Endothelial dysfunction and oxidative stress in children with sleep disordered breathing: Role of NADPH oxidase

Endothelial Dysfunction and Oxidative Stress DuringEstrogen Deficiency in Spontaneously Hypertensive RatsSven Wassmann, MD*; Anselm T. Bäumer, MD*; Kerstin Strehlow, MD; Martin van Eickels, MD;

Christian Grohé, MD; Katja Ahlbory, MS; Renate Rösen, MD; Michael Böhm, MD; Georg Nickenig, MD

Background—Postmenopausal estrogen deficiency is associated with an increased cardiovascular risk, hypertension, andoxidative stress. Angiotensin type 1 (AT1) receptor regulation is involved in the pathogenesis of atherosclerosis. Tocharacterize vascular function, oxidative stress, and AT1 receptor regulation during estrogen deficiency, ovariectomizedspontaneously hypertensive rats (SHR) were investigated in comparison with sham-operated animals and withovariectomized rats receiving estrogen replacement therapy with 17b-estradiol.

Methods and Results—Arterial blood pressure was similar in all 3 groups investigated. Five weeks after ovariectomy,endothelial dysfunction in aortic rings was observed, which was reversed by estrogen replacement therapy. Estrogendeficiency led to an enhanced vasoconstriction by angiotensin II. Vascular superoxide production was significantlyincreased compared with that in sham-operated rats, as measured by lucigenin chemiluminescence assays. Estrogensubstitution normalized the production of free radicals in the vessel wall. Vascular AT1 receptor expression wassignificantly upregulated by estrogen deficiency, as shown by quantitative reverse transcription–polymerase chainreaction, whereas endothelial NO synthase mRNA expression and NO release were unchanged. Five-week treatment ofthe animals with the AT1 receptor antagonist irbesartan prevented endothelial dysfunction in ovariectomized rats andnormalized the vascular production of free radicals.

Conclusions—In SHR, estrogen deficiency leads to increased vascular free radical production and enhanced angiotensinII–induced vasoconstriction via increased vascular AT1 receptor expression, resulting in endothelial dysfunction.Estrogen replacement therapy and AT1 receptor antagonism prevent these pathological changes. Therefore, estrogendeficiency–induced AT1 receptor overexpression and oxidative stress may play an important role in cardiovasculardiseases associated with menopause.(Circulation. 2001;103:435-441.)

Key Words: angiotensinn atherosclerosisn hormonesn endothelium

Females suffer less frequently from cardiovascular dis-eases during their reproductive years than do their male

counterparts. This tendency disappears after menopause, thenatural state of estrogen deficiency.1,2 Estrogen replacementtherapy potentially prevents the development of cardiovascu-lar diseases in postmenopausal women.3–5 The vascular ef-fects of estrogens are not completely understood. Estrogenslower plasma lipoproteins,3 influence the renin-angiotensinsystem,6,7 exert antioxidative properties,8 and may act ascalcium-blocking agents.9 In addition, estrogens exert directeffects on the vessel wall, such as an increase of vascular NOproduction and modulation of endothelial NO synthase(eNOS [NOS III]) expression.10–12

Increased NO production and the modulation of the lipid profilemay in part underlie the well-recognized beneficial effects ofestrogens on endothelial dysfunction, a prerequisite of atheroscle-

rosis.13,14However, it is currently thought that endothelial dysfunc-tion is not based on reduced production but is evoked by a decreasedbioavailability of NO.15,16The latter is decisively influenced by thelevel of reactive oxygen species (ROS), such as superoxide, in thevessel wall. An increased production of superoxide putatively leadsto the scavenging of NO and to the cellular damage associated withendothelial dysfunction.15,16

Angiotensin type 1 (AT1) receptor activation is a predom-inant source of free radical production in the vasculature.17,18

Recently, it has been shown that estrogen deficiency causesAT1 receptor overexpression in vivo, leading to enhancedbiological effects of the renin-angiotensin system that couldin part serve as an explanation for the increase in cardiacevents after menopause in women.19

We hypothesized that a lack of estrogens could induceenhanced oxidative stress via AT1 receptor overexpression,

Received June 21, 2000; revision received July 31, 2000; accepted August 4, 2000.From the Medizinische Klinik und Poliklinik, Innere Medizin III, Universitatskliniken des Saarlandes, Homburg/Saar, Germany, and the Institut für

Pharmakologie (R.R.), Universität zu Köln, Köln, Germany. Dr Grohé is now at Medizinische Klinik II, Universität Bonn, Bonn, Germany. Dr van Eickelsis now at Molecular Cardiology Research Institute, Tufts University, Boston, Mass.

