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Chronic estradiol-17 exposure increases superoxide productionin the rostral ventrolateral medulla and causes hypertension:reversal by resveratrol

Madhan Subramanian,1 Priya Balasubramanian,2 Hannah Garver,2 Carrie Northcott,2 Huawei Zhao,2Joseph R. Haywood,2 Gregory D. Fink,2 Sheba M. J. MohanKumar,2 and P. S. MohanKumar11Departments of Pathobiology and Diagnostic Investigation and 2Pharmacology and Toxicology, Colleges of Veterinary,Osteopathic and Human Medicine, Michigan State University, East Lansing, MichiganSubmitted 10 January 2011; accepted in final form 10 March 2011

Subramanian M, Balasubramanian P, Garver H, Northcott C, ZhaoH, Haywood JR, Fink GD, MohanKumar SM, MohanKumar PS.Chronic estradiol-17 exposure increases superoxide production in the rostralventrolateral medulla and causes hypertension: reversal by resveratrol. Am JPhysiol Regul Integr Comp Physiol 300: R1560R1568, 2011. First pub-lished March 16, 2011; doi:10.1152/ajpregu.00020.2011.Women are ex-posed to estrogen in several forms, such as oral contraceptive pills andhormone replacement therapy. Although estrogen was believed to becardioprotective, lately, its beneficial effects are being questioned.Recent studies indicate that oxidative stress in the rostral ventrolateralmedulla (RVLM) may play a role in the development of hypertension.Therefore, we hypothesized that chronic exposure to low levels ofestradiol-17 (E2) leads to hypertension in adult-cycling femaleSprague Dawley (SD) rats potentially through generation of superox-ide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old)female SD rats were either sham-implanted or implanted (subcutane-ously) with slow-release E2 pellets (20 ng/day) for 90 days. A groupof control and E2-treated animals were fed lab chow or chow con-taining resveratrol (0.84 g/kg of chow), an antioxidant. Rats wereimplanted with telemeters to continuously monitor blood pressure(BP) and heart rate (HR). At the end of treatment, the RVLM wasisolated for measurements of superoxide. E2 treatment significantlyincreased mean arterial pressure (mmHg) and HR (beats/min) com-pared with sham rats (119.6 0.8 vs. 105.1 0.7 mmHg and371.7 1.5 vs. 354.4 1.3 beats/min, respectively; P 0.0001).Diastolic and systolic BP were significantly increased in E2-treatedrats compared with control animals. Superoxide levels in the RVLMincreased significantly in the E2-treated group (0.833 0.11 nmol/minmg) compared with control (0.532 0.04 nmol/minmg; P 0.05). Treatment with resveratrol reversed the E2-induced increases inBP and superoxide levels in the RVLM. In conclusion, these findingssupport the hypothesis that chronic exposure to low levels of E2induces hypertension and increases superoxide levels in the RVLMand that this effect can be reversed by resveratrol treatment.

oxidative stress; blood pressure; estrogen; brain stem

CARDIOVASCULAR DISEASE REMAINS one of the leading causes ofmorbidity and mortality in women. Postmenopausal increasesin blood pressure (BP) in women make hypertension moreprevalent in women compared with men of the same age (6,42). Because premenopausal women have lower blood pres-sure compared with age-matched men, estrogens were thoughtto play a protective role against hypertension. Estrogens are

reported to improve lipid profile (52), decrease vascular resis-tance (28), and modulate the activity of brain nuclei involvedin cardiovascular regulation (18, 47). However, recent reportsfrom the Womens Health Initiative study, National Institutesof Health have provided evidence that hormone replacementtherapy (HRT) using estrogen alone or a combination ofestrogen and progestin does not confer cardiovascular protec-tion and may actually increase the risk for coronary heartdisease among postmenopausal women (30). These clinicalstudies have brought to light the importance of clinical andbasic research in understanding the role of estrogen in BPregulation (57). Oral estrogen administration, either givenalone or in combination with progestins, was found to promotesystolic blood pressure in postmenopausal women (34, 46).Although the magnitude of the increase in BP was onlybetween 1 and 2 mmHg, similar increases in systolic BP andpulse pressure are known to be associated with a higher rate ofprogression of coronary atherosclerosis (35) and developmentof cardiovascular events (34) in large clinical trials.

