Effects of ovariectomy and 17β-estradiol treatment on the renin–angiotensin system, blood pressure, and endothelial ultrastructure

International Journal of Cardiology 130 (2008) 196 – 204 www.elsevier.com/locate/ijcard

Effects of ovariectomy and 17β-estradiol treatment on the renin–angiotensin system, blood pressure, and endothelial ultrastructure Xia Xu a , Jing-Chuan Xiao a , Li-Fang Luo b , Shan Wang b , Jie-Ping Zhang a , Jian-Jun Huang a , Mei-Lian Liu a , Chen-Geng Liu a , Ke-Qian Xu a , Yuan-Jian Li b , Hui-Ping Song a,⁎ a

Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha, 410078, PR China b Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, 410078, PR China Received 3 March 2007; received in revised form 31 May 2007; accepted 3 August 2007 Available online 20 February 2008

Abstract The purpose of this study was to determine whether the renin–angiotensin system (RAS), nitric oxide (NO), atrial natriuretic peptide (ANP), blood pressure (BP), ultrastructural characteristics, and endothelium-dependent relaxation of thoracic aorta were modulated by the estrogen level. Rats were divided into 3 groups: ovariectomized (OVX); not ovariectomized (sham); and ovariectomized and treated with subcutaneous 17β-estradiol (15 μg/kg/day, OVX + E2) (n = 15–17 per group). For 13 weeks after surgery, blood pressure, serum estrogen, NO, plasma angiotensin II (Ang II), ANP, and renin activity levels were monitored. Thirteen weeks after surgery, the vasodilator responses of the aortic rings to acetylcholine and the ultrastructural characteristics of the thoracic aorta were determined. In the 9th and 13th week, OVX rats had a significantly higher blood pressure than the other two groups (p b 0.05). Ovariectomy led to a significant decrease in plasma Ang II level and a significant increase in renin activity in OVX rats compared to sham rats; this effect could be reversed by estrogen treatment. In the 5th, 9th, and 13th weeks, the serum NO level was significantly lower in the OVX group than in the sham group (p b 0.05); this effect could be reversed by estrogen treatment. Plasma ANP levels in the 9th and 13th weeks were significantly lower in the OVX group (p b 0.05), and plasma ANP levels could be completely restored by estrogen treatment. Ovariectomy markedly reduced endothelium-dependent relaxation in response to acetylcholine in isolated rat thoracic aortic rings; chronic estrogen treatment significantly restored endothelium-dependent relaxation in response to acetylcholine. Under electron microscopy, the endothelial cells in OVX rats were swollen, even necrosed; estrogen treatment inhibited these changes. These results strongly suggest that estradiol protects rats from the development of hypertension and has a protective effect on the endothelium by increasing NO and ANP levels while decreasing renin activity. However, there was a discordance between the effects that estradiol had on angiotensin II and on blood pressure. This might be the result of negative feedback that ultimately results in the overall suppression of the RAS. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovariectomy; 17β-Estradiol; RAS; NO; ANP; Endothelium; Blood pressure

1. Introduction Cardiovascular diseases (CVD) are the leading cause of death in women, and the vast majority of CVD occurs in ⁎ Corresponding author. Department of Biochemistry, School of Biological Science and Technology, Central South University, Xiang-Ya Road #110, Changsha 410078, PR China. Tel.: +86 731 4839137; fax: +86 731 2355199. E-mail address: [email protected] (H.-P. Song). 0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.08.041

postmenopausal women. The loss of estrogen associated with menopause can increase the prevalence of various cardiovascular disease risk factors, including elevated blood pressure, hypertriglyceridemia, and diabetes. It has long been suspected that the level of circulating estradiol has a protective role on the cardiovascular system [1]. However, the protective actions of estrogen appear to contradict the hormone's effects on several components of the renin–angiotensin system, which is one of the key regulatory systems for the control of blood pressure and cardiovascular pathologies [2]. In particular, the promoter

