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Cardiovascular Research 1998 37(3):738-747; doi:10.1016/S0008-6363(97)00268-X
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Impairment of endothelium-dependent vasorelaxation in experimental atherosclerosis is dependent on gender

Georg Kojdaa,*, Björn Hüsgena, Andreas Hackera, Dorothea Peringsa, Ebbo Michael Schnaithb, Eva Kottenberga and Eike Noacka

aInstitut für Pharmakologie, Heinrich-Heine-Universität, Moorenstr. 5, 40225 Düsseldorf, Germany
bPraxis für Laboratoriumsdiagnostik, Am Vechtufer 9, 48529 Nordhorn, Germany

* Corresponding author. Tel. (+49-211) 811 2518; Fax (+49-211) 811 4781.

Received 17 June 1997; accepted 25 September 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Nitric oxide (NO) has been suggested to have antiatherosclerotic effects. It has also been demonstrated that there is a greater basal release of endothelium derived relaxing factor (EDRF) 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 (0.75%) 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 (females: P<0.0008, males: P<0.0002). Importantly, the cholesterol diet induced a significantly (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 (from 84.4±1.2% to 60.7±8.5%). Both male gender (P<0.0001) and the extent of impairment of endothelium-dependent relaxation (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.

KEYWORDS Atherosclerosis; Endothelial dysfunction; Rabbit; Isosorbide mononitrate; Pentaerythritol tetranitrate; Nitric oxide; Gender; Superoxide


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The endothelium derived relaxing factor (EDRF) was discovered in rabbit aorta [1]and identified as NO [2–4]or a closely related compound [5]. It is synthesized in endothelial cells by a specific membrane bound enzyme [6]converting L-arginine to NO and citrulline [7]. NO stimulates vascular soluble guanylate cyclase to produce cyclic guanosine monophosphate, the intracellular second messenger promoting vasorelaxation [8, 9]. In certain diseases endothelium-dependent vasorelaxation is severely impaired [10]. Atherosclerosis was the first disease shown to be associated with impaired endothelium dependent vasorelaxation in rabbits, [11, 12]primates [13]and also humans [14]. In addition, impaired endothelium dependent vasorelaxation is an early pathological event in human atherosclerosis [15]that may occur even in the absence of clinically detectable atherosclerotic lesions [16].

An increasing body of evidence suggests that NO has protective effects against development of atherosclerosis [17]. Treatment of cholesterol-fed rabbits with L-arginine, the precursor of NO biosynthesis, reduced the severity of aortic and coronary atherosclerosis [18, 19], whereas long-term inhibition of endogenous NO synthesis had the opposite effect [20]. In addition, organic nitrates reduce the formation of atherosclerotic intima lesions and improve endothelium-dependent vasorelaxation in cholesterol-fed rabbits [21]. 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 [22], suggesting that endothelial function may exhibit gender differences. It is known that the atherogenic process in humans is dependent on gender [23]. 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 [24]. Furthermore, pre- and postmenopausal estrogen intake is associated with a reduced incidence of coronary heart disease [25].

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 (SNAP) or pentaerythritol tetranitrate (PETN) and isosorbide 5-nitrate (ISMN), respectively.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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 18–20°C, a humidity of 50–60% and a day-night-rhythm of 12 h and received water ad libitum. Both female and male rabbits were randomly divided in two groups and fed 40 g/kg per day of a standard or a cholesterol-enriched (0.75%) 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 (see 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 (26.4203.1-24/92 and 23.4203.1-48/93) and the experiments were performed according to the guidelines for the use of experimental animals as given 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 vessel segments
After a 24-h fast rabbits were anesthetized by injection of a mixture of xylazine (5 mg/kg) and ketamine (25 mg/kg) 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 (95% O2+5% CO2) Krebs–Henseleit solution (pH 7.4) of the following composition (mmol/l): Na+ 143.07, K+ 5.87, Ca2+ 1.6, Mg2+ 1.18, Cl– 125.96, HCO3– 25.00, H2PO4– 1.18, SO42– 1.18 and glucose 5.05. Four ring segments (4 mm width) of thoracic aorta were mounted between stainless steel triangles in a water jacketed organ bath (37°C) for measurement of tension-development as recently described [26]. Previous experiments with KCl (10–60 mmol/l) revealed an optimal resting tension of 2 g for development of contractile function in the vessels. After equilibration (1 h), contractile function of aortic segments was tested by application of KCl (60 mmol/l). This was followed by a cumulative application of phenylephrine (0.01–10 µmol/l), which resulted in a maximal tension of approximately 12 g (see Section 3.5). Function of endothelium was then examined by cumulative addition of acetylcholine (0.01–10 µmol/l) following precontraction with a single dose of phenylephrine. A comparable submaximal aortic tension within the different groups was achieved by adding 0.5 µmol/l phenylephrine to rings from female rabbits (standard diet: 5.9±0.6 g; cholesterol diet: 5.1±0.5 g) and by adding 0.2 µmol/l of phenylephrine to aortic rings of male rabbits (standard diet 5.1±0.6 g; cholesterol diet 4.2±0.4 g). Addition of 0.5 µmol/l phenylephrine to aortic rings from male rabbits resulted in a significantly stronger tension (standard 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 µmol/l oxyhemoglobin, which scavenges extracellular NO (2 rings of each animal), and 200 U/ml superoxide dismutase (2 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 (1 nmol/l–10 µmol/l), ISMN (0.1 µmol/l–1 mmol/l) and PETN (1 nmol/l–100 µmol/l) were studied following precontraction with phenylephrine (3 µmol/l). In rings of male rabbits the vasorelaxant activity of SNAP was also evaluated in presence of superoxide dismutase (200 U/ml). 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.

Preliminary experiments revealed that the maximal achieved concentration of dimethylsulfoxide (0.01%), 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 pD2 value of the dose-dependent relaxation caused by SNAP showed a marked decrease in the presence of 1 µM oxyhemoglobin (from 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 4000xg. The resulting supernatants were directly used for enzymatic determination of cholesterol, high density lipoproteins (HDL) and triglycerides according to published methods [27–29]. Frozen plasma samples were thawed and used for determination of estradiol by gas chromatography/mass spectrometry (GC/MS). The analyte and the internal standard were extracted from the plasma samples (1 ml) by liquid/liquid extraction and derivatized to their pentaflourobenzoyl derivatives. These were chromatographed on a DB-5 fused silica capillary column (J&W Scientific, Folcom, CA, USA) in a Carlo Erba Mega series gas chromatograph (Carlo Erba, Italy). Detection was by negative ion mass spectrometry (Nermag R 10-10, France) after chemical ionisation (NICI-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 [21]. The tissues were fixed in formol solution (10 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 4°C. 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 (atherosclerotic area) related to the whole intimal surface was determined in each segment by use of a laser-scanning apparatus (Molecular Dynamics Densitometer, model 300 A, Sunnyvale, CA 94086) and calculated by use of Image Quant, Version 3.15. The aortic segments were photographed under reproducible conditions (camera: Mamiya RZ 67 professional, objective: Mamiya Sekor 2.8/110 mm, film: Kodak TMX 6025, 6x7 cm negatives, black-and-white). Both negatives and developed photographs were easily scanned and yielded comparable results. The percentage values presented in Section 3.4are based on scanned negatives.

2.5 Substances and solutions
SNAP was synthesized in our laboratory according to Field et al. [30]. The reaction product was recrystallized once in methanol. Analysis of SNAP included a thin-layer chromatography on Merck® RP18 plates with methanol/water (7:3, v/v) 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 (25%). 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 acid/{alpha}-naphthylamine 250 mg each in 30% acetic acid. This procedure resulted in a concentration-dependent increase of the colored area. The preparation decomposed at 148–150°C. In addition, the NO liberating property of synthesized SNAP was determined using the oxyhemoglobin assay [31]. With 1 mmol/l of SNAP, the rate of release of NO was 1.28±0.01 µmol/l per min (n=3). Furthermore, the spontaneous release of NO from SNAP (at 37°C and pH 7.4) was confirmed by direct measurement of NO using an NO electrode (ISO-NO, WPI-Instruments, Berlin, Germany). A concentration of 100 µmol/l SNAP produced a peak concentration of 221 nmol/l NO.