In addition to women who are on HRT, younger women whotake oral contraceptives are also at increased risk for poten-tially developing cardiovascular disorders. Oral contraceptiveshave been used worldwide for more than 30 years. Mostwomen taking oral contraceptives have been found to havesmall elevations in BP of2 mm of Hg (56). In addition,5%of patients who take a preparation containing more than 50 gof estradiol-17 (E2) have significant elevations in BP greaterthan 140/90 mmHg (56). Estrogen rather than progesterone,appears to be the primary cause for increases in BP observedwith oral contraceptives because women taking progestin-onlycontraceptives do not have significant elevations in BP (21).Therefore, it is important to elucidate the mechanisms bywhich chronic exposure to E2 increases BP.

The central nervous system plays an important role in thedevelopment and maintenance of hypertension (4, 9, 25, 26).The cortex, limbic system, hypothalamus, brain stem, andthe autonomic nervous system are all known to be involvedin the maintenance of BP. In the brain stem, the rostralventral lateral medulla (RVLM) is a critical area involved inthe regulation of sympathetic activity and BP. The RVLMintegrates sympathetic outflow and provides excitatory inputto preganglionic sympathetic cells in the spinal cord (51).The RVLM maintains basal vasomotor tone, and an increasein RVLM activity is associated with hypertension and heartfailure (26). Recently, oxidative stress in the RVLM hasbeen suggested to increase sympathetic nervous system

Address for reprint requests and other correspondence: P. S. MohanKumar,Neuroendocrine Research Laboratory, B-336, Life Sciences Bldg., Dept.Pathobiology and Diagnostic Investigation, College of Veterinary Medicine,Michigan State Univ., East Lansing, MI 48824 (e-mail: [email protected]).

Am J Physiol Regul Integr Comp Physiol 300: R1560R1568, 2011.First published March 16, 2011; doi:10.1152/ajpregu.00020.2011.

0363-6119/11 Copyright 2011 the American Physiological Society http://www.ajpregu.orgR1560

activity and BP in several animal models of hypertension (4,9, 25). Therefore, we hypothesized that E2-induced increasein blood pressure could be mediated by oxidative stress-related changes in RVLM.

To test this idea, young, adult cycling female Sprague-Dawley rats were exposed to low levels of E2 on a chronicbasis. We attempted to reduce oxidative stress in these animalsby treating them with resveratrol, an antioxidant.

MATERIALS AND METHODS

Experimental animals and treatment. All of the protocols fol-lowed in this experiment were approved by the Institutional Ani-mal Care and Use Committee at Michigan State University. Adult

(3 4 mo old) female Sprague-Dawley (SD) rats (Harlan Sprague-Dawley, Indianapolis, IN) were housed in temperature (23 2C)-and light-controlled (14:10-h light-dark cycle) rooms with adlibitum food and water. Estrous cycles were monitored, as de-scribed previously (22) for 2 wk, and animals that were cyclingregularly were used in the experiments. In experiment 1, animalswere divided into two groups (n 4/group); sham-implanted(control); or implanted with E2 (20 ng/day, 90-day slow-releasepellet; Innovative Research America, Sarasota, FL). After 60 days,sham and E2-treated rats were implanted with radiotelemetrytransmitters to monitor BP, as described below. After 90 days ofexposure to E2, all of the animals were killed at noon on the dayof estrus. Most of the E2-treated animals were in persistent estrusafter 90 days of treatment. The control animals were killed on the

Fig. 1. Effect of chronic E2 exposure onblood pressure parameters. Line graphs de-picting mean arterial pressure (MAP;mmHg) (A), heart rate (HR; beats/min) (B),systolic blood pressure (SBP) (C), and dia-stolic blood pressure (DBP; mmHg) (D),respectively. Solid circles () represent E2pellet implanted (20 ng/day, 90-day slow-release pellets), and open circles () repre-sent control rats. EH: bar graphs showingthe average values of the BP parametersshown in AD, between E2-implanted andcontrol rats. *Significant difference (P 0.05) from control rats.