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region in the angiotensinogen gene is responsive to estrogen. The administration of exogenous estrogen to postmenopausal women increases plasma concentrations of this renin substrate. Furthermore, estrogen administration has been shown to increase plasma renin activity [3,4]. However, estrogen has also been shown to suppress components of the RAS, including angiotensin converting enzyme and angiotensin II type 1 receptor (AT1), which may attenuate angiotensin II formation or decrease Ang II bioavailability [2,5–7]. For nearly 50 years, estrogen replacement therapy (ERT) and hormone replacement therapy (HRT, estrogen/progestin) have been extensively used to prevent cardiovascular disease in postmenopausal women [8]. However, the Women's Health Initiative (WHI) studies of HRT and ERT failed to verify the cardiovascular protective action of such treatments. In fact, HRT recipients were found to have higher cardiovascular disease risks [9–11]. Mendelsohn et al. thought that resolving this controversy would require a more complete understanding of the differences in the vascular biology that exist between premenopausal and older women [12]. As the guardian of the vascular wall, the endothelium is the “first-responder” to multiple physical, biochemical, and cellular events that occur in the lumen. Endothelial dysfunction is associated with several pathologic conditions, including hypertension and diabetes [13]. The clinical effects of ERT and HRT on blood pressure and the endothelium are still controversial. To assess the beneficial and harmful effects of estrogen replacement on the endothelium and blood pressure, systematic studies were done to elucidate the effects of ovariectomy and 17βestradiol on the ultrastructural characteristics, the endothelium-dependent relaxation of the thoracic aorta, the blood pressure, and the expression of circulating RAS components, as well as related mediators, including ANP and NO. 2. Methods 2.1. Reagents 17β-Estradiol was purchased from Sigma-Aldrich Biotechnology (USA). Sesame oil (purity: 100%), from Xiwang biological company (Wuhan, China), was used as a vehicle to dissolve 17β-estradiol. The RIT kits for the ANP, Ang II, and renin activity assays were obtained from the Beijing North Institute of Biological Technology (Beijing, China). The RIT kit for the 17β-estradiol assay was purchased from the Jiuding Biological Company (Tianjing, China). The nitric oxide assay kit was obtained from the Ju-Li Biological Medical Engineering Institute (Nanjing, China). 2.2. Animals Female Sprague–Dawley rats (5 months old) were obtained from the Animal Center of the Institute of Science and Technology (Shanghai, China) and were housed in the animal facility located at Central South University (Chang-

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sha, China) for 2 weeks prior to the start of the experiments. The rats were housed two or three per cage and maintained at 22–24 °C with a 12-h light–dark cycle and free access to water. Animals received humane care in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ published by the US National Institutes of Health (NIH publication 86-23, revised 1986). 2.3. Experimental protocols Three groups of rats were studied using a slight modification of the protocol previously described by Liu et al. [8]. Sham group (n =15): animals were subjected to a surgical procedure without doing an ovariectomy and were treated with 0.1 ml vehicle (pure sesame oil, sc). OVX group (n= 17): animals were subjected to an ovariectomy at the beginning of the experiment and were treated with 0.1 ml vehicle (sc). OVX + E2 group (n =17): animals were subjected to an ovariectomy at the beginning of the experiment and received E2 treatment (15 μg/ kg sc). All three groups received treatment at the same time everyday. All rats were weighed weekly during the experimental period. Their body length was measured when the animals were anaesthetized preoperatively and 13 weeks after surgery. The body weight and body length were used to determine the body mass index (BMI, kg/m2) =body weight/ length2. Tail vein blood samples were collected regularly from fasting rats to determine serum nitrite/nitrate levels, plasma ANP and Ang II levels, and plasma renin activity. Thirteen weeks after surgery, 8–10 rats/group were randomly selected for the aortic contraction–relaxation experiment, and 5 rats/ group were randomly selected to obtain specimens for aortic transmission electron microscopy. 2.4. Systolic blood pressure measurement During the 13-week postoperative period, systolic blood pressure (SBP) measurements were performed at 2–4 week intervals in the sham (n = 15), OVX (n = 17), and OVX + E2 (n = 17) groups. The SBP was measured using the tail cuff method (RBP-I rat tail blood pressure systems for rats and mice, Sino-Japan Friendship Institute of Medical Sciences, China). Each systolic blood pressure was the mean of at least five consecutive recordings. 2.5. Serum 17β-estradiol assay Blood samples were incubated at 37 °C for 30 min. This was followed by spinning to separate the serum. The serum 17β-estradiol level was determined using a solid-phase 125Iradioimmunoassay technique according to the manufacturer's instructions. 2.6. Plasma angiotensin II and renin activity assay Blood was collected into prechilled tubes that contained ethylenediaminetetraacetate and angiotensinase inhibitor