*Drs Wassmann and Bäumer contributed equally to this study.Correspondence to Dr Georg Nickenig, Medizinische Klinik und Poliklinik, Innere Medizin III, Universitatskliniken des Saarlandes, 66421

Homburg/Saar, Germany. E-mail [email protected]© 2001 American Heart Association, Inc.

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which could ultimately lead to endothelial dysfunction. Totest this hypothesis, we investigated spontaneously hyperten-sive rats after ovariectomy with and without concomitantestrogen replacement therapy. The significance of AT1 recep-tor activation was substantiated by an additional treatmentregimen with the AT1 receptor antagonist irbesartan.

MethodsMaterialsAngiotensin II, lucigenin, Taq DNA polymerase, nucleotides, salts,and other chemicals were purchased from Sigma Chemical Co.Moloney murine leukemia virus reverse transcriptase was obtainedfrom GIBCO-BRL. Nitroglycerin was purchased from Solvey. RNAclean was purchased from AGS. Irbesartan was a gift from Sanofi-Synthelabo (Berlin, Germany).

AnimalsFemale spontaneously hypertensive rats (SHR) were put on astandard chow and were ovariectomized or sham-operated (controlgroup) 16 weeks after birth. For treatment, 17b-estradiol pellets(containing 1.7 mg estradiol each, 60-day release, Innovative Re-search) were administered subcutaneously with a 10-gauge trochar.Irbesartan treatment was started 2 weeks after ovariectomy at 50mgzkg-1zd-1 by adding the drug to the drinking water.20 The rats werekilled by decapitation. Animal experiments were performed inaccordance with the German animal protection law. Tissue sampleswere harvested 5 weeks (7 weeks for the irbesartan group) aftersurgery.

Blood Pressure MeasurementAnimals were anesthetized (100 mg/kg body wt IP ketamine and 5mg/kg body wt IP xylazine), and a stretched PE catheter was insertedinto the femoral artery and exteriorized at the neck. The animalswere allowed to recover from anesthesia for 48 hours before theblood pressure measurements were performed by connecting thesaline-filled catheter to a pressure transducer. Measurements tookplace in conscious animals 5 times for 10 minutes each on 2consecutive days. Thereafter, the animals were anesthetized asdescribed above and killed by decapitation, and the organs wereexplanted.

Aortic Ring Preparations and Tension RecordingAfter excision of the descending aorta, the vessel was immersed inchilled modified Tyrode’s buffer (pH 7.4) composed of (mmol/L)NaCl 136.9, KCl 5.4, CaCl2 1.8, MgCl2 1.05, Na-EDTA 0.05,NaH2PO4 0.42, NaHCO3 22.6, andD(1)glucose 5.5, which containedadditional ascorbic acid (0.28 mmol/L) and indomethacin (0.01mmol/L). Adventitial tissue was carefully removed. Five-millimeterrings were mounted for recording of isometric tension in organ bathsfilled with modified Tyrode’s buffer (37°C), which was continuouslyaerated with 95% O2/5% CO2. The preparations were attached to aforce transducer, and isometric tension was recorded on a polygraph.Aortic rings were allowed to equilibrate for 60 minutes. A restingtension of 1 g was maintained throughout the experiment. Drugswere added in increasing concentrations to obtain cumulativeconcentration-response curves: KCl (20 and 60 mmol/L), angioten-sin II (0.01 nmol/L to 1mmol/L), phenylephrine (0.1 nmol/L to 10mmol/L), carbachol (0.1 nmol/L to 100mmol/L), and nitroglycerin (1nmol/L to 10mmol/L). The drug concentration was increased whenvasoconstriction or vasorelaxation was completed (on average, 3 to6 minutes for each step). Drugs were washed out before the nextsubstance was added.

mRNA Isolation and PCRsAortas were isolated, quickly frozen in liquid nitrogen, and homog-enized with a motorized homogenizer. RNA was isolated with RNAclean according to the manufacturer’s protocol to obtain total cellularRNA. Aliquots (1mg) were electrophoresed through 1.2% agarose–

0.67% formaldehyde gels and stained with ethidium bromide toverify the quantity and quality of the RNA. Isolated total RNA (1mg)and an AT1 receptor mutant mRNA (10 pg) were mixed and reverse-transcribed by using random primers and Moloney murine leukemia virusreverse transcriptase for 60 minutes at 42°C and 10 minutes at 75°C. Thesingle-stranded cDNA was amplified by polymerase chain reactions (PCRs)by using Taq DNA polymerase. Twenty-eight cycles were performed underthe following conditions: 30 seconds at 94°C, 45 seconds at 55°C, and 45seconds at 72°C. The sequence for AT1 receptor sense and antisense primerswere 59-ACCCTCTACAGCATCATCTTTGTGGTGGGG-39 and 59-GGGAGCGTCGAATTCCGAGACTCATAATGA-39, respectively. Thesame cDNA samples were used for GAPDH cDNA amplification (22cycles) to confirm that equal amounts of RNA were reverse-transcribed.The primers used were 59-ACCACAGTCCATGCCATCAC-39and 59-TCCACCACCCTGTTGCTGTA-39. PCR amplification gave 479-, 191-,and 452-bp fragments that originated from AT1 receptor wild-type mRNA,mutated AT1 receptor mRNA, and GAPDH mRNA, respectively. Ampli-fication of a 340-bp fragment of eNOS cDNA was carried out with primerpairs 59-TTCCGGCTGCCACCTGATCCTAA-39 and 59-AACATA-TGTCCTTGCTCAAGGCA-39for 35 cycles under the following condi-tions: 30 seconds at 94°C, 30 seconds at 60°C, and 60 seconds at 72°C. Forsemiquantification, PCR conditions were chosen so that the reaction waswithin the linear exponential phase with respect to the amount of cDNAtemplate and number of cycles performed. Equal amounts of reversetranscription (RT)-PCR products were loaded on 1.5% agarose gels, andoptical densities of ethidium bromide–stained DNA bands were quantified.AT1 receptor mRNA expression is expressed as the ratio of AT1 receptorwild-type and AT1 receptor mutant (internal standard) PCR signal of eachsample.

Measurement of ROSFor measurement of superoxide release of intact vessel segments,aortas were excised carefully and placed in chilled modified Krebs-HEPES buffer (pH 7.4) composed of (mmol/L) NaCl 99.01, KCl4.69, CaCl2 1.87, MgSO4 1.20, Na-HEPES 20.0, K2HPO4 1.03,NaHCO3 25.0, andD(1)glucose 11.1. Connective tissue was re-moved, and aortas were cut into 5-mm segments. The aortic ringswere placed in Krebs-HEPES buffer aerated with 95% O2/5% CO2

and were incubated for 30 minutes at 37°C. Then the samples weretransferred into scintillation vials containing 2 mL Krebs-HEPESbuffer with 5 mmol/L lucigenin. Chemiluminescence was assessedover 10 minutes in a scintillation counter (Berthold Lumat LB 9501)at 1-minute intervals. Background signals were subtracted. Thevessel segments were then dried, and dry weight was determined.Superoxide release is expressed as relative chemiluminescence permilligram aortic tissue.

NO MeasurementExcised and prepared aortic segments were placed in oxygenated(PO2 150 mm Hg) 10 mmol/L HEPES buffer. The vessel waslongitudinally opened and placed in an organ bath with the luminalface turned upward. An NO-sensitive electrode (ISO-NO electrode,World Precision Instruments) was placed at a fixed distance of 1 mmabove the aortic lumen. Beforehand, the electrode was calibratedwith a standardized NO solution. Substances were added at the sameplace in the organ bath, and NO release of the aortic segment wasmeasured.

Statistical AnalysisData are presented as mean6SEM obtained in at least 3 separateexperiments. Statistical analysis was performed by ANOVA (posthoc Scheffé procedure) and Mann-WhitneyU test with SSPS 6.0software. A value ofP,0.05 indicates statistical significance.

ResultsEstrogen Plasma ConcentrationsEstrogen plasma levels dropped in ovariectomized rats (1.660.5pg/mL) compared with sham-operated rats (35.7612 pg/mL)and recovered after estrogen substitution (61621 pg/mL).

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Effect of Estrogen Deficiency on BloodPressure in SHRBlood pressure was evaluated intra-arterially in conscious ani-mals. Blood pressure levels (4 weeks after ovariectomy) werenot significantly different between groups: systolic blood pres-sures were 16064 mm Hg for sham-operated rats, 170612mm Hg for ovariectomized rats, and 17869 mm Hg for ovari-ectomized rats with estrogen replacement (n55 per group).

Effect of Estrogen Deficiency on AorticVasorelaxation and VasoconstrictionAortic rings were isolated 5 weeks after ovariectomy, andtheir functional performance was assessed in organ cham-ber experiments (n55 with 15 rings per group). Figure 1

shows the endothelium-dependent vasorelaxation on in-creasing concentrations of carbachol and the endothelium-independent relaxation exerted by nitroglycerin. Whereasthe endothelial cell–independent vasorelaxation was notaltered by ovariectomy, the carbachol-induced vasodilata-tion was impaired during estrogen deficiency, suggesting adecremental effect of estrogen deficiency on endothelialfunction in SHR (force of contraction 18.664.8% versus3.461.0% for control of phenylephrine-induced vasocon-striction; carbachol, 100mmol/L; P,0.05 versus control).Endothelial function was improved after estrogen replace-ment therapy of ovariectomized rats, which supports thenotion that estrogen selectively influences endothelialfunction. Nitroglycerin-induced vasodilatation at concen-trations of 10 nmol/L and 1mmol/L nitroglycerin wasimpaired during estrogen replacement therapy (P,0.05versus control). However, ED50 values and maximal effi-cacy remained unaltered.

The contraction of the aortas was assessed during exposureto increasing concentrations of either phenylephrine or an-giotensin II. Figure 2 reveals that the angiotensin II–inducedvasoconstriction was selectively increased after ovariectomy(force of contraction 2.060.1 versus 1.460.1 mN for control;angiotensin II, 0.1mmol/L; P,0.05 versus control). Thishypercontractility on angiotensin II stimulation was com-pletely abolished by estrogen replacement treatment. In

Figure 1. Effect of estrogen deficiency and AT1 receptor block-ade on aortic vasorelaxation. Sixteen-week-old female SHRwere ovariectomized or sham-operated. In one group, 17b-estradiol pellets were administered subcutaneously. Anothergroup was treated with irbesartan for 5 weeks. Aortic segmentswere isolated 5 weeks after ovariectomy, and their functionalperformance was assessed in organ chamber experiments.Drugs were added in increasing concentrations. Endothelium-dependent vasorelaxation was tested with carbachol (A),whereas endothelial cell–independent relaxation was investi-gated with nitroglycerin (B). Graphs show force of contraction,expressed in percentage of maximum phenylephrine-inducedvasoconstriction. Data are shown as mean6SEM (n55 with 15rings per group). *P,0.05 vs sham and irbesartan. Ovarex indi-cates ovariectomized SHR. AT1-B indicates irbesartan-treatedanimals.

Figure 2. Effect of estrogen deficiency on aortic vasoconstric-tion. Vasoconstriction of aortic rings was investigated withangiotensin II (A) and phenylephrine (B), expressed as absoluteforce of contraction. Data are shown as mean6SEM (n55 with15 rings per group). *P,0.05 vs sham. Ovarex indicates ovariec-tomized SHR.

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contrast, a-adrenoreceptor–mediated vasoconstriction in-duced by phenylephrine was not altered significantly.

Effect of Estrogen Deficiency on VascularSuperoxide ProductionThe increased vascular responsiveness on angiotensin II inovariectomized SHR could possibly lead not only to en-hanced vasoconstriction but also to an enhanced level of freeradicals in the vessel wall, which could cause the observedendothelial dysfunction. Therefore, the vascular productionof ROS was assessed by lucigenin chemiluminescence assaysin intact isolated aortic segments (n510 per group). Figure 3illustrates that estrogen deficiency induced a significantincrease of superoxide production in the vessel wall to160627% of control levels (P,0.05 versus control), whichwas completely prevented by concomitant estrogen replace-ment therapy (P,0.05 versus ovariectomy).

Effect of AT1 Receptor Blockade on EndothelialFunction and Superoxide Release DuringEstrogen DeficiencyThe above-mentioned findings suggest that enhanced AT1

receptor activation causes endothelial dysfunction as well asenhanced oxidative stress. To further support this notion,ovariectomized SHR were treated with the AT1 receptorantagonist irbesartan for 5 weeks. Vasomotion of aortic ringpreparations was assessed in organ chamber experiments(n55 with 15 rings per group). Figure 1 reveals that AT1

receptor antagonism completely normalized endothelial dys-function in estrogen-deficient rats (P,0.05 versus ovariecto-my). Nitroglycerin-induced vasorelaxation was similar be-tween the groups. Endothelial function in either sham-operated or estrogen-treated animals was not altered (data notshown).

Endothelial function is likely to be improved by thereduction of oxidative stress. Figure 3 demonstrates that thetreatment with irbesartan significantly decreased vascularsuperoxide production in ovariectomized SHR (P,0.05 ver-sus ovariectomy).

Effect of Estrogen Deficiency on Vascular AT1Receptor and eNOS mRNA ExpressionEstrogen deficiency of SHR caused an increase of angioten-sin II–induced vasoconstriction and vascular ROS produc-tion. Both effects are prominently mediated through AT1

receptor activation. Therefore, it was reasonable to assumethat estrogens directly influenced vascular AT1 receptorexpression. Vascular AT1 receptor mRNA concentrationswere assessed by means of quantitative RT-PCR in RNAisolated from aortic segments of all SHR groups. Figure 4Ashows the densitometric analysis (n55 per group), revealingthat AT1 receptor mRNA expression was significantly up-

Figure 3. Effect of estrogen deficiency and AT1 receptor block-ade on vascular production of ROS. Superoxide production inintact isolated aortic segments was assessed by lucigeninchemiluminescence assays (5 mmol/L lucigenin). Superoxiderelease is expressed as relative chemiluminescence (RLU) permilligram of aortic tissue (mean6SEM, n57 to 10 per group).*P,0.05 vs sham; **P,0.05 vs ovariectomized SHR (Ovarex).

Figure 4. Effect of estrogen deficiency on vascular AT1 receptorand eNOS expression. AT1 receptor mRNA expression in aorticpreparations of sham-operated control rats and estrogen-deficient SHR was assessed by quantitative RT-PCR; expres-sion of eNOS and GAPDH mRNA was quantified by semiquanti-tative RT-PCR. Densitometric analysis was expressed asmean6SEM (n55 per group) of ratio of AT1 receptor wild-type(AT1-R WT) and AT1 receptor mutant (AT1-R mutant) PCR signal(A). Densitometric analysis of amplified GAPDH (B) and eNOSDNA fragments (C) is expressed as mean6SEM (n55 pergroup). *P,0.05 vs sham; **P,0.05 vs ovariectomized SHR(Ovarex).

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regulated to 177626% of control in ovariectomized SHR(P,0.05 versus control). Treatment of ovariectomized ratswith estrogens reversed this AT1 receptor overexpression(P,0.05 versus ovariectomy). Figure 4B demonstrates theunaltered GAPDH expression (n55 per group). In addition,eNOS mRNA expression was assessed in the same samplesvia semiquantitative RT-PCR. Figure 4C illustrates the den-sitometric results of these experiments (n55 per group).Expression of eNOS mRNA remained unchanged betweengroups.

Effect of Estrogen Deficiency on VascularNO ReleaseEstrogen-induced increase of vascular NO release could alsoaccount for the worsening of endothelial dysfunction duringestrogen deficiency. Therefore, the NO release of aorticsegments was selectively measured with an NO electrode.Figure 5 shows that carbachol-induced NO release was notstatistically different between groups (n57 per group), sug-gesting that estrogen-induced modulation of NO release orproduction was not involved in the detected alterations ofvascular function.

DiscussionEstrogens have been suggested to exert vasoprotectiveeffects, leading to a considerable lowering of cardiacevents.21 This notion is based on several epidemiological,clinical, and molecular findings: The incidence of cardio-vascular disease is low in premenopausal women, but itincreases steadily in postmenopausal women. Addition-ally, postmenopausal hormone replacement therapy mayreduce this rise of cardiovascular events, as suggested byretrospective studies.1–5 Earlier reports attributed the ben-eficial vascular effects of estrogens mainly to their influ-ence on serum lipid concentrations.3 Recently, evidence isaccumulating that direct effects of estrogens on bloodvessels may contribute significantly to their cardioprotec-tive effects.21 This involves long-term effects on cellulargene expression programs, which are thought to be medi-

ated by genomic effects of the activated steroid receptors.In addition, nongenomic rapid effects in vascular cells,which obviously occur independently of modulation ofgene expression, have been reported. One of the mostprominent features seems to be a rapid vasodilatory effectof estrogen, which is elicited through endothelium-dependent and endothelium-independent mechanisms.Current data suggest that estrogens enhance the bioavail-ability of NO through stimulation of eNOS (NOS III) andNO release and potentially also through antioxidantproperties.10 –12,21