R1561ESTROGEN AND HYPERTENSION

AJP-Regul Integr Comp Physiol VOL 300 JUNE 2011 www.ajpregu.org

day of estrus after 90 days of sham implantation for comparison tothe treatment group. The brains and brain stem were removed,frozen on dry ice, and stored at 70C. The trunk blood wascollected, and serum was separated and stored at 70C untilprocessed.

In experiment 2, animals were divided into four groups (n 6/group): sham-implanted (control); implanted with E2 slow-releasepellets (E-90; 20 ng/day for 90 days) (22); control fed with chowcontaining 0.84 g resveratrol/kg of chow (Res) (3); and E2-implantedrats fed with chow containing resveratrol (ResE-90). Resveratroltreatment was started 7 wk after pellet implantation. Animals wereimplanted with telemeters on the 9th wk, as described below. Foodintake, water intake, and body weight were monitored weeklythroughout the experimental period. The animals were killed at theend of 90 days, while in the state of estrus.

BP measurement. In the 9th wk of the treatment period, radio-telemetry transmitters (model TA11-PA-C40; Data Sciences Interna-tional, St. Paul, MN) were implanted, as described previously (24).Briefly, the tip of the transmitter catheter was placed in the abdominalaorta through the femoral artery under general anesthesia. The body ofthe transmitter was placed in a subcutaneous pocket in the abdomen.Ten days later, BP data from the transmitter were collected continu-ously for 11 days in experiment 1, and 14 days in experiment 2 (24h/day; 10-s averages collected every 10 min). Data were stored andanalyzed using the Dataquest A.R.T. software (Data Sciences Inter-national, St. Paul, Minnesota).

Brain microdissection. Palkovitss microdissection procedure(32) was used to isolate the RVLM. Briefly, 300-m serial sectionsof brain stem were obtained using a cryostat (Slee Mainz, London,UK). The sections were transferred to microscope slides and placedon a cold stage maintained at 10C. The RVLM of the brain stemwas microdissected from sections obtained 12.00 mm to 12.48 mmposterior to the bregma using a 500-m-diameter punch, using therat brain stereotaxic atlas as a reference (41). The punches werestored immediately at 70C until processed for superoxide mea-surement.

Superoxide measurement. Superoxide (O2) levels from the RVLMregion were measured using a lucigenin O2 chemiluminescence assayadapted from Rey et al. (43). RVLM punches were placed in HEPESbuffer (in mmol): 119 NaCl, 20 HEPES, 4.6 KCl, 1.0 MgSO4, 0.15Na2HPO4, 0.4 KH2PO4, 5 NaHCO3, 1.2 CaCl2, 5.5 glucose (pH 7.4)].Diethyldithiocarbamate (DDC; a superoxide dismutase inhibitor; 10mmol) was added and incubated at 37C for 30 min. Lucigenin (5mol/l) was added, and the samples were incubated for 10 min at37C. Chemiluminescence measurements were obtained using amodel TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA) for10 readings (30 s/reading). Tiron (10 mmol/l; an O2 scavenger;Sigma, St. Louis, MO) was added and incubated for 15 min at 37C,and 10 additional readings were taken. The relative amount of O2 wasdetermined by taking the average of 2nd9th readings prior to theaddition of tiron, and subtracting the average of the 7th10th readingsafter the addition of tiron. O2 generated was reported as change inchemiluminescence per minute per milligram tissue weight.

Serum estradiol. Estradiol levels in serum separated from trunkblood were measured by double antibody radioimmunoassay by Dr.A. F. Parlow, National Hormone and Pituitary Program, NationalInstitute of Diabetes and Digestive and Kidney Diseases. Sampleswere assayed in duplicate.

Statistics. Daily BP measurements and weekly food intake, waterintake, and body weight were analyzed by repeated-measuresANOVA followed by post hoc Fischers LSD test. Average BPparameters, serum estradiol and O2 data were analyzed by ANOVA,followed by post hoc Fischers LSD test. Results were consideredsignificant when P 0.05. Average food intake, water intake, bodyweight, and heart-body weight ratio were analyzed by ANOVAfollowed by post hoc Fishers LSD test. Results were consideredsignificant when P 0.05.

RESULTS

Chronic exposure to estradiol-17 (E2) causes hypertension. Thedaily average profiles and the average mean arterial pressure(MAP), systolic blood pressure (SBP), diastolic blood pressure(DBP) and heart rate (HR) during the 1011th wk of treatmentin control and E2-treated rats are shown in Fig. 1. As seen inFig. 1A, the MAP in control animals remained steady over theentire period of observation. In contrast, E2 treatment increasedMAP significantly (P 0.01; Fig. 1A). The average MAP(means SE) measured during the 1011th wk of observationin control rats was 105.1 0.7 mmHg. In contrast, E2 expo-sure increased MAP significantly to 119.6 0.8 (P 0.0001;Fig. 1E). The HR profile of E2-treated rats had a tendency toincrease compared with control rats during the entire period ofobservation (Fig. 1B). The average HR (means SE, beats/min; Fig. 1F) during the 1011th wk of treatment in E2-treatedrats (371.7 1.5) was significantly elevated compared withcontrol rats (354.5 1.4; P 0.0001). Similarly, the SBP andDBP profiles in E2-treated rats were significantly elevated inE2-treated rats compared with control rats (P 0.0001; Fig. 1, C

Fig. 2. Effect of chronic E2 exposure on superoxide in the rostral ventrolateralmedulla (RVLM). A: schematic coronal brain stem section showing thelocation of micropunches of RVLM (within circles) taken from Paxinos andWatson rat brain atlas, 6th ed. The section coordinate (12.24-mm bregma)represents the distance caudal to the bregma. ROb, raphe obscurus nucleus; rs,rubrospinal tract; sp5, spinal trigeminal tract; IRt, intermediate reticular nu-cleus; Sol, nucleus of the solitary tract. B: bar graph denoting O2 levelsmeasured in RVLM brain stem punches of E2-treated (20 ng/day, 90-dayslow-release pellets) and sham-implanted female Sprague-Dawley (SD) rats(n 4 per group) using a luminometer. *Significant difference (P 0.05)from control rats.

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and D). E2 exposure also increased the average SBP and DBP(means SE; 140.0 0.7 and 101.1 0.8 mmHg, respectively)significantly compared with control rats (125.4 0.7 and 88.00.7 mmHg, respectively; P 0.0001; Fig. 1, G and H).

Chronic exposure to E2 increases O2 levels in RVLM. ChronicE2 exposure significantly elevated O2 levels (means SE) in theRVLM of E2-treated rats (0.833 0.1 nmol/minmg) comparedwith control rats (0.532 0.04 nmol/minmg; P 0.01) (Fig. 2).

Fig. 3. Effect of chronic E2 exposure and resveratrol on food intake, water intake, body weight, and heart weight. AC: line graphs showing food intake (g), waterintake (ml), and body weight (g) between different groups of female SD rats (n 6 per group); open circles () shows control, solid circle () shows E2 pelletimplanted (E-90), open inverted triangle () shows resveratrol (Res) treatment alone, and closed triangle shows resveratrol treatment on E2-implanted rats(ResE-90). *Significant difference from control (P 0.05). D: bar graphs showing the average values of heart weight (g) and heart-to-body weight ratio inthe different treatment groups (n 6 per group). EG: bar graphs showing the average values of food intake, water intake, and body weight shown in AC,respectively. E: *Significant difference (P 0.05) from control and resveratrol-treated groups. F: *Significant difference (P 0.005) from the rest of the groups;a denotes significant difference from control (P 0.05). G: *Significant difference (P 0.05) from control.



Chronic E2 exposure increases food intake, water intake and bodyweight. The average weekly food intake (g/wk; means SE),water intake (ml/wk; means SE), and body weight (g;means SE) of control, E2, resveratrol, resveratrolE2 ani-mals is shown in Fig. 3. There was no difference in food intakebetween control and E2-treated groups at the beginning of theexperiment. However, food intake in the E2-treated groupappeared to increase on the 2nd wk (141.7 4.7 g/wk), the 5thwk (132.1 5.3 g/wk), and the 7th wk (127.6 3.8 g/wk)compared with control rats (123.8 2.8 g/wk, 117.7 2.3g/wk, and 117.6 2.5 g/wk, respectively; P 0.05). Theaverage food intake was significantly higher in E2-treated rats(134.6 1.7 g/wk) compared with control rats (122 2.3g/wk) and those treated with resveratrol alone (123.1 1.3g/wk; P 0.05; Fig. 3E). Feeding chow containing resveratroldid not affect food intake in the control and E2-treated groups.

The average weekly water intake in control and experimen-tal animals is given in Fig. 3B. There was a tendency forincreased water intake in E2-treated rats compared with controlrats in the 1st wk, and this trend continued throughout theperiod of observation, though the change was not statisticallysignificant. However, the average water intake (Fig. 3F) wassignificantly higher in the E2-treated group (316.5 5.6ml/wk) compared with control rats (288.2 5.5 ml/wk),sham-implanted resveratrol-treated group (279.6 2.9 ml/wk)and the resveratrol and E2-treated group (257.5 5.8 ml/wk;P 0.005). The E2-treated group fed chow containing res-veratrol consumed less water (257.5 5.8 ml/wk) comparedwith the control group (288.2 5.5 ml/wk; P 0.05).

Changes in weekly body weight among the different groupsare shown in Fig. 3C. There was no difference in body weightbetween the treatment groups at the beginning of the experi-ment. Although there was a tendency for body weight toincrease with time, the average weekly body weight in the E2group was not statistically different from that of control rats.Feeding control and E2 rats with chow containing resveratrolfor 2 wk did not produce any change in body weight. Theaverage body weight over the entire period of observation (Fig.3G) was significantly higher in the E2 group (297.4 2.7 g)compared with control rats (287.3 1.6 g; P 0.005).Feeding resveratrol did not alter the average body weight incontrol and E2-treated rats. Body weight in E2-treated rats fedchow containing resveratrol (299.6 0.4 g) was significantlyhigher compared with control (P 0.05).

There were no significant changes in heart weight or theratio of heart weight to body weight in any of the treatedgroups compared with the control group (Fig. 3D).

Serum E2. Serum E2 levels (means SE) at the end of 90days of E2 exposure and resveratrol treatment are shown inFig. 4. Exposure to E2 resulted in significant increases in serumE2 of animals that are treated with E2 alone (68.2 4.07pg/ml) or treated with E2 and resveratrol (59.4 1.5 pg/ml)compared with control rats (46.7 2.9 pg/ml) or rats treatedwith resveratrol alone (47.4 4.4 pg/ml, P 0.0005). Therewas no difference in E2 levels between the E2-treated groups(with or without resveratrol).

Resveratrol reverses chronic E2-induced hypertension. Thedaily average profiles and the average MAP, SBP, DBP, andHR during the entire period of observation in control, E2,resveratrol and resveratrol E2-treated rats are shown inFig. 5. Similar to what was observed in Fig. 1, exposure to E2

for 90 days significantly elevated MAP, HR, SBP, and DBPcompared with control rats. Feeding chow containing resvera-trol to sham-implanted control rats did not alter MAP, HR,SBP, and DBP. In contrast, feeding chow containing resvera-trol to E-90 rats completely reversed E2-induced increase inMAP, HR, SBP, and DBP in E2-implanted rats (P 0.01).

Resveratrol attenuates oxidative stress in RVLM. Changes insuperoxide levels (means SE, nmol/minmg) in the RVLMof control, E2, resveratrol and resveratrolE2 groups areshown in Fig. 6. As seen in Fig. 2, superoxide levels increasedsignificantly in E-90 rats (0.608 0.052 nmol/minmg) com-pared with control rats (0.454 0.007 nmol/minmg; P 0.005). When sham-implanted control rats were fed chowcontaining resveratrol alone, it did not alter superoxide levels(0.431 0.007 nmol/minmg) compared with control rats(0.454 0.007 nmol/minmg). In contrast, feeding E2-treatedrats with chow containing resveratrol completely reversedE2-induced increase in superoxide levels in the RVLM(0.431 0.007 vs. 0.608 0.052 nmol/minmg; P 0.0005).DISCUSSION

The results from the present study provide evidence thatchronic exposure to low levels of E2 for 3 mo increasessuperoxide levels in RVLM and results in hypertension. Treat-ment with an antioxidant, resveratrol, reversed E2-inducedincreases in superoxide levels in the RVLM and reversed theincrease in BP, indicating that chronic exposure to low levelsof E2 is capable of causing hypertension, possibly by increas-ing superoxide generation in the RVLM.