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and 11.0 mM glucose). One of the ring ends was connected to a force transducer. The aortic ring was stretched with a 2 g resting force and equilibrated for 60 min, and then precontracted with KCl (60 mM). After a maximal response to KCl was obtained, the rings were washed repeatedly with Krebs' solution and equilibrated again for 30 min. In order to measure the vasodilator responses, the rings were contracted with phenylephrine (10− 9–10− 6 M) to 40–50% of their maximal contraction. After the contraction stabilized, the cumulative concentration–response curve to acetylcholine (3 × 10− 9–10− 6 M) or sodium nitroprusside (10− 7 M) was determined. The relaxations were expressed as the percentage reduction in the phenylephrine-induced contraction. Fig. 1. Effect of OVX and E2-treatment on body weight. Week 0: the week prior to surgery. Values are expressed as means ± S.E.M. (n = 15–17/group). ⁎p b 0.05 sham vs. OVX; #p b 0.05 OVX vs. OVX + E2.

(0.1 ml Bestatin Solution). After centrifugation, plasma samples were stored at − 70 °C until analysis. Plasma renin activity was determined by quantitation of the radioimmunoassay for the amount of angiotensin I generated after incubating the plasma at 37 °C for 1 h. With this method, the amount of angiotensin I generated in vitro in the plasma specimens under controlled conditions is used as an index of renin activity in vivo. Ang II was measured using a competitive radioimmunoassay kit according to the manufacturer's instructions. 2.7. Serum nitrite/nitrate concentrations assay The serum level of nitric oxide was determined indirectly by measuring the nitrite and nitrate levels. The serum nitrite/ nitrate level was measured as previously described [14]. Briefly, nitrate was converted to nitrite with aspergillus nitrite reductase, and the total nitrite was measured using the Griess reagent. The absorbance was determined at 540 nm with a spectrophotometer. 2.8. Plasma ANP assay Plasma ANP was determined using a solid-phase 125Iradioimmunoassay technique according to the manufacturer's instructions. 2.9. Organ chamber experiments The rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). After blood samples were collected from an artery, the upper thoracic aorta was rapidly isolated and cut into 3–4-mm-long rings. The rings were suspended horizontally between two stainless steel wires and mounted in a 5-ml organ chamber filled with warmed (37 °C) and oxygenated (95% O2 and 5% CO2) Krebs' solution (119.0 mM NaCl; 25.0 mM NaHCO3; 4.7 mM KCl; 1.2 mM KH2PO4; 1.2 mM MgSO4·7H2O; 2.5 mM CaCl2;

2.10. Transmission electron microscopy Upper thoracic aortas were fixed in 2.5% glutaraldehyde in a phosphate buffer (pH 7.4) for 24 h. The samples were postfixed in 2% osmium tetroxide, and dehydrated through a graded acetone series. Then, the samples were infiltrated and embedded in Epon embedding medium, and sectioned (about 500 Å) using an ultramicrotome (LKB-III, Sweden). Ultrasections were positioned on 200 mesh grids, stained with uranyl acetate and lead citrate, then examined using a transmission electron microscope (Hitachi-7500, Japan). The morphometric parameters were computed using image analyzer Software (Visual New Technology developing Company, China). The following parameters were quantified: the thickest measurement to the thinnest measurement of the internal elastic lamina (IEL) located in one vascular circle, the endothelial cell (EC) area, the area of mitochondria and the area of the nucleus in the EC.