Oxyhemoglobin was freshly prepared every day as described previously [31]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 (4.404 mmol/l) was available in isotonic glucose solution and used as stock solution. Stock solutions (10 mmol/l) of acetylcholine, ISMN and phenylephrine in distilled water, those of SNAP and PETN (100 mmol/l) 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 (pD2 values) were calculated from the individual concentration-effect-curves as proposed by Hafner et al. [32]and are related to the maximal achieved vasoconstriction. Vasorelaxation due to treatment with the drugs is expressed as percentage of the contractile response achieved with phenylephrine (0.2, 0.5 or 3 µmol/l) at the beginning of the experiments. The concentrations of acetylcholine or the nitrovasodilators for half-maximal inhibition of phenylephrine-induced preconstriction (pD2 values) were calculated as described above. All data were analyzed by one-way analysis of variance (ANOVA) with subsequent Student–Newman Keuls test (SAS PC Software 6.04, PROC ANOVA, also used to calculate the correlations and the pD2 values) and are expressed as mean values and standard error of the mean (S.E.M.). The pD2 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
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Body weight
At the beginning of the experiments the average body weight of the rabbits was 1996±48 g (females) and 2201±44 g (males). Daily feeding with 40 g/kg of the different diets resulted in a weekly increase in body weight of approximately 35–50 g (standard diet) and 50–65 g (cholesterol 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 (1358±111 mg/dl) and females (907±85.8 mg/dl, P<0.0001). The cholesterol diet did not affect the concentration of HDL and triglycerides (data not shown). The cholesterol concentration in standard-fed rabbits showed no significant difference (males: 46±5.1 mg/dl; females: 69.8±10.0 mg/dl). 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 levels of estradiol
The concentration of estradiol in the plasma of cholesterol-fed female and male rabbits, as determined by gas chromatography/mass spectrometry was always below the detection limit of 5 pg estradiol/ml plasma. In a few samples extrapolation beyond the detection limit yielded estradiol concentrations of 1.2–1.9 pg/ml. In experiments using plasma spiked with 30 pg/ml estradiol the recovery rate was >90%.

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 (Table 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.


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  		Table 1 Atherosclerotic lesion area in the intima of cholesterol fed rabbits

 
3.5 Vascular smooth muscle contractile activity
The contractile activity of thoracic ring segments was evaluated by cumulative application of phenylephrine. The maximal contractile response occurred at a concentration of 10 µmol/l (Fig. 1). Preliminary experiments (data not shown) and experiments with aorta from standard-fed rabbits (Fig. 1) showed that rings from males respond significantly stronger to low concentrations of phenylephrine than rings from females (at 0.1 µmol/l 1.80±0.28 g vs. 1.0±0.2 g, respectively). There was a significant difference (P<0.0001) between the pD2 values of phenylephrine evaluated in cholesterol-fed female (6.12±0.05) and male rabbits (6.45±0.03), whereas in the standard fed groups this value was comparable (females: 6.25±0.02; males 6.28±0.05). The maximal tension induced by 10 µmol/l phenylephrine was identical in each group (Fig. 1).


Figure 1
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  		Fig. 1 The vasocontractile activity of phenylephrine in isolated thoracic aorta of standard-fed (upper panel) and cholesterol-fed (lower panel) female and male New Zealand White rabbits. Each concentration–response curve was plotted by taking the respective mean values of nine separate experiments (S.E.M. indicated by bars). The respective pD2 values of phenylephrine in aortic rings of cholesterol-fed rabbits are significantly different (P<0.05, data see Section 3.5).

 
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 (see Section 2.2). Both, cholesterol diet (P<0.0001) and male gender (P<0.025) significantly reduced acetylcholine induced vasorelaxation.