Estrogen was originally believed to prevent hypertensionbased on the observation that postmenopausal women have ahigher incidence of hypertension compared with age-matchedmen (17, 31, 40), and, therefore, HRT containing estrogenicpreparations were believed to reduce BP in these women (48).Several possible mechanisms by which estrogens could de-crease BP have been suggested. There is evidence that estrogenincreases ACh-induced, nitric oxide-mediated relaxation ofaorta in male spontaneously hypertensive rats (23) throughupregulation of endothelial nitric oxide synthase (eNOS) (20).Also, estrogen is believed to act on some brain stem autonomiccenters to decrease sympathetic nervous activity (54, 55),thereby causing reduced vasomotor tone and lowered BP.

Contrary to the belief that estrogens can lower BP, there isa large body of evidence, indicating that repeated exposure to

Fig. 4. Serum E2 levels. Bar graphs showing serum E2 levels in all the groups(n 57 per group). *Significant difference (P 0.05) from control and thegroup treated with resveratrol alone.



low levels of estrogens, as in the case of oral contraceptives,can cause an increase in BP. Numerous human clinical studieshave associated chronic use of contraceptive pills with increasein BP (12, 29, 56). Similar results have been seen in animalstudies, where exposure to a combination of 1 g of ethinylestradiol and 10 g of norgestrel or ethinylestradiol alone for10 wk resulted in concomitant increases in systolic and mean

arterial BP (7, 36, 38). In another study, female SD ratsinjected with 0.2 g of ethinyl estradiol exhibited significantincreases in systolic BP of 17 mmHg in 6 wk and 32 mmHg in12 wk (7). Different mechanisms have been proposed for oralcontraceptive-induced hypertension. These include impairedrenal handling of water resulting in volume-dependent hyper-tension (37) and hyperactivity of the renin-angiotensin system

Fig. 5. Effect of resveratrol on chronic E2-induced hypertension. AD: line graphs depicting MAP (mmHg), HR (beats/min), SBP and DBP (mmHg), respectively.Solid circles () represent E2 pellet implanted (20 ng/day, 90-day slow-release pellets), and open circles () represent control female SD rats, solid triangles ()represent rats fed with chow containing 0.84 g/kg resveratrol only, and open inverted triangles () represent E2 pellet-implanted (20 ng/day, 90-day slow-releasepellets) rats fed with chow containing 0.84 g/kg resveratrol. #Significant difference from control. *Significant difference from all the other groups. EH: bargraphs showing the average values of the BP parameters shown in AD. *Significant difference (P 0.05) from all the other groups, a represents significantdifference from control and ResE-90, and b represents significant difference from control.



(7, 38). Increased water intake as observed in the present studycould be another contributing factor to volume expansion.However, it is not clear whether this is associated with theincreased food intake and body weight observed in E2-treatedanimals. Interestingly, there were no measureable increases inheart weight, or the ratio of heart weight to body weight, inE2-treated rats. This is perhaps due to the rather mild increasein BP caused by E2 and to the known protective effect of E2against pressure-induced cardiac hypertrophy (50).

The increase in MAP, HR, SBP, and DBP with chronic E2exposure that was observed in the present study is supported byother reports (7, 36, 38). The main difference between theseand the present study is the dose of E2 used and duration ofexposure. Although the doses used in other studies ranged from0.2 to 10 g of various estrogenic preparations, we were ableto observe increases in cardiovascular parameters with 10-foldless concentration of E2 (0.02 g or 20 ng). In contrast to ourpresent study, Brosnihan et al. (5), have shown that 3 wk ofestrogen treatment decreased MAP and significantly decreasedANG-II-induced pressor response in female ovariectomizedrats, which was accompanied by significant reduction inplasma ANG II levels (5). However, the dose of E2 used intheir study was 1.5 mg/day, which yielded plasma concentra-tions of 190 20 pg/ml. With a similar dosage of E2, Gimenezet al. (16) demonstrated enhanced hypotensive responses toadministration of an ACE inhibitor in E2-treated 18-mo-oldfemale SHR rats (16). When using 1.5 mg/day, the serum E2concentrations were2.5 fold higher than those that have beenfound in the present study, where serum E2 concentrationswere 68.2 4.07 pg/ml in animals that were treated with E2alone. Previous studies also used a much shorter E2 exposuretime frame than those used in the present study, thus compli-cating the comparisons. In the present study, we monitoredchanges in BP only 9 wk after E2 implantation. Further studiesare needed to determine exactly when the changes in BPbecome apparent in these animals.