Table 1 Characterization of the animal models

n BWbefore (g) BWafter (g) BMIbefore (kg/m2) BMIafter (kg/m2) HW (mg) HW/BW (mg/g) Uterus weight (g) Lung weight (g) Kidney weight (g) E2before (pg/ml) E2after (pg/ml) BPbefore (mmHg) BPafter (mmHg)

Sham

OVX

OVX + E2

10–15 248.0 ± 3.4 294.3 ± 5.7 5.55 ± 0.33 5.54 ± 0.22 1044.9 ± 41.5 3.50 ± 0.13 0.789 ± 0.067 1.836 ± 0.127 1.764 ± 0.040 90.63 ± 20.97 82.80 ± 26.05 104.2 ± 2.2 112.6 ± 1.8

12–17 249.0 ± 3.4 339.4 ± 8.5⁎ 5.51 ± 0.32 6.22 ± 0.27⁎ 1114.6 ± 47.1 3.30 ± 0.14 0.153 ± 0.015⁎ 1.967 ± 0.117 1.799 ± 0.050 92.58 ± 19.68 11.20 ± 2.65⁎ 103.4 ± 2.3 119.8 ± 1.1⁎

12–17 247.2 ± 5.1 282.3 ± 5.6# 5.43 ± 0.32 5.42 ± 0.23# 1014.9 ± 34.7 3.61 ± 0.11 0.756 ± 0.089# 1.941 ± 0.132 1.763 ± 0.029 97.80 ± 24.13 105.66 ± 9.15# 103.6 ± 2.5 112.1 ± 1.8#

BWbefore and BWafter: body weight prior to surgery or 13 weeks post-surgery, respectively; BMIbefore and BMIafter: body mass index prior to surgery or 13 weeks post-surgery, respectively; E2before and E2after: 17β-estradiol serum level prior to surgery or 13 weeks post-surgery, respectively; BPbefore and BPafter: blood pressure prior to surgery or 13 weeks post-surgery, respectively; HW: heart weight. Values are expressed as means ± S.E.M. ⁎p b 0.05 sham vs. OVX. # p b 0.05 OVX vs. OVX + E2.

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gen-treated rats were evaluated using ANOVA and the Dunnett post hoc analysis (StatMate). All other data were analyzed using Student's t-test, and figures were constructed using sigma plotting. The significance level was chosen as p b 0.05. 3. Results 3.1. Characterization of the animal models

Fig. 2. SBP measured at 2–4 week intervals over the 13-week postoperative period in sham (n=15), OVX (n=17), and OVX +E2 (n=17) rats. Week 0: the week prior to surgery. Values are expressed as means±S.E.M. (n=8–10). ⁎pb 0.05, sham vs. OVX; #pb 0.05, OVX vs. OVX+E2; +pb 0.05, vs. baseline.

2.11. Statistical analysis All measurements are expressed as means ± S.E.M. Comparisons among the sham, ovariectomized, and estro-

The success of the ovariectomy procedure was confirmed by examining the rats' body weight, uterus weight, and plasma estradiol level. At the beginning of the study, the body weights did not differ among the three groups. However, starting at the 3rd postoperative week, the OVX rats had significantly higher body weights than the other two groups, and this finding persisted throughout the whole experimental period. In the 13th week, the body weight of the OVX rats increased by 36.3% ± 2.9%, from 249 ± 3 g at baseline to 339 ± 9 g at 13th weeks (Fig. 1). The BMI of the OVX rats increased significantly; this could be reversed by estrogen treatment. The absolute heart weight, as well as the relative heart weight compared to the body weight, was not

Fig. 3. Effect of OVX and E2-treatment on the plasma angiotensin II level (A), renin activity (B), the nitrite/nitrite level (C), and the plasma ANP level (D). Week 0: the week prior to surgery. Values are expressed as means±S.E.M. (n=8–10/group). ⁎pb 0.05, sham vs. OVX; #pb 0.05, OVX vs. OVX+E2; +pb 0.05, sham vs. OVX +E2.

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different among the 3 groups in the 13th week. The lung weight and the kidney weight were also not different among the 3 groups. E2 treatment maintained uterus weight; the uterus weight was significantly higher in the OVX + E2 rats than in the OVX rats, and similar to that in the sham rats. Serum estradiol levels were significantly lower in the OVX rats than in the sham and OVX + E2 rats (p b 0.05); there was no difference between the sham and OVX + E2 rats (p N 0.05) (Table 1). 3.2. Systolic blood pressure At the beginning of the study, there were no differences in the systolic blood pressure among sham, OVX, and OVX + E2 rats. However, 9 and 13 weeks postoperatively, SBP was significantly higher in OVX rats than in the other two groups, though the SBP was within the normal range in all groups. All rats had a significant increase in SBP over the 13-week period, which is similar to that previously reported [15] (Fig. 2).

