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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:782-792

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:782.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Dehydroepiandrosterone Retards Atherosclerosis Formation Through Its Conversion to Estrogen

The Possible Role of Nitric Oxide

Toshio Hayashi; Teiji Esaki; Emiko Muto; Hatsuyo Kano; Yukako Asai; Navin Kumar Thakur; Daigo Sumi; Muthuvel Jayachandran; Akihisa Iguchi

From the Department of Geriatrics, Nagoya University School of Medicine, Nagoya, Japan.

Correspondence to Toshio Hayashi, Department of Geriatrics, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550 Japan. E-mail hayashi{at}med.nagoya-u.ac.jp


*    Abstract
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Abstract—Dehydroepiandrosterone (DHEA) is speculated to have an antiatherosclerotic effect, although the mechanism of action remains unclear. The objective of the current study was to determine whether the antiatherosclerotic effect of DHEA is related to its conversion to estrogen and to define the role of nitric oxide (NO) in the antiatherosclerotic effect of DHEA. Forty-eight oophorectomized rabbits were divided into 5 groups and fed the following diets for 10 weeks: group 1, a regular rabbit diet plus 1% cholesterol (a high-cholesterol diet [HCD]); group 2, an HCD plus 0.3% DHEA; group 3, an HCD plus 0.3% DHEA and fadrozole (2.0 mg · kg-1 · d-1), a specific aromatase inhibitor; group 4, an HCD plus 17ß-estradiol (20 µg · kg-1 · d-1); and group 5, a regular diet. Atherosclerotic lesions, lipid deposition in aortic vessels, and basal and stimulated NO release were measured in the aforementioned groups of rabbits. NO release was measured by using an NO-selective electrode as well as by measuring vascular responses and the plasma NO metabolites nitrite and nitrate. The plasma total cholesterol level was increased, but there were no significant differences in lipid profile in the 4 groups of rabbits that were fed the HCD. The area occupied by atherosclerosis in the thoracic aorta was diminished by {approx}60% in the DHEA-treated rabbits (group 2) compared with the HCD group of rabbits (group 1); there was a corresponding 80% decrease in the estradiol group (group 4) but only a 30% decrease in the DHEA plus fadrozole group (group 3). In the aortas of rabbits from groups 1 and 3, the acetylcholine-induced and tone-related basal NO-mediated relaxations were diminished compared with those of the controls (group 5). However, these relaxations were restored in the aortas of group 2 and 4 rabbits, and an increase in NO release was observed in groups 2 and 4 compared with groups 1 and 3, as measured by an NO-selective electrode. Injection of neither solvent (20% ethanol/distilled water) nor fadrozole significantly affected the atherosclerotic area or the NO-related responses described above. We conclude that {approx}50% of the total antiatherosclerotic effect of DHEA was achieved through the conversion of DHEA to estrogen. NO may also play a role in the antiatherosclerotic effect of DHEA and 17ß-estradiol.


Key Words: dehydroepiandrosterone • atherosclerosis • nitric oxide • estrogen • aromatase


*    Introduction
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 *Introduction
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Dehydroepiandrosterone (DHEA) and its sulfate ester DHEA-S are weak androgens produced primarily by the adrenal gland. Although their plasma concentrations by far exceed those of any other adrenal product, their physiological roles have not yet been determined. In plasma, where the major portion of these hormones is present in the sulfate form, it is possible that DHEA-S serves as a reservoir for DHEA, since various tissues have been shown to contain steroid sulfatases.1 The peak plasma levels of DHEA and DHEA-S occur at approximately age 25 years, decrease progressively thereafter, and diminish by 95% around the age of 85 years. Epidemiological evidence has shown that adult men with high plasma DHEA-S levels are less likely to die of cardiovascular disease.2 A study indicated that administration of DHEA reduced aortic fatty streak formation and cholesterol accumulation by {approx}30% to 40% in cholesterol-fed rabbits.3 Another report has shown a 50% reduction in aortic atherosclerosis in rabbits that underwent balloon aortic injury and were then fed a high-cholesterol diet with DHEA.4 These studies suggest that higher levels of DHEA and DHEA-S might be protective against the development of atherosclerosis.