In vessel segments of all standard-fed rabbits, acetylcholine (1 nmol/l–0.5 µmol/l) produced vasorelaxations, which were similar between males and females (Fig. 2). In accordance, the pD2 values of acetylcholine in rings of female (6.91±0.03) and male rabbits (6.99±0.10) were identical.


Figure 2
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  		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 (S.E.M. indicated by bars). Maximal vasorelaxation of atherosclerotic aorta is significantly greater in females as compared to males.

 
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 (Fig. 2, P<0.0001). Importantly, this effect was much more pronounced in rings of male compared to rings of female rabbits (Fig. 2). In both cholesterol-fed groups, the extent of endothelium-dependent vasorelaxation was inversely correlated with the degree of atherosclerotic intima-lesions (Fig. 3, Table 2) demonstrating that in both groups these events are directly related.


Figure 3
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  		Fig. 3 Correlation between the percentage of maximal vasodilatation induced by acetylcholine in segments of isolated thoracic aorta of female (A) and male (B) New Zealand White rabbits (four 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 (females) and P<0.0002 (males). Mean values of maximal vasorelaxation are plotted in Fig. 2. Mean values of the area of luminal atherosclerotic lesions are shown in Table 1 (a=slope, b=interception, r=correlation coefficient).

 

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  		Table 2 Correlation coefficients for the linear relationships

 
3.7 Effects of nitrovasodilators
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 cholesterol-fed rabbits SNAP was significantly less potent than in the respective female and male controls (Fig. 4, Table 3, P<0.0002). In addition, we found in both cholesterol-fed groups a significant direct correlation between the pD2 values of SNAP evaluated in each animal and the corresponding maximal relaxation to acetylcholine (Fig. 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 (P<0.0001; pD2 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 (Table 3): Neither gender nor the feeding protocol showed a significant influence on the efficacy of PETN. ISMN induced a significantly more potent vasorelaxation in aortic rings of cholesterol-fed female rabbits (Table 3, P<0.02).


Figure 4
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  		Fig. 4 The vasorelaxing activity of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in isolated segments of thoracic aorta of female (A) and male (B) 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 (S.E.M. indicated by bars).

 

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  		Table 3 The vasorelaxant efficacy of ISMN, PETN and SNAP in isolated segments of thoracic aorta from female and male rabbits fed different diets

 

Figure 5
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  		Fig. 5 Correlation between the efficacy (pD2 values) of the spontaneous NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in segments of isolated thoracic aorta of female (A) and male (B) New Zealand White rabbits (four 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 (females) and P<0.03 (males). The mean pD2 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 (a=slope, b=interception, r=correlation coefficient).

 
3.8 Effects of superoxide dismutase
Superoxide dismutase (200 U/ml) significantly improved the dose-dependent relaxation of aortic rings of standard-fed male rabbits elicited by SNAP as indicated by the increase of the pD2 value from 6.76±0.1 to 7.06±0.1 (n=9). In aortic rings of cholesterol-fed animals superoxide dismutase (200 U/ml) 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 pD2 values in the absence (6.22±0.15, see Table 3) and presence of this enzyme (6.25±0.2).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
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 (Table 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 (Fig. 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 [33]. 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 cholesterol-feeding. This would indicate that endogenous hormone status in addition to exogenous hormone replacement may 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 [34–36]. In preliminary studies (see Section 2.2), we were able to confirm previous studies showing that SNAP spontaneously released NO at a rate of 1.28±0.01 µmol/l 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 [37]. 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 [38, 39]. 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 [40].

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 Cu/Zn SOD (200 U/ml). While this had a small effect in normal male animals, there was no effect in the cholesterol-fed rabbits. This does not exclude a role for superoxide in this process. Because of its hydrophilicity, Cu/Zn 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 [38, 41].

Previously, it has been suggested that vessels from female animals produce more NO under basal conditions than do vessels from male animals [22]. Others have reported that estradiol decreases superoxide production in endothelial cells [42]. 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 (5 pg/ml). Others have reported similarly low levels of estradiol in rabbits [43].

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 (Table 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 [44].

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 similar 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.

Time for primary review 36 days.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft SFB 242; Projekt A11.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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