maximal tension was 1.534 ± 0.124 g, 1.714 ± 0.120 g, and 1.468 ± 0.177 g for the sham, OVX, and OVX + E2 groups, respectively (n = 8–10/group) (Fig. 4A). 3.7. Endothelium-dependent and -independent relaxation After the tension was increased with phenylephrine, acetylcholine was used to induce a concentration-dependent vasorelaxation in the isolated rat thoracic aortas. The vasodilator responses to acetylcholine were significantly decreased in OVX rats compared to sham rats (p b 0.05). Chronic estrogen replacement significantly restored the vasodilator response to acetylcholine in OVX rats ( p b 0.05) (Fig. 4B). On the other hand, there were no differences among the three groups in the aortic responses to sodium nitroprusside in phenylephrine-contracted rings (p N 0.05). The relaxation was 101.58% ± 2.02%, 102.30% ± 6.84%, and 97.06% ± 1.68% for the sham, OVX, and OVX + E2 groups, respectively (n = 8– 10/group).

3.3. Plasma angiotensin II and renin activity levels On the 5th postoperative week, there were no significant differences in the plasma angiotensin II and renin activity levels among the three groups. On the 9th and 13th postoperative weeks, OVX rats had a significant decrease in plasma angiotensin II concentration and a significant increase in plasma renin activity compared to sham rats ( p b 0.05); with estrogen treatment, both levels were restored to the levels in sham rats (p N 0.05) (Fig. 3A and B). 3.4. Serum nitrite/nitrate concentration The serum nitrite/nitrate level was significantly lower in OVX rats than in sham rats (p b 0.05). Estrogen replacement resulted in a significant increase in nitrite/nitrate levels in the 5th, 9th, and 13th postoperative weeks compared to OVX rats, even though the level was lower than in the sham group in the 5th week (p b 0.05). Estrogen treatment completely restored the nitrite/nitrate level in OVX rats in the 9th and 13th postoperative weeks (Fig. 3C). 3.5. Plasma atrial natriuretic peptide At the beginning of the study and in the 5th postoperative week, there were no differences in the plasma ANP level among the sham, OVX, and OVX + E2 rats. However, in the 9th and 13th postoperative weeks, the plasma ANP concentration was significantly decreased in OVX rats compared to sham and OVX + E2 rats (p b 0.05) (Fig. 3D). 3.6. Phenylephrine-induced contractions Phenylephrine was added to increase the smooth muscle tone in the rat aortic rings. The phenylephrine concentration–contraction curves were similar in the three groups; the

Fig. 4. Phenylephrine concentration curves (A) and acetylcholine relaxation curves (B) in rings from sham, OVX, and OVX + E2 rats. Values are expressed as means ± S.E.M. (n = 8–10). ⁎p b 0.05, sham vs. OVX; #p b 0.05, OVX vs. OVX + E2.

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3.8. Ultrastructural characteristics of aortic endothelial cells Under transmission electron microscopy, the ultrastructure of the thoracic aorta in the sham rats consisted of normal endothelial cells, smooth muscle cells, and uniform internal elastic lamina (Fig. 5A and B). In the OVX group, the endothelial cells were swollen, with mitochondrial swelling and cytoplasmic vacuolization (Fig. 5C). Some endothelial

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cells were necrosed and exfoliated; the thickness of the internal elastic lamina was irregular, with some thin parts (Fig. 5D). In the OVX + E2 group, the endothelial cells, the smooth muscle cells, and the internal elastic lamina were normal (Fig. 5E and F). Quantitative analysis of the endothelial cells of OVX rats showed that there was a significant increase in area, which could be reversed by estrogen treatment. In the OVX rats, the mitochondria had a significantly increased mean area. The

Fig. 5. Ultrastructural characteristics of the endothelial cells of the thoracic aorta under transmission electronic microscopy. Normal endothelial cells, smooth muscle cells, and uniform internal elastic lamina are seen in sham rats (A and B). In the OVX group, swollen endothelial cells, swollen mitochondria, and cytoplasmic vacuolization are seen (C). Furthermore, the endothelial cells are necrosed and have exfoliated, and the internal elastic lamina is irregular (D). Endothelial cells, smooth muscle cells, and internal elastic lamina are normal in OVX + E2 rats (E and F).