DHEA has been shown to reduce serum LDL cholesterol levels in humans of average health,5 although no relationship was reported between the plasma cholesterol level and the beneficial effect of elevated plasma DHEA.6 Furthermore, none of these results can explain the drastic antiatherosclerotic effects of DHEA with regard to a high-cholesterol diet–induced atherosclerosis, because no remarkable lipid profile changes were observed in these kinds of animal models after DHEA treatment.3 4 Information regarding the receptor-mediated action of DHEA is available only in caudal epididymal spermatozoa and activated human T lymphocytes.7 8

On the other hand, it is well known that hormone replacement therapy with estradiol decreases the risk of coronary events in postmenopausal women.9 10 An abundance of epidemiological data confirms this atheroprotective effect of estradiol9 and has also prompted recommendations for the widespread use of estrogen replacement therapy for the primary prevention of ischemic cardiovascular disease in postmenopausal women.11 The antiatherosclerotic effects of estradiol were thought to be partly attributable to changes in plasma lipid levels (ie, the increase in HDL cholesterol and decrease in LDL cholesterol).12 However, the contribution of these changes to the total antiatherosclerotic effect of estrogen is only {approx}50%, based on multiple regression analyses.12

The direct action of estrogen on the vessel wall has been studied vigorously over the past decade.13 14 15 16 Recently, estrogen receptors have been found in the vascular endothelium and smooth muscle cells, as well as in blood cells such as monocytes.13 14 17 The antiatherosclerotic effect of estrogen could be induced by a direct effect on the vessel wall, such as the inhibition of smooth muscle cell proliferation and migration or of LDL oxidation.18 19 20 Furthermore, estrogen was able to inhibit cholesterol accumulation in the aorta as well as in the coronary arteries of cholesterol-fed rabbits and monkeys, and this was partly explained by suppression of the arterial uptake and/or degradation of LDL.16 21 23 Whereas the direct antiatherogenic effect of estrogen per se on the vessel wall is poorly understood, interest has focused on the role of nitric oxide (NO) because NO has antiatherosclerotic effects such as inhibition of monocyte adherence to endothelial cells; inhibition of smooth muscle cell chemotaxis, proliferation, and relaxation; and inhibition of platelet aggregation.24 25 26 27 Estrogen has also been shown to increase endothelial NO synthase (eNOS) protein and activity and NO production by receptor-mediated mechanisms in cultured endothelial cells.28 It has therefore been hypothesized that the ability of estrogen to increase NO bioavailability is involved in the antiatherogenic effect of estrogen.29 30 More recently, direct evidence was shown that NO mediates the antiatherosclerotic effect of estrogen.31 DHEA is known to convert to estradiol in vivo. Therefore, the current study was undertaken to determine whether the antiatherosclerotic effect of DHEA is related to its conversion to estrogen and to define the role of NO in the antiatherosclerotic effect of DHEA.


*    Methods
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Chemicals and Solutions
L-Arginine hydrochloride, acetylcholine chloride (ACh), prostaglandin F2{alpha} (PGF2{alpha}), calcium ionophore A-23187, hemoglobin, indomethacin, and NG-monomethyl-L-arginine acetate (L-NMA) were all purchased from Sigma Chemical Co. Nitroglycerin (NTG, 10% wt/wt triturate in lactose) was from Nihon Kayaku Co Ltd. Fadrozole, a specific aromatase inhibitor, was the gift of Novartis Pharma, Japan Co, Ltd (Tokyo, Japan). Krebs-Henseleit solution (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 11 mmol/L glucose, and 0.002 mmol/L EDTA; pH 7.4) was saturated with 95% O2/5% CO2. The composition of the depolarizing KCl solution was similar to that of Krebs-Henseleit buffer, except for the replacement of NaCl by an equimolar amount of KCl. All concentrations shown for the in vitro studies are those in the final bath.

Animals
Experiment 1
A total of 48 female New Zealand White rabbits, 12 or 13 weeks old and weighing 2.0 to 2.4 kg, were obtained from Kitayama Rabbits (Ina, Nagano, Japan). The rabbits were housed individually in stainless-steel cages at 20±3°C with a 12-hour light/dark cycle and with free access to water. All animals were initially fed a regular rabbit diet (Oriental Yeast Co, Ltd) for 2 weeks. The rabbits were then divided into 5 groups (each group having >8 rabbits) during the 6 weeks after bilateral oophorectomy. Group 1 received a high-cholesterol diet (regular rabbit diet with 1% cholesterol [HCD]). Group 2 received the HCD supplemented with 0.3% DHEA. Group 3 received the HCD supplemented with 0.3% DHEA and a daily injection of fadrozole (2.0 mg · kg-1 · d-1), an aromatase-specific inhibitor. Group 4 received the HCD plus a daily injection of 17ß-estradiol (20 µg · kg-1 · d-1), and group 5 received a regular rabbit diet. Fadrozole and 17ß-estradiol were each dissolved in 20% ethanol/distilled water. Final concentrations of fadrozole and 17ß-estradiol were 10 mg/mL and 50 µg/mL, respectively. The rabbits were treated for 10 weeks under the conditions described above.