These effects are of special interest with respect to thepathogenesis of atherosclerosis. Namely, increased release ofNO has been associated with improved endothelial dysfunc-tion and inhibition of cell growth. Endothelial dysfunction isnot only a prerequisite of atherosclerosis but seems to servealso as a potent predictor of cardiac event rates.22–25 BesidesNO, ROS are thought to be involved in the onset anddevelopment of endothelial dysfunction. Endothelial cells andvascular smooth muscle cells are known to be potent sourcesof ROS.15,17,18,26 It has recently been shown that thesemolecules participate in the proliferation of vascular smoothmuscle cells, promote the development of hypertension, andinfluence the apoptosis of vascular cells,17,26–28which may berelated to either oxidative scavenging of NO or to directcellular effects of free radicals.15 Recent findings suggest thatan overwhelming production of ROS, such as superoxide andhydrogen peroxide, rather than a decreased production of NOmay be decisively involved in the initiation and the acceler-ation of vascular damage.16

AT1 receptor activation induces vasoconstriction andcellular growth and leads to free radical release in thevessel wall.29 This receptor is highly regulated, amongothers, by angiotensin II, lipoproteins, growth factors, andinsulin.30 –33 It has recently been reported that estrogencauses downregulation of the vascular AT1 receptor andthat estrogen deficiency is accompanied by AT1 receptoroverexpression.19 On the basis of these findings, wereasoned that estrogen deficiency could lead to increasedoxidative stress and endothelial dysfunction via AT1 re-ceptor regulation.

Indeed, the present study indicates that estrogen defi-ciency causes endothelial dysfunction in SHR, which ispresumably mediated through increased oxidative stress,as assessed by the enhanced superoxide production in thevessel wall. Expression of eNOS and NO release were notaltered by ovariectomy or estrogen replacement therapy,suggesting that not a decrease in NO synthesis but ratheran enhanced production of free radicals such as superoxideunderlies the observed endothelial dysfunction. The lattermay be evoked by AT1 receptor overexpression duringestrogen deficiency, which was reversed by estrogentherapy. The prevented AT1 receptor overexpression dur-ing estrogen supplementation led to decreased oxidativestress and to an improved endothelial function.

The presented data suggest that vascular eNOS expressionand NO release are not influenced by estrogens in this model;these findings seem to be contradictory to the aforementionedfindings on estrogen-induced NO release.10–12 Whereas our

Figure 5. Effect of estrogen deficiency on vascular NO release.NO release of aortic segments was measured with NO elec-trode. Aortic rings were opened longitudinally and placed inorgan bath. NO electrode was placed closely above aorticlumen. Carbachol-induced NO release was measured for allgroups. Data are expressed as mean6SEM (n57 per group,P5NS). Ovarex indicates ovariectomized SHR.

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results are derived from a long-term animal model, data onestrogen-evoked NO release are mostly derived from short-term in vitro studies, which may explain the contrastingfindings. Moreover, it has been reported that estrogens didnot enhance eNOS expression and activity in mouse and ratmodels or in cultured endothelial cells,34–36 which supportsour presented data.

To further explore the role of AT1 receptor activation inthe setting of estrogen deficiency, ovariectomized ratswere concomitantly treated with an AT1 receptor antago-nist. This treatment not only normalized vascular superox-ide production but also reversed the endothelial dysfunc-tion associated with estrogen deficiency withoutreplacement of estrogens. This strongly suggests that thedetected AT1 receptor overexpression in the absence ofestrogens may play a decisive role in the enhancedvascular damage after ovariectomy. This is also docu-mented by the fact that angiotensin II caused a profoundlyincreased vasoconstriction in the ovariectomized animals.According to our data, the antioxidant properties of estro-gens could at least in part be mediated through thedownregulation of AT1 receptor gene expression.

Our findings are in good agreement with a recentlypublished study that showed, in comparison with anantihypertensive regimen, a more potent reduction ofblood pressure by AT1 receptor antagonism in postmeno-pausal women.37 Especially in the light of the Heart andEstrogen/Progestin Replacement Study (HERS) trial, aprospective secondary prevention study that did not showa beneficial influence of estrogen replacement therapy oncardiovascular mortality in postmenopausal women,38 al-ternative treatment strategies for women at coronary riskafter menopause need to be evaluated. AT1 receptoroverexpression in the pathophysiological setting of estro-gen deficiency and the profound antihypertensive effect ofAT1 receptor antagonists provide new mechanistic insightsand medical tools that could help to introduce a moresuccessful prevention of cardiovascular events in post-menopausal females.

AcknowledgmentsThis work was supported by the Deutsche Forschungsgemein-

schaft, the Deutsche Herzstiftung, and a research grant from Sanofiand Bristol Myers Squibb.

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