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Table 2 Quantitative analysis of endothelial cells and the internal elastic lamina of the thoracic aorta

n IEL thickest (μm) thinnest (μm) EC area (μm2) Nucleus area (μm2) mitochondrial area (μm2)

Sham

OVX

OVX + E2

5 1.78 ± 0.09 1.37 ± 0.07 10.22 ± 0.87 3.23 ± 0.22 0.032 ± 0.002

5 1.90 ± 0.32 0.70 ± 0.05⁎ 33.95 ± 14.91⁎ 3.76 ± 0.38 1.090 ± 0.246⁎

5 1.73 ± 0.07 1.35 ± 0.03# 8.37 ± 1.89# 2.82 ± 0.36 0.030 ± 0.002#

IEL: internal elastic lamina; EC: endothelial cell. ⁎p b 0.05 sham vs. OVX. # p b 0.05 OVX vs. OVX + E2.

area of the nucleus in endothelial cells was not different among the three groups. The thickness of the thinnest part of the internal elastic lamina in one vascular circle was markedly and significantly decreased in OVX rats, and this effect could be reversed by estrogen treatment. No morphological alterations were observed in the thickest part of the internal elastic lamina, and the parameter analyzed does not show significant variations (Table 2). 4. Discussion In the present study, the effects of ovariectomy and chronic estrogen replacement on the RAS, NO level, and ANP level, as well as ultrastructural changes and endothelium-dependent relaxation of the thoracic aorta, were assessed in female SD rats. The present study confirms that chronic 17β-estradiol treatment elicits a protective effect on endothelium and protects from the development of hypertension. Previous investigations have indicated that postmenopausal women are more than twice as likely to be hypertensive as premenopausal women [16]. In the present study, the systolic blood pressure in the OVX rats was within normal limits, though it was significantly higher than in sham rats; the increase in SBP could be reversed by estrogen treatment. This is consistent with previous reports that transdermal estrogen can lower blood pressure, while the effects of oral estrogens tend to be neutral [17,18]. In agreement with previous reports, it was also found that estrogen deficiency significantly increased weight gain in ovariectomized rats; this effect could be attenuated by estrogen treatment. These results support the hypothesis that the change in the estrogen status might be related to the development of obesity. An increasing level of obesity could partly contribute to the risk of hypertension and ischemic heart disease [19]. Obesity is also accompanied by an increase in sympathetic activity, particularly in the kidney, which leads to an increase in renin release that could contribute to hypertension. Therefore, although blood pressure increases in most postmenopausal women, obese postmenopausal women have a greater predisposition to hypertension than thin postmenopausal women [20].

Estrogen affects the renin–angiotensin system through several pathways. In the present study, ovariectomy decreased the plasma angiotensin II level and increased plasma renin activity. This effect could be reversed by estrogen treatment. However, the effects of estradiol on the vasoconstrictor, angiotensin II, and on BP were discordant. The decrease in angiotensin II did not result in lower BP in OVX rats. The reasons for this discordance are unclear. One possible explanation is that the increased generation of angiotensin II that occurs with estrogen exerts a negative feedback that eventually results in the overall suppression of the RAS through inhibition of renin secretion [21]. Ovariectomy in normotensive female rats induced a reduction in plasma angiotensin II, but its actions may be counteracted by downregulation of vasodilators, such as ANP and NO, as found in the present study. This hypothesis would explain the very modest blood pressure elevation noted in the absence of estrogen and the BP reduction noted in some postmenopausal women given ERT [22]. However, this effect was only found in healthy postmenopausal women receiving oral estradiol for 4 weeks. In contrast to the results of the present study, both Ang II and renin activity increased with estradiol, and this was thought to contribute to higher cardiovascular event rates reported in recent ERT trials [23]. The mechanism that would explain these discordant results needs to be further elucidated. Angiotensin II is not only a potent vasoconstrictor factor, but is also the principle regulator of aldosterone secretion. There is normally a good correlation between circulating Ang II and aldosterone levels. However, it has been shown that Ang II is essential for normal aldosterone production; aldosterone secretion and Ang II levels can become dissociated under certain conditions [24]. A study involving cultured bovine adrenal cells found that Ang II caused a decrease in the number of AT1 binding sites and their corresponding messenