It is unlikely that this affected the
degree of relaxation to acetylcholine. All concentrations
were submaximal and others have shown that the degree of
relaxations to endothelium-dependent agonists are similar
over these ranges of submaximal constriction w44x.
In summary, our findings suggest that male and female
animals respond differently in terms of the impairment of
endothelium-dependent vascular relaxations which develop
during hypercholesterolemia. This occurs in spite of simi-
746
G. Kojda et al.r CardioÕascular Research 37 (1998) 738–747
lar degrees of development of atherosclerosis. Our findings
suggest that these differences may be due to enhanced
degradation of NO in the vessels of male animals. This
may reflect either increased production of reactive oxygen
species or decreased radical scavenging mechanisms in
these vessels.
w16x
w17x
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft SFB 242; Projekt A11.
w18x
w19x
w20x
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