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Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats

Francesco Squadrito , Domenica Altavilla , Giovanni Squadrito , Antonino Saitta , Domenico Cucinotta , Letteria Minutoli , Barbara Deodato , Marcella Ferlito , Giuseppe M. Campo , Antonio Bova , Achile P. Caputi
DOI: http://dx.doi.org/10.1016/S0008-6363(99)00359-4 454-462 First published online: 14 January 2000

Abstract

Background: We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. Methods: Female mature Sprague–Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17β-estradiol (20 μg/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM–10 μM) and sodium nitroprusside: (SN 15–30 nM) induced relaxation of aortic rings as well as NG-l-arginine (L-NMA: 10–100 μM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. Results: Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 μM: Sham OVX=2.1±0.2 g/mg tissue; OVX=1.7±0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17β-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. Conclusions: Our data suggest that genistein and 17β-estradiol show overlapping effects on experimental endothelial dysfunction.

Keywords
  • Endothelial function
  • Hormones
  • Nitric oxide

Time for primary review 28 days.

1 Introduction

A large body of evidences suggests that estrogens play a protective role on the cardiovascular system [1]. Postmenopausal estrogen therapy protects against cardiovascular disorders [2]. However, the mechanism(s) by which this protection is mediated remain largely unresolved, because beneficial effects of estrogen on the blood lipid profile account only 20–30% of the overall protection [3]. Growing evidence suggests that estrogen has direct effects on the blood vessel wall indicating that vascular endothelium may play a key role in mediating these effects by producing several vasoactive factors and more specifically nitric oxide (NO) [4]. In fact estrogen replacement therapy (HRT) in postmenopausal women improved the impaired endothelial-dependent relaxation [5].

However, estrogens also have adverse effects on the reproductive system of females [6] and males [7] that limit their therapeutic potential.

Recently several compounds that are estrogen agonists for bone, liver and cardiovascular system that appear to have no estrogen agonist effect on the female reproductive system have been studied [8–12]. Phytoestrogens represent a family of plant compounds that have been shown to have both estrogenic and antiestrogenic properties and fall under two main categories: isoflavones and lignans. For example soy and flax products are particularly good sources of isoflavones and lignans, respectively.

Soybean phytoestrogens have also no activity on the reproductive system in male or female animals [10] and, therefore, it is reasonable to explore the therapeutic potential of these naturally occurring plant estrogens in the prevention of cardiovascular dysfunction.

Genistein is a dietary-derived isoflavonoid bearing an isoflavonoid structure that enhances the dilator response to acetylcholine of atherosclerotic arteries [13]. These data predict for genistein a favorable impact on the cardiovascular system.

Using an experimental model of endothelial dysfunction induced by ovariectomy in female rats, that resembles the impairment in endothelium-dependent vasodilatation observed in postmenopausal women, we investigated whether genistein and 17β-estradiol exert the same degree of protection against the decline in vascular function in ovariectomized rats. This experiment should contribute to understand whether phytoestrogens and more specifically genistein might substitute for estrogens in the prevention of vascular dysfunction.

2 Methods

2.1 Animals

Female mature Sprague–Dawley rats (Charles River Laboratories; 250–280 g) were subjected to a bilateral ovariectomy (OVX rats). Sham operated animals (Sham OVX rats) were used as controls. All rats were maintained at constant humidity (60±5%), temperature (24±1°C) and light cycle (06:00–18:00 h) and were given free access to standard laboratory chow and drinking water. Experiments were approved by the Ethical Committee of the University of Messina and were consistent with the Guide for the Care and Use of Laboratory Animals (NIH Publication No 85-23, revised 1996).

2.2 Estrogen and genistein supplementation

Three weeks after surgery OVX rats and sham OVX animals were randomly assigned to one of the four treatment groups. The first group received daily injections of 17β-estradiol (20 μg/kg in 100 μl cottonseed oil s.c. daily), the second group received the 17β-estradiol vehicle (100 μl cottonseed oil s.c. daily); the third group was given with genistein [0.2 mg/kg in 100 μl of a mixture of DMSO (1.25%) and PEG-400 (98.75%) s.c. daily] and the fourth group received the genistein vehicle [100 μl of a mixture of DMSO (1.25%) and PEG 400 (98.75%) s.c. daily]. The treatment lasted 4 weeks.