Experiment 2
To investigate the effect of solvent or fadrozole, solvent was injected into oophorectomized rabbits under the same conditions as used for group 1, 2, and 5 rabbits (n=6 animals in each group), and fadrozole (2.0 mg · kg-1 · d-1) was injected into oophorectomized rabbits fed the regular rabbit chow (n=5) and rabbits fed the HCD (n=6).

In both experiments, feeding was restricted to 100 g/d. Fadrozole or 17ß-estradiol was administered daily at 9 AM by intramuscular injection. Blood was collected 24 hours after the last feeding. The general appearance of the rabbits was observed daily, and they were weighed every 4 weeks. All experiments were conducted in accordance with institutional guidelines for animal studies.

Lipid and Sex Steroid Hormone Concentration Assays
Total and free cholesterol levels were determined by using cholesterol oxidase (Wako Pure Chemical Industries, Ltd).32 Triglyceride levels were measured by enzymatic techniques as described previously.33 HDL cholesterol was initially measured after precipitation with phosphotungstate-MgCl2.34 The plasma concentrations of DHEA, DHEA-S, pregnanediol, testosterone, and 17ß-estradiol were measured as described previously.35

Histological Evaluation of Atherosclerosis
The descending thoracic aorta was taken from the portion of the orifice at the left first costal artery ({approx}4 cm distal to the aortic valve) to 3 cm above the portion enclosed by the diaphragm ({approx}7 cm distal to the aortic valve). Morphometric analysis was performed as described by Weiner et al,36 with slight modifications. Six blocks were taken from the descending thoracic aorta of each rabbit. Each block was adjacent to another segment taken for evaluation of endothelium-dependent responses and was stained with hematoxylin-eosin to examine the endothelial lining and with van Gieson’s elastic stain to determine the surface involvement of atherosclerotic lesions (fatty streaks and fibrous plaques) and the area occupied by the atherosclerotic lesion as defined below. The first complete section of each block was projected onto a vertical surface with a projecting microscope. The contours of the lumen and the internal elastic lamina were traced, and the tracings were digitized (PC-9801 ES, NEC) by using a graphics tablet. The mean surface involvement by atherosclerotic lesion per vessel per animal was calculated by summing all results obtained after dividing the lesion circumference by the circumference of the internal elastic lamina and then dividing the sum by the number of sections studied (n=6 for 1 vessel). Circumferences of lesions and normal portions were defined as circumferences of internal elastic lamina where intimal thickening was observed and where normal intima was observed, respectively. The area occupied by atherosclerotic lesions was defined as the percent area bounded by the lumen and the internal elastic lamina for the ideal luminal area. The ideal luminal area was calculated from the perimeter of the internal elastic lamina on the assumption that the true shape of the vessel was circular and to exclude the artificial effect due to tissue fixation in 10% formalin solution. The mean area occupied by the lesions per vessel per animal was calculated by summing the areas occupied by lesions of all sections and dividing the sum by the number of sections per vessel (n=6 for 1 vessel). Data were transferred to a minicomputer (Macintosh Quadra 700, Apple, Ltd) for further analysis.

Determination of Aortic Cholesterol Content
The segment of the aortic arch (2 cm distal to the aortic valve) to the bifurcation of the left subclavian arteries was removed, weighed, minced, and homogenized in 10 volumes of sucrose-Tris buffer with a motor-driven, glass homogenizer at 0°C to 2°C. The homogenates were used for total lipid extraction.37 The extracted lipids were used for the examination of total cholesterol,32 free cholesterol,32 and esterified cholesterol by the method of Badimon et al.37

Isometric Tension Measurement
After 10 weeks of diet treatment, the rabbits were killed by exsanguination after being anesthetized with pentobarbital (50 mg/kg IV). The thoracic aortas were removed carefully to protect the endothelial lining, cleaned of adhering fat and connective tissue, and cut into 2-mm-wide transverse rings. The optimal passive load for both control and atherosclerotic aortas was determined as the contractile response to 122 mmol/L KCl. Before the experiments, the rings were stretched to their predetermined optimal tension, mounted on stainless-steel hooks in 20-mL-capacity muscle chambers, and bathed in Krebs-Henseleit solution, pH 7.4, at 37°C for 1 hour. Tension was measured isometrically by using a force displacement transducer (model DSA-603, Minebea Co, Ltd) and was displayed on a multipen recorder (model R-60, Rika Denki Co, Ltd). Experiments were conducted to determine the responsiveness of endothelium-intact aortic rings to the endothelium-dependent vasodilator ACh or A-23187. The responsiveness of endothelium-denuded aortic rings, which were prepared by gently rubbing the luminal surface with a swab moistened with Krebs-Henseleit solution, to the endothelium-independent vasodilator NTG was also measured. In these experiments, PGF2{alpha} initially induced submaximal tension (2.6x10-6 mol/L). To investigate the tone-related release of NO from endothelium-intact aortic rings, moderate vascular tone (35% to 50% of the contractile response obtained with 122 mmol/L KCl) was induced by low PGF2{alpha} concentrations (0.8x10-6 mol/L). Concentration-related contractile responses to L-NMA (1 to 100 µmol/L) were also assessed.29 In some experiments, indomethacin (5 µmol/L) was added to the muscle chambers for 60 minutes before the start of the experiment to rule out the contribution of prostanoids.

Measurement of NO by an NO Electrode
The function of the NO meter (model NO501, Intermedic Co, Ltd) is the measurement of the redox current between a working electrode and a counterelectrode by using a microsensor as modified by the method of Malinski and Taha.38 The working electrode was made of a Pt/Ir alloy (0.2 magnetomotive Force) coated with a 3-layer membrane consisting of KCl, an NO-selective resin, and normal silicone membranes. The counterelectrode was made of carbon fiber. The KCl membrane was deposited to suppress overvoltages in the discharge of NO. The NO-selective resin was made of a nitrocellulose/pyroxylin lacquer. An outer membrane was used to avoid nonspecific ionic effects and electrochemical reactions. Polarographic current was detected with a current-voltage converter circuit. The current increased linearly from 1.5 to 305 pA when the S-nitroso-N-acetyl-dl-penicillamine (SNAP) concentration increased from 5 nmol/L to 1 µmol/L.39 We measured ACh (1 µmol/L)–stimulated NO release in ring segments from rabbit aortas (3 mm wide) cut longitudinally and mounted on a silicone plate in the Krebs-Henseleit tissue bath that was saturated with 95% O2/5% CO2. With the use of a micropositioner (0.2-mm x-y-z resolution), the microsensor was placed at a 0.2-mm distance from the surface of endothelial cells in the aorta. NO release from the aorta was expressed as pA per mm2 of luminal surface area of rabbit aorta segment. Three picoamperes was assumed to be the amount of NO released from 10 nmol/L SNAP.

Measurement of NO2-/NO3-
Concentrations of nitrite and nitrate (NO2-/NO3-) in plasma were measured in an automated NO detector–high-performance liquid chromatography system (ENO10, Eicom Co) as previously reported.40 41 In brief, samples were collected in an automated sample injector connected to an automated NO detector. NO2- and NO3- in each sample were diluted 5 times with mobile-phase buffer (10% methanol containing 0.15 mol/L NaCl/NH4Cl and 0.5 g/L sulfanilamide with 0.25 g/L N-naphthylethylenediamine) and were separated on a reverse-phase separation column packed with polystyrene polymer (NO-PAK, 4.6x50 mm, Eicom), and NO3- was reduced to NO2- in a reduction column packed with copper-plated cadmium fillings (NO-RED, Eicom). NO2- was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The absorbance of the color of the product dye at 540 nm was measured by a flow-through spectrophotometer (NOD-10, Eicom). The mobile phase was delivered by a pump at a rate of 0.33 mL/min with mobile-phase buffer. The Griess reagent (0.5% sulfonamide, 0.025% N-naphthylethylethylenediamine dihydrochloride, and 1.25% HCl) was employed to form a purple azo dye.

Measurement of cGMP
The concentration of cyclic GMP (cGMP) in homogenates of aortic tissue was determined by a specific radioimmunoassay.42 In brief, 4 aortic rings (derived from the abdominal aorta from the portion enclosed by the diaphragm to 3 cm just above the orifice of the right renal artery; wet weight each, 30±1 mg) were incubated in test tubes containing Krebs-Henseleit buffer saturated with 95% O2/5% CO2 for 30 minutes to exclude the hypoxic effect induced during sacrifice of the rabbits. The rings were promptly frozen in LN2 and stored at -80°C. To determine basal cGMP levels, the rings were homogenized in 1 mL of 6% trichloroacetic acid at 4°C and centrifuged at 12 000g for 5 minutes. The supernatant was washed 4 times with 4 mL of water-saturated ethyl ether. Liquid samples were then frozen at -80°C and lyophilized overnight. The lyophilized sample was resolubilized in 1 mL of 0.05 mol/L sodium acetate buffer, and 50-µL aliquots were placed in test tubes. The cGMP contents in the samples were determined by an enzyme-linked immunoassay in a commercial assay kit (RPN226, Amersham). Solids remaining from the initial homogenization step were digested in 1 mL of 0.1N NaOH overnight, and total protein was determined by the method of Lowry et al.43

Data Analysis
Relaxation was measured as the percent decrease in tension below that evoked by PGF2{alpha} (2.6x10-6 mol/L) in arterial rings. Contraction in response to L-NMA (1 to 100 µmol/L) was measured as the percent increase in tension above that evoked by PGF2{alpha} (0.8x10-6 mol/L) in arterial rings. Data are expressed as mean±SEM. Means were compared by ANOVA with repeated measurements. When a significant F value was found, Scheffe’s test for multiple comparisons was used to identify differences among groups. A level of P<0.05 was considered statistically significant.


*    Results
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 *Results
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Status of Lipids and Sex Steroid Hormones
There were no significant differences in total serum protein among the 4 experimental groups and the control group over the course of the study (data not shown). The addition of 1% cholesterol to the diet (groups 1, 2, 3, and 4) increased total cholesterol and triglyceride levels but had no significant effect on total protein or HDL cholesterol compared with the baseline values (Table 1Down). Body weight was not changed among the 5 groups (Table 1Down). The application of DHEA or 17ß-estradiol, in addition to the 1% cholesterol in the diet, did not affect the plasma lipid levels. In experiment 2, injection of solvent into group 1, 2, and 5 rabbits did not affect the lipid levels significantly (Table 2Down). Injection of fadrozole into the HCD or the regular-diet rabbit group also had no significant effect (Table 2Down). The plasma concentrations of DHEA and the sex steroid hormones as metabolite products are shown in Table 3Down. No significant increase in testosterone was demonstrated, although 17ß-estradiol was increased by DHEA treatment (group 2) but not by DHEA plus fadrozole treatment (group 3). DHEA and DHEA-S levels increased significantly after DHEA treatment, with or without fadrozole. Injection of solvent into group 1, 2, and 5 rabbits (groups 1b, 2b, and 5b) and injection of fadrozole into group 1 and 5 rabbits had no significant effect on the data described above. Fatty livers were observed in groups 1, 2, 3, and 4; however, no liver function abnormality was detected in the plasma samples from these groups (data not shown). Figure 1Down shows the metabolite map of DHEA.


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  		Table 1. Plasma Lipid Levels and Body Weights After Consumption of an HCD


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  		Table 2. Data From Experiment 2


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  		Table 3. Plasma Steroid Levels After Consumption of an HCD



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  		Figure 1. Metabolite map of DHEA-related products and the action point of fadrozole.

Histological Evaluation of Atherosclerosis and Assays for Tissue Cholesterol Content
Histological examination of the thoracic aorta revealed that there were more atheromatous lesions in group 1 than in groups 2, 3, 4, or 5. The area of atherosclerosis in the thoracic aortas of the DHEA group (group 2) was reduced by {approx}60%, the estradiol group (group 4) by 80%, and the DHEA plus fadrozole group (group 3) by only 30% when compared with group 1 (HCD group; Figure 2Down). Total and esterified cholesterol concentrations in the vessels showed the same tendency as that of atherosclerotic area. Free cholesterol was increased by feeding with the HCD (groups 1, 2, 3, and 4), without any significant differences among these 4 groups (Figure 3Down). Injection of solvent into group 1, 2, and 5 rabbits and injection of fadrozole into group 1 and 5 rabbits had no significant effect on the above-mentioned parameters (Table 2Up).



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  		Figure 2. Top, Histological evaluation of atherosclerotic areas of rabbit thoracic aortas as indicated by the mean percentage of luminal encroachment (% surface involvement) and the mean lesion area (% area occupied by lesion). Cross sections of descending thoracic aorta were stained with van Gieson’s elastic stain. The atherosclerotic areas were calculated from the thoracic aortas of rabbits fed an atherogenic diet (group 1), an atherogenic diet plus DHEA (group 2), an atherogenic diet plus DHEA and fadrozole (group 3), an atherogenic diet plus 17ß-estradiol (group 4), or a regular diet (group 5). Error bars indicate SEM. These data are from experiment 1. Bottom, Representative photomicrographs of descending thoracic aortas (original magnification x20). Bar=500 µm.



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  		Figure 3. The content of total, free, and esterified cholesterol in the rabbit aortic arch. Lipid contents were measured from the aortic arch of rabbits fed an atherogenic diet (group 1), an atherogenic diet plus DHEA (group 2), an atherogenic diet plus DHEA and fadrozole (group 3), an atherogenic diet plus 17ß-estradiol (group 4), or a regular diet (group 5). Error bars indicate SEM. These data are from experiment 1.

Endothelium-Dependent and -Independent Relaxation
In all experimental groups, the endothelium-dependent vasodilator ACh and A-23187 (data not shown) produced concentration-dependent relaxation of precontracted aortic rings with an intact endothelium (Figure 4Down). No significant differences in ACh-induced, endothelium-dependent relaxation were observed among the aortic rings obtained from the normocholesterolemic group (group 5) or from the hypercholesterolemic animals administered DHEA only (group 2) or 17ß-estradiol (group 4). The magnitude of relaxation of the aortic rings from the hypercholesterolemic animals without DHEA (group 1) or with DHEA plus fadrozole (group 3) was significantly diminished compared with the aortic rings from normolipidemic animals (group 5). The endothelium-independent vasodilator NTG produced a concentration-dependent relaxation in precontracted, endothelium-denuded aortic rings. There were no significant differences among the 5 groups in terms of the relaxation response to NTG in the aortic rings (Figure 5Down). Inhibition of NOS by L-NMA (100 µmol/L) led to a contractile response in the aortic rings precontracted with PGF2{alpha} (Figure 6Down). This contractile response was concentration dependent, and its magnitude was decreased in endothelium-intact aortic rings obtained from HCD-fed rabbits (group 1) when compared with rings obtained from control rabbits (group 5). The contractile response of aortic rings from atherosclerotic rabbits treated with DHEA or 17ß-estradiol (group 2 or group 4) did not differ significantly from that of control animals (group 5). The contractile response of animals treated with DHEA plus fadrozole (group 3) was almost the same as that of untreated, atherosclerotic rabbits (group 1). Preincubation of the aortic rings with indomethacin did not affect endothelium-dependent relaxation, indicating that prostanoids did not alter the effect of DHEA in endothelium-dependent relaxation. Injection of solvent into group 1, 2, and 5 rabbits (groups 1b, 2b, and 5b) or fadrozole treatment alone for group 1 or 5 rabbits did not significantly affect the above-mentioned results (data not shown).



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  		Figure 4. Cumulative concentration-response curves to ACh during contraction evoked by PGF2{alpha} (2.6x10-6 mol/L) in the thoracic aorta of rabbits fed an atherogenic diet (group 1), an atherogenic diet plus DHEA (group 2), an atherogenic diet plus DHEA and fadrozole (group 3), an atherogenic diet plus 17ß-estradiol (group 4), or a regular diet (group 5). Relaxation is expressed as a percent decrease in tension from the contraction evoked by PGF2{alpha} (2.6x10-6 mol/L) alone. These data are from experiment 1. Data are shown as mean±SEM. *Significant difference vs group 5 at P<0.05.



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  		Figure 5. Cumulative concentration-response curves to NTG during contraction evoked by PGF2{alpha} (2.6x10-6 mol/L) in the thoracic aorta of rabbits fed an atherogenic diet (group 1), an atherogenic diet plus DHEA (group 2), an atherogenic diet plus DHEA and fadrozole (group 3), an atherogenic diet plus 17ß-estradiol (group 4), or a regular diet (group 5). Relaxation is expressed as a percent decrease in tension from the contraction evoked by PGF2{alpha} alone. These data are from experiment 1. Data are shown as mean±SEM. There was no significant difference in the NTG response in the vessels among the groups.



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  		Figure 6. Cumulative concentration-response curves to L-NMA during contraction evoked by PGF2{alpha} (0.8x10-6 mol/L) in the thoracic aorta of rabbits fed an atherogenic diet (group 1), an atherogenic diet plus DHEA (group 2), an atherogenic diet plus DHEA and fadrozole (group 3), an atherogenic diet plus 17ß-estradiol (group 4), or a regular diet (group 5). These data are from experiment 1. Data are shown as mean±SEM. *Significant difference at P<0.05 vs group 5.

Measurement of NO by NO Electrode
The release of NO from normolipemic rabbit aortas (group 5) and from hypercholesterolemic rabbit aortas (groups 1, 2, 3, and 4) was determined by using an NO-selective electrode. The release of NO from endothelial cells of each aorta stimulated by 1 µmol/L ACh is shown in Figure 7Down and Table 4Down. NO release in the groups treated with 17ß-estradiol or DHEA increased only in comparison with that in the group of HCD rabbits (group 1); however, NO release in the group treated with DHEA plus fadrozole (group 3) was not increased. Preincubation of aortic rings with L-NMA abolished the NO release stimulated by ACh (data not shown).



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  		Figure 7. Representative traces of the release of NO from rabbit aortas from groups (Gp) 1 (atherogenic diet), 2 (atherogenic diet plus DHEA), and 3 (atherogenic diet plus DHEA with fadrozole). These data are from experiment 1.


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  		Table 4. NO and NO-Related Product Levels After Consumption of an HCD

Measurement of NO2-/NO3-
Plasma concentrations of NOx (the sum of [NO2-] and [NO3-]) were increased in groups 2 and 4 compared with those in groups 1, 3, and 5 (Table 4Up).

Measurement of cGMP
In homogenate samples of rabbit aortas, animals treated with DHEA (group 2) or 17ß-estradiol (group 4) showed an increased level of cGMP compared with those of hypercholesterolemic animals (group 1) or animals treated with DHEA plus fadrozole (group 3; Table 4Up).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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DHEA has been shown to have an antiatherosclerotic effect in animal models,3 4 although its mechanism of action is not yet known. Although its plasma concentration usually far exceeds that of any other sex steroid hormone, little information is available on its direct action in blood vessels.2 8 We focused on the metabolite of DHEA as the candidate responsible for the antiatherosclerotic effect, since DHEA converts to estrogen, a major metabolite that is well known for its antiatherosclerotic effect.21 Fadrozole (CGS 16946A) was developed recently as an anti–breast cancer drug and is 1 of the strongest specific aromatase inhibitors.44 45 Its potency as an aromatase inhibitor is 400 times stronger than that of aminoglutethimide and 30 times stronger than that of 4-hydroxyandrostenedione, the more commonly used aromatase inhibitor.45 Fadrozole was reported to effectively inhibit estrogen synthesis without affecting the levels of corticosterone or testosterone in rats and humans.44 45

This is the first animal study to attempt to determine the atheroprotective effect of DHEA in oophorectomized rabbits. The plasma lipid levels did not differ among the 4 groups receiving an HCD diet (groups 1, 2, 3, and 4 ). Although pharmacological doses of DHEA may lower plasma lipoprotein levels in humans,5 this was not the case in this study. Another mechanism may be responsible for the antiatherosclerotic effect of DHEA. The area of atherosclerosis in the thoracic aortas of the DHEA group (group 2), in comparison with that of the HCD group (group 1),was reduced by {approx}60%; that of the estradiol group (group 4) was reduced by 80%; and that of the DHEA plus fadrozole group (group 3) was reduced by only 30%. The antiatherosclerotic effect of DHEA was almost the same as that of a physiological concentration of 17ß-estradiol. Fadrozole treatment inhibited {approx}50% of the antiatherosclerotic effect of DHEA, although it did not have a direct effect on atherosclerosis. The antiatherosclerotic effect of DHEA in the current study may be due to its conversion to estrogen, because inhibition of aromatase (estrogen-converting enzyme) by fadrozole reduced by 50% the antiatherosclerotic effect of DHEA.

The plasma estradiol concentration was increased by DHEA treatment, although testosterone concentration was not significantly increased. Epidemiological and pharmacological studies suggest that even a low plasma concentration of estradiol can achieve an antiatherosclerotic effect.46 Our previous reports suggested that the rate of progression of atherosclerosis was much slower in female than in male rabbits and that an identical HCD led to a similar increase in serum lipoprotein concentrations.30 This suggests that a higher concentration of plasma estrogen in female rabbits (basal plasma estradiol concentration of 23.5±5.5 pg/mL) than in male rabbits (8.4±8.3 pg/mL) under control conditions might cause this greater basal release of NO in females in the initial stages of atherosclerosis.29 30 However, the precise mechanism by which endogenous estrogen inhibited the progression of atherosclerosis was not fully addressed, because the plasma estradiol concentration level was identical in males and females after 10 weeks of HCD-induced hyperlipidemia.30 It has been shown that a considerable part of the plasma lipid–independent, antiatherogenic effect of estrogen is mediated through its effect on endothelial NO in cholesterol-fed rabbits.31 Based on these studies, to attain a physiological plasma estradiol concentration in this study, we treated the rabbits with 20 µg · kg-1 · d-1 of 17ß-estradiol (group 4) and compared its antiatherosclerotic effect with that of DHEA (group 2). The increase in plasma estrogen level in group 2 may partially account for the antiatherosclerotic effect of DHEA, which was partially abolished by concomitant treatment with fadrozole. DHEA can be converted into many steroids other than estrogens, including a number of more potent androgens.47 There was no significant increase in testosterone or pregnanediol (data not shown) in the fadrozole-treated group (group 3). However, the effect of testosterone on atherosclerosis remains controversial.48 49 In response to aromatase, rabbit testes have been shown to secrete DHEA and convert it to testosterone and testosterone-related metabolites, including estrogen.50 We used oophorectomized rabbits to establish a simple endocrinological model, although previous experiments on DHEA have either disregarded the sex of the rabbits or used males only.3 4 Therefore, we suggest that these studies can be applied to assessing the effect of DHEA on male rabbits, although more specific investigations will be necessary. Because fadrozole appeared to have no direct effect on HCD-induced atherosclerosis in this study, the possibility is remote that the slight fluctuation in testosterone level or that of other steroids due to fadrozole treatment affected the formation of atherosclerotic lesions.

A direct action of estrogen on the arterial wall is important for a considerable part of the plasma lipid–independent, antiatherogenic effect of DHEA. Estrogen seems able to inhibit cholesterol accumulation and intimal hyperplasia, independent of changes in plasma lipoproteins in ovariectomized cynomolgus monkeys and rabbits fed an atherogenic diet.21 22 23 Suppression of the arterial uptake and/or degradation of LDL by estrogen may explain these effects.23 However, these mechanisms are not able to work when the endothelial cell layer is seriously damaged.51 Estrogen can normalize abnormal vasomotor responses in atherosclerotic coronary arteries of cholesterol-fed monkeys.52 From these results, one might speculate that endothelium-derived NO may play an important role in the antiatherosclerotic effect of estrogen. NO released from eNOS was shown to regulate blood flow and suppress a number of processes involved in atherosclerosis.24 25 26 27 Several studies have shown that enhancing arterial NO synthesis retards the progression of atherosclerosis53 54 and that inhibiting NOS in vessels promoted the progression of atherosclerosis.55 Estrogen has been reported to increase eNOS activity by an endothelial receptor–mediated system in vitro.28 We speculate that the ability of estrogen to increase NO bioavailability is involved in the antiatherogenic effect of estrogen, especially in the initial stages of a cholesterol diet–induced atherosclerosis.30 More recently, Holm et al31 directly showed that inhibition of NOS reduced the antiatherosclerotic effect of estrogen. Thus, our attention was attracted to the role of NO in the antiatherosclerotic effect of DHEA. The aortas of DHEA-treated animals (group 2) showed restoration of the ACh-induced and tone-related basal NO-mediated relaxation, whereas it was diminished in the aortas of group 1 rabbits. The vascular response in animals treated with DHEA plus fadrozole (group 3) was not reversed significantly. The increase in plasma NO2-/NO3- concentrations and tissue cGMP levels in DHEA-treated rabbits showed the increasing effect of an NO-dependent mechanism. This was supported by increases in basal NO release with the improvement of a tone-related basal NO response in the arteries. These effects were diminished in the group treated with DHEA plus fadrozole (group 3). The present observations suggest that DHEA retards atherosclerosis formation by means of an NO-dependent system, which may be partially related to the conversion of DHEA to estrogen. The level of cGMP was increased in the aortas of atherosclerotic rabbits (group 1) compared with control rabbits (group 5). These observations are consistent with a previous report that the action of NO was decreased, but the release of NO and O2- increased, in atherosclerosis.56

DHEA has been reported to induce a consistent and reversible morphological change in cultured endothelial cells derived from human umbilical vein.57 Ultrastructurally, multilamellar lysosomal lipid structures were formed, and these changes were thought to be related to the effect of peroxisomes.57 DHEA is known to induce peroxisomes, peroxisome-associated enzymes, and microsomal enzymes in the liver of rats and mice.58 Because the effects of hypolipidemic drugs such as clofibrate, which is known as a weak peroxisome proliferator, appear to be sex dependent in rats, DHEA may have some effects on the plasma lipid profile through the peroxisomes or related enzymes.58 However, the likelihood of such an effect is small, because DHEA treatment did not change the plasma lipid profile in the current study; it seemed to act on the vessels directly.

The binding activity of DHEA has been shown to be higher in an activated human T-lymphocyte line in vitro.8 This bound portion may play a role as a receptor, although that report did not show a change in function caused by DHEA binding.8 DHEA was also reported to antagonize the suppressive effects of dexamethasone on T- and B-lymphocyte proliferation in mice.59 T lymphocytes are known to be present and to play a certain role in atherosclerosis60 ; however, that role has not been fully established. Although the population and distribution of T cells in an atherosclerotic lesion after DHEA treatment did not show any change in our preliminary observations, the possibility of an antiatherosclerotic effect of DHEA through the receptor-mediated system of activating T lymphocytes remains to be elucidated.

Many possible mechanisms may be involved in the atheroprotective effect of DHEA, although certainly 1 of these is the conversion of DHEA to estrogen, and NO seems to play a specific role in the antiatherosclerotic effect of both estrogen and DHEA. The contribution of estrogen to the atheroprotective effect of DHEA was {approx}50% in our study of an HCD-induced atherosclerosis in a rabbit model.


*    Acknowledgments
 
This study was supported in part by grant-in-aid No. 09470166 from the Japanese Ministry of Education. We also thank Norie Kametsuta and Yuriko Kato for their excellent technical assistance.

Received September 22, 1998; accepted October 14, 1999.


*    References
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