The mechanism by which exposure to low doses of E2induces hypertension is not clear. Several brain sites, such asthe paraventricular nucleus of the hypothalamus, the subforni-cal organ, nucleus tractus solitarius, and the RVLM are knownto be involved in the regulation of blood pressure (8). We

chose to focus specifically on the RVLM because it is knownto be involved in the maintenance of basal vasomotor tone viaregulation of sympathetic activity (45). Moreover, brain stemneurons in the RVLM contain estrogen receptor mRNA (1, 27,49), indicating that this is a potential site of E2 action. Also,stimulation of the RVLM is known to activate sympatheticpreganglionic neurons in the intermediolateral cell column ofthe spinal cord and increase sympathetic activity resulting inhypertension (14). An E2-induced increase in O2 levels in theRVLM observed in the present study may act through similarmechanisms. Other studies have shown an association betweenO2 in the RVLM and hypertension in multiple animal models(9, 19, 25, 39). Moreover, reversal of hypertension by increas-ing the expression of superoxide dismutase using gene transferand adenoviral vectors or decreasing O2 in the RVLM sub-stantiates the role of oxidative stress in the pathogenesis ofhypertension (10, 11, 13, 15). Because our results clearlyindicated that E2 treatment increases O2 production in theRVLM, we wanted to explore the possibility of reversing thiseffect using the antioxidant, resveratrol.

Resveratrol is a red wine polyphenol, which is known topossess antioxidant and O2-scavenging properties (53) andexerts strong antioxidant effects in the brain (33). In the currentstudy, treatment with resveratrol reduced MAP, HR, SBP, andDBP induced by E2 treatment with complete quenching of O2in the RVLM. Resveratrol has been shown to reduce SBP inobese Zucker rats by increasing eNOS expression in the aorta(44). Resveratrol treatment also prevented hypertension causedby a high-fat diet in female rats (2). In the present study,treatment of E2-treated rats with resveratrol reversed E2-in-duced increase in O2 in the RVLM and also decreased BP,raising the possibility that E2s effects on BP could be medi-ated through increased production of O2.

In conclusion, our results support the hypothesis that chronicexposure to low levels of E2 causes hypertension in young SDfemale rats. This hypertensive effect of E2 may be related toincreased O2 production in the RVLM. Although, this studyexamines the effect of E2 on O2 generation in the RVLM, theinvolvement of other brain sites and peripheral tissues, such asthe vasculature and kidney, should also be considered. Collec-tively, this study provides valuable insights into the mecha-nisms by which chronic low-dose exposure to E2 causeshypertension and draws attention to potential cardiovascularrisks faced by women on long-term oral contraceptives.

ACKNOWLEDGMENTS

We thank Ms. Katrina Linning for her technical help and Dr. A. F. Parlow,National Hormone and Peptide Program, National Institute of Diabetes andDigestive and Kidney Diseases for performing the estradiol radioimmunoas-say.

GRANTS

This work was supported by National Institutes of Health Grant AG027697,Michigan Agricultural Experiment Station, Companion Animal Fund, Collegeof Veterinary Medicine, Michigan State University and the Pearl J. AldrichEndowment in Aging-Related Research.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

Fig. 6. Effect of resveratrol on chronic E2-induced oxidative stress in RVLM.Bar graph showing superoxide levels measured in RVLM punches fromcontrol, E2 treated (20 ng/day, 90-day slow-release pellets), rats fed with chowcontaining 0.84 g/kg resveratrol only, and rats implanted with E2 pellet (20ng/day, 90-day slow-release pellets) fed with chow containing 0.84 g/kgresveratrol (n 46 per group). *Significant difference (P 0.05) from allthe other groups.



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