2.3 Mean arterial blood pressure, heart rate, body weight and uterine assay

Mean arterial blood pressure (MAP) and heart rate (HR) were measured uncruently using the tail cuff method at baseline conditions and after 4 weeks of treatment. Body weight (mg) was also monitored at the same time points. At the end of experiment the uteri were removed immediately after perfusion fixation and weights were subsequently measured.

2.4 Plasma estradiol, plasma total cholesterol levels and plasma genistein levels

Plasma were collected at the end of experiment when the animals were killed. Plasma 17β-estradiol levels were determined by radioimmunoassay with a commercially available kit. Assay sensitivity was 8.0 pg/ml, and intra-assay and inter-assay coefficients of variation for estradiol were 5.3 and 6.4%, respectively. The analysis of cholesterol in the blood was determined by an enzymatic cholesterol esterase/cholesterol oxidase using an automatic analysis technique on a chemical analyzer.

In order to evaluate genistein plasma levels blood samples (0.5 ml) were collected in polypropylene tubes containing 50 μl of heparin (50 000 IU) and after centrifugation at 3000×g at 4°C for 10 min each sample was stored at −70°C until analysis. The assay was performed by using an HPLC method with UV detection [14] with some modifications. Briefly, 0.5 ml of plasma were added in polycarbonate tubes containing 4-hydroxybenzophenone, used as an internal standard, and terbutylmethyl ether. After shaking, the organic layer was recovered and evaporated in a vacuum concentrator system (Heto La Equipment, Denmark). The extract was reconstituted with methanol 0.05 M ammonium acetate buffer, pH 4.5 (35:65, v/v), and 50 μl of the solution were injected into the HPLC apparatus. The column used was a 3-μ Luna C8, 150×4.6 mm I.D. (Phenomenex, Terrance, CA). Chromatography was carried out at room temperature using a mobile phase of acetonitrile–0.05 ammonium formate buffer, pH 4.0 (28:75, v/v) at a flow-rate of 1.0 ml/min. The HPLC equipment consisted of a solvent delivery module (Mod 422 Master, Kontron Instruments, Everett, USA), a programmable variable wavelength detector (Spectromonitor 4100, Thermo Separation Products, Florida, USA), connected to an automatic integrator (Mod. CR-3A, Shimadzu, Kyoto, Japan). The UV detector was set at a wavelength of 260 nM. The concentration of plasma genistein was expressed in nmol/l.

2.5 Vessel reactivity studies

At the end of the treatment period heparinized rats were euthanized with an overdose of sodium pentobarbital (75 mg/kg, i.p.). Thoracic aortas were removed and placed in cold Krebs solution of the following composition (nM): NaCl 118.4, KCl 4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NAHCO3 25.0 and glucose 11.7. Then, aortas were cleaned of adherent connective and fat tissue and cut into rings approximately 2 mm in length. Rings were then placed under 1 g of tension in an organ bath containing 10 ml Krebs solution at 37°C and bubbled with 95% O2 and 5% CO2 (pH 7.4). All experiments were carried out in the presence of indomethacin (10 μM) in order to exclude the involvement of eicosanoids and their metabolites. Developed tension was measured with an isometric force transducer and recorded on a polygraph (Ugo Basile, Varese, Italy). After an equilibration period of 60 min during which time the rings were washed with fresh Krebs solution at 15–20-min intervals and basal tension was readjusted to 1 g, the tissue was exposed to phenylephrine (PE, 100 nM). When the contraction was stable, the functional integrity of endothelium was assessed by a relaxant response to acetylcholine (ACh, 100 nM). The tissue was then washed occasionally for 30 min.

Endothelium response was evaluated with cumulative concentrations of ACh (10 nM–1 μM) in aortic rings precontracted with phenylephrine (PE; 100 nM). Endothelium independent response was investigated by analyzing the relaxant effects of sodium nitroprusside (SN 15–30 nM) in endothelium denuded aortic rings. Relaxation of the rings was calculated as percentage decrease of contractile force. Some rings were precontracted with phenylephrine and then incubated with NG-l-arginine (L-NMA 10–100 μM) and the results were expressed as g of tension×mg of tissue.

2.6 Nitric oxide synthase activity

To measure nitric oxide synthase (NOS) activity the lungs were removed at the end of experiments and immediately frozen in liquid nitrogen and later assay according to the method of Bredt and Snyder [15] with slight modifications. Briefly, tissues were homogenized on ice in 10 vols. of homogenizing buffer (composition in mM): Tris–HCl 50, DTA 0.1, Pefabloc 1, and dithiothreitol 1. A 50-μl sample of the homogenate was incubated with 50 μl of incubation buffer for 20 min at 37°C. The incubation buffer contained the following cofactors: l-arginine 10 μM, [3H]l-arginine 5 pmol, NADPH 1 mM, calmodulin 30 nM, tetrahydrobiopterin 5 μM, calcium 2 mM, l-valine 60 mM in a Tris buffer at a pH of 7.4. To determine calcium independent NOS activity a buffer replacing the calcium with EGTA (5 mM) was used. Non-specific conversion of arginine to citrulline was determined in the absence of NADPH and a separate buffer containing NG-l-arginine methylester (L-NAME) (1 mM) provided further evidence that NOS was being measured.

After the 20-min incubation, the reaction was stopped by the addition of 1 ml of ice-cold stop buffer (containing Hepes 20 mM, EGTA 2 mM, and EDTA 2 mM at pH 5.5). The reaction mixture was applied to 1-ml Dowex columns (NA+ form, 50 W, mesh size 100–200) which had previously been equilibrated with 1 ml of stop buffer. The eluate was collected and the columns washed twice with 0.75 ml deionized water to retrieve any residual activity. A 400-μl sample of the eluate was taken for scintillation counting.

The protein concentration of the homogenates was determined by the Bradford assay [16] using bovine serum albumin (fraction V) as a standard.

2.7 Statistical analysis

Data are expressed as means±S.D. Comparison between the means of the two groups was performed using ANOVA followed by Bonferroni's test and considered significant at the P<0.05 level.

3 Results

3.1 Mean arterial blood pressure, heart rate, body weight and uterine assay

Bilateral ovariectomy did not modify mean arterial blood pressure, heart rate and body weight. Furthermore, supplementation with either 17β-estradiol or genistein did not cause any significant change in the above-indicated parameters throughout the study in sham ovariectomized or ovariectomized rats. Mean arterial blood pressure and heart rate ranged from 94±10 to 103±13 mmHg and from 339±10 to 356±12 beats/min, respectively.

Changes in body weight were similar among groups and ranged from 275±15 to 300±13 g.

Uterine weights were decreased by greater than 35–40% in the OVX control (vehicle) group compared to sham OVX rats and genistein-supplemented OVX rats showed a similar decline (Fig. 1). The OVX rats treated with 17β-estradiol had a significantly greater mean uterine weight compared to either vehicle or genistein-supplemented OVX rats (Fig. 1).

Fig. 1

Uterine weights in OVX rats treated daily with genistein (0.2 mg/kg s.c. in 100 μl of a mixture of DMSO and PEG-400), 17β-estradiol (20 μg/kg s.c. in 100 μl cottonseed oil) or their vehicle (100 μl of a mixture of DMSO and PEG-400 or 100 μl s.c. cottonseed oil). Mean weights were determined for each treatment group and compared to untreated sham OVX rats. Each point represents the mean±S.D. of six experiments. # P<0.05 vs. vehicle.

3.2 Plasma 17β-estradiol, plasma total cholesterol levels and plasma levels of genistein

Plasma 17β-estradiol levels were significantly reduced in ovariectomized rats (OVX rats) when compared to sham operated rats (sham OVX rats) (Table 1). Estrogen replacement therapy markedly increased plasma 17β estradiol to the levels resembling those observed in sham operated rats (Table 1). In contrast genistein supplementation did not cause any change in the circulating hormone levels (Table 1) either in OVX rats or sham OVX rats.

View this table:
Table 1

Plasma 17β-estradiol, plasma cholesterol and plasma genistein levels in ovariectomized (OVX) or sham ovariectomized (Sham OVX) rats treated with genistein (Gen), 17β-estradiol (E) and their respective vehicles (Gen vehicle and E vehicle)a

Group17β-Estradiol (pg/ml)Genistein (nmol/l)Cholesterol (mmol/l)
Controls (unoperated rats)33±8<12.3±0.1
Sham OVX+Gen vehicle35±7<13.2±0.3
Sham OVX+E vehicle32±5<13.1±0.5
Sham OVX+Gen29±816±2*3.7±0.2
Sham OVX+ E56±6<12.9±0.6
OVX+Gen vehicle4±0.9*<12.7±0.4
OVX+E vehicle3±0.7*<12.8±0.5
OVX+Gen6±1.1*18±3*3.1±0.6
OVX+E28±7*<12.9±0.4
  • a Each point represents the mean±S.D. of six experiments. Animals received daily subcutaneously genistein (0.2 mg/kg in 100 μl of a mixture of DMSO and PEG-400), the genistein vehicle (0.2 mg/kg in 100 μl of a mixture of DMSO and PEG-400)), 17β-estradiol (20 μg/kg in 100 μl cottonseed oil) and the 17β-estradiol vehicle (100 μl cottonseed oil). The treatments lasted 4 weeks.

  • * P<0.001 vs. sham OVX and controls; *P<0.001 vs. OVX+vehicle.

Plasma total cholesterol levels were not significantly changed in ovariectomized rats when compared to sham operated rats. Furthermore neither estrogen replacement therapy or genistein supplementation produced changes in this lipid parameter in both OVX rats and sham OVX rats (Table 1).

Plasma genistein levels were less than 1 nmol/l in sham OVX or OVX rats treated either with vehicle or 17β estradiol. In contrast genistein supplementation significantly increased the plasma levels of the phytoestrogen in both sham OVX and OVX rats (18±3 nmol/l) (Table 1).

3.3 Vessel reactivity

Endothelium response (acetylcholine: ACh; 10 nM–10 μM) and endothelium independent relaxation (sodium nitroprusside: SN 15–30 nM) of thoracic aortas precontracted with phenylephrine (PE 100 nM) were studied at the end of treatment. The effect of sodium nitroprusside were investigated in endothelium denuded aortic rings (E). Contractile responses to phenylephrine ranged from 1.3±0.3 to 1.5±0.2 g/mg tissue and were not different between the several groups. Ovariectomy markedly reduced the relaxant effect of acetylcholine (Fig. 2A and B) while did not produce any significant change in the relaxant effect caused by sodium nitroprusside (Fig. 3A and B). Estrogen replacement therapy or genistein supplementation did not modify the relaxant effects of either acetylcholine or sodium nitroprusside in sham operated rats (Figs. 2 and 3). In contrast both treatments succeeded in improving the impairment in endothelium dependent relaxation of ovariectomized rats and no significant difference was observed between these two active treatments. (Fig. 2).

Fig. 3

(A) Relaxant effect of sodium nitroprusside (15 and 30 nM) in endothelium denuded (E) aortic rings (contracted with phenylephrine 100 nM) from Sham OVX and OVX rats treated daily with genistein (0.2 mg/kg s.c. in 100 μl of a mixture of DMSO and PEG-400) or its vehicle (100 μl s.c. of a mixture of DMSO and PEG-400). Each point represents the mean±S.D of six experiments. The Y-axis shows the percent change in tension from the contraction to phenylephrine. (B) Relaxant effect of sodium nitroprusside (15 and 30 nM) in endothelium denuded (E) aortic rings (contracted with phenylephrine 100 nM) from Sham OVX and OVX rats treated daily, with 17β-estradiol (20 μg/kg s.c. in 100 μl cottonseed oil) or its vehicle (100 μl s.c. cottonseed oil). Each point represents the mean±S.D of six experiments. The Y-axis shows the percent change in tension from the contraction to phenylephrine.

Fig. 2

(A) Relaxant effect of acethylcholine (ACh) in aortic rings (contracted with phenylephrine, 100 nM) from Sham OVX and OVX rats treated daily with genistein (0.2 mg/kg s.c. in 100 μl of a mixture of DMSO and PEG-400) or its vehicle (100 μl of a mixture of DMSO and PEG-400). The Y-axis shows the percent change in tension from the contraction to phenylephrine. * P<0.001 vs. Sham OVX; # P<0.005 vs. OVX+vehicle. (B) Relaxant effect of acethylcholine (ACh) in aortic rings (contracted with phenylephrine 100 nM) from Sham OVX and OVX rats treated daily with 17β-estradiol (20 μg/kg s.c. in 100 μl cottonseed oil) or its vehicle (100 μl s.c. cottonseed oil). Each point represents the mean±S.D of six experiments. The Y-axis shows the percent change in tension from the contraction to phenylephrine. * P<0.001 vs. Sham OVX; # P<0.005 vs. OVX+vehicle.

Addition of NG-l-arginine (L-NMA 10–100 μM) to the organ bath caused a significant contraction of the aortic rings precontracted with phenylephrine (Fig. 4A and B). L-NMA induced vasoconstriction was significantly blunted in ovariectomized rats when compared to sham operated rats. Estrogen replacement therapy or genistein supplementation markedly increased the constrictor response elicited by L-NMA and no significant difference was observed between these two therapeutical regimens. Finally both the active treatments did not change L-NMA induced responses in sham ovariectomized rats (Fig. 4A and B).

Fig. 4

(A) Effects of L-NMA (10 and 100 μM in aortic rings) on maximal contractile response to phenylephrine in aortic rings taken from Sham OVX and OVX rats treated daily with genistein (0.2 mg/kg s.c. in 100 μl of a mixture of DMSO and PEG-400) or its vehicle (100 μl s.c. of a mixture of DMSO and PEG-400). * P<0.05 vs. Sham OVX; # P<0.05 vs. OVX+vehicle. Each point represents the mean±S.D. of six experiments. (B) Effects of L-NMA (10 and 100 μM in aortic rings) on maximal contractile response to phenylephrine in aortic rings taken from Sham OVX and OVX rats treated daily with 17β-estradiol (20 μg/kg s.c. in 100 μl cottonseed oil) or its vehicle (100 μl s.c. cottonseed oil). * P<0.05 vs. Sham OVX; # P<0.05 vs. OVX+vehicle. Each point represents the mean±S.D. of six experiments.

3.4 Nitric oxide synthase activity

Ca2+-dependent NO synthase (cNOS) activity was investigated in lung homogenates at the end of treatment (Table 2). Ovariectomized rats showed a marked decrease in NO synthase activity in comparison with sham operated animals (Table 2). Estrogen replacement therapy or genistein supplementation markedly enhanced the activity of NO synthase in lung homogenates and no significant difference was observed between these two treatments. Neither estrogen therapy or genistein treatment modified cNOS activity in sham ovariectomized rats. Furthermore the addition of NG-l-arginine methylester (L-NAME) to the incubation buffer effectively inhibited NOS activity in lung, thus providing further evidence that NOS was being measured.

View this table:
Table 2

Calcium-dependent (cNOS) and calcium independent (iNOS) nitric oxide synthase activity in lung of ovariectomized (OVX) or sham ovariectomized (Sham OVX) rats treated with genistein (Gen), 17β-estradiol (E) and their respective vehicles (Gen vehicle and E vehicle)a

GroupcNOSINOSTotal NOS+L-NAME
Controls (unoperated rats)19.5±4.52.1±0.92.5±1.3
Sham OVX+Gen vehicle21.7±3.41.9±0.82.9±1.7
Sham OVX+E vehicle18.6±6.62.5±2.22.4±0.9
Sham OVX+Gen20.7±5.52.1±1.22.8±1.3
Sham OVX+ E23.8±6.72.8±1.51.9±1.2
OVX+Gen vehicle10.5±2.5*2.3±1.11.1±0.2
OVX+E vehicle11.1±3.8*2.4±1.61.3±0.4
OVX+Gen17.7±4.5*2.2±1.42.1±0.7
OVX+E19.3±2.8*2.3±1.71.9±0.8
  • a NOS activity was expressed as pmol citrulline/20 min per mg protein and shown as mean±S.D. of six experiments. Animals received daily subcutaneously genistein (0.2 mg/kg in 100 μl of a mixture of DMSO and PEG-400), the genistein vehicle (0.2 mg/kg in 100 μl of a mixture of DMSO and PEG-400)), 17β-estradiol (20 μg/kg in 100 μl cottonseed oil) and the 17β-estradiol vehicle (100 μl cottonseed oil). The treatments lasted 4 weeks.

  • * P<0.001 vs. sham OVX and controls; *P<0.001 vs. OVX+vehicle.

4 Discussion

Genistein is a naturally occurring plant-derived estrogen-like compound. The precursors of this biologically active compound originate in soybean products, whole grain cereal food, seeds, and berries and nuts and they are converted by intestinal bacteria into hormone-like compounds with weak antioxidative and estrogenic activity [17]. Plant derived estrogens have been show to mimic many of the biological activities of 17β-estradiol. The research findings of Barnes et al. [18], Setchell et al. [19] and other investigators [20] have suggested that genistein acts via estrogen receptors, but these and other workers have more recently reported that higher doses of genistein have multiple cellular effects such as inhibition of protein kinase [21].

Diets containing soy product have been shown to reduce several cardiovascular disease risk factors in both human and non-human primates [10]. Furthermore, experimental evidence suggests that the soy isoflavones rather than the protein component itself are responsible for this protective effect [10–22]. Genistein, the principal isoflavone found in soy [23], is structurally similar to 17β-estradiol and diethylbestrol, binds to estrogen receptors and exhibits estrogenic properties in some tissue [24].

It has been suggested that one of the mechanisms by which estrogen replacement therapy may reduce the cardiovascular risk among postmenopausal women is by improving vascular reactivity [25–27]. This improvement in vascular reactivity is independent of estrogen's beneficial effects on plasma lipids and lipoproteins [25] and may be a major factor in the decrease in coronary events observed in women taking estrogen replacement therapy [26].

More specifically it has been hypothesized that the protective effects of estrogen supplementation on the cardiovascular apparatus may be mediated by an increased production of endothelial nitric oxide (NO) from the vascular endothelium. The rationale for this hypothesis is that NO release is reduced in cardiovascular diseases such as hypertension and atherosclerosis [28], and NO has several actions that are vasoprotective such as vasodilatation [29,30], inhibition of platelet adhesion and aggregation [31] and inhibition of smooth muscle cell proliferation and migration [32].

Several studies support the NO hypothesis for estrogen-induced cardiovascular protection. It has been shown that 17β-estradiol acutely attenuated abnormal coronary vasomotor responses to acetylcholine [33] and more recently Gerhard et al. [5] confirmed, using a chronic treatment, that 17β-estradiol restored the impaired endothelium-dependent vasodilatation in postmenopausal women. Further support for the NO hypothesis is found in the fact that 17β-estradiol stimulates the constitutive NO synthase activity by increasing the transcription of the endothelial NO synthase gene [4].

Genistein protective effects on the cardiovascular apparatus have been mainly ascribed to its cholesterol lowering effects [34]. Besides this activity, another potential mechanism has recently emerged. In fact it has been suggested that the isoflavone enhances the dilator response to acethylcholine in atherosclerotic arteries [13] and relaxes noradrenaline, potassium chloride and calcium chloride precontracted arterial rings [35].

With this background, we attempted to determine if the protective effects of genistein supplementation on the cardiovascular apparatus might be mediated, at least in part, by an increased production of endothelial nitric oxide (NO) from the vascular endothelium. To test this NO hypothesis for the genistein beneficial activity we performed a comparison study with 17β-estradiol on endothelial dysfunction induced by bilateral ovariectomy in female rats.

Our results suggest that ovariectomized rats developed a typical endothelial dysfunction resembling that observed in postmenopausal women. More specifically they had a markedly reduced relaxant response to acetylcholine, an endothelium and NO-dependent relaxant agent and unchanged response to sodium nitroprusside, an endothelium and NO-independent relaxing compound. In addition, aortic rings prepared from ovariectomized rats showed a reduced contraction in response to L-NMA, an inhibitor of NO synthase. All these data, taken together, would suggest that ovariectomized rats show a marked impairment in the l-arginine/NO pathway characterized by a blunting production of endothelial NO. This blunted endothelial NO release seems to be the consequence of a decreased activity of the enzyme devoted to its regulation, the constitutive NO synthase. Indeed lung homogenates prepared from ovariectomized rats showed a reduced constitutive NO synthase activity, when compared to sham operated rats. This disturbance in the l-arginine/NO pathway has already been proposed to explain the endothelial dysfunction observed in postmenopausal women [5]. In contrast, the plasma levels of cholesterol were unchanged in ovariectomized rats, thus suggesting that the present model, at least under this experimental conditions (4 weeks of estrogen deficiency), causes a selective impairment in the l-arginine/NO pathway (endothelial dysfunction) without this other important confounding alteration. The major finding of this paper is that genistein supplementation succeeded in reverting the impaired endothelial dysfunction observed in ovariectomized rats. The phytoestrogen was able to restore the endothelium response likely through an increased production of basal endothelial NO release: in fact genistein treatment increased the constrictor response elicited by L-NMA. This strongly supports the idea that the isoflavone treatment was able to enhance the depressed l-arginine–NO pathway. From a mechanistic point of view it can be proposed that genistein increased the activity of the endothelial NO synthase: indeed lung homogenates prepared from phytoestrogen-supplemented ovariectomized rats showed an increased activity of this enzyme isoform. As far as we known this represents the first report indicating that this natural occurring phytoestrogen stimulates in vivo the activity of endothelial NO synthase. However, previously in vitro published data showed that neither the removal of endothelium nor the inhibition of nitric oxide synthesis had any effect on the genistein-induced relaxation responses [35]. This would suggest that perhaps there is a genomic and non-genomic effect of the genistein similar as to genomic and non-genomic effect of estrogen, and that there is no relationship between the exact concentrations of genistein which would be in the in vitro experiment compared to that in the present study which is an in vivo experiment. Indeed we measured the plasma levels of the phytoestrogen following a 4-week period of genistein supplementation. The plasma genistein levels averaged 18±3 nmol/l, a concentration quite different from that present in the organ bath of the in vitro study [35]. In this regards, it has been suggested that genistein behaves as a tyrosine kinase inhibitor at higher doses while at lower doses (as the dose used in the present study) it exerts estrogenic activity [21]. Finally, the lower doses may be easily reached from a nutritionally-based treatment since the concentration of genistein in most soy food ranges from 1–2 mg/g protein.

The second important finding of this paper is that genistein and 17β-estradiol showed overlapping effects, as far as endothelial dysfunction is concerned. In fact, at least under our experimental conditions, the well-known ability of estrogen replacement therapy to revert endothelial dysfunction during estrogen deficiency was nicely reproduced by genistein supplementation. No statistical differences were, in fact, observed between these two active treatments. In contrast, while 17β-estradiol significantly modify uterine weights, the beneficial effects of genistein on the cardiovascular apparatus was not accompanied by any change in the uterus weight. These data confirm that genistein does not affect the reproductive system and led us to hypothesize that genistein may represent a good candidate to substitute estrogens in the treatment of postmenopausal endothelial dysfunction.

Interestingly, addition of either 17β-estradiol or genistein to ovarian intact animals did not affect endothelium-dependent relaxation and NOS activity, thus suggesting that addition of either genistein or 17β-estradiol in the presence of endogenous estrogen is not able to further improve endothelial function.

Estrogen receptors are present in the blood vessel wall [36] and genistein acts as estrogen agonist on these structures [37]. It could therefore be argued that genistein may increase the activity of the endothelial NO gene, as previously shown for 17β-estradiol [4]. However at this time we have no direct evidence to support such an idea.

In conclusion our findings suggest that genistein-induced augmentation of NO production may contribute to its beneficial cardiovascular actions.

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View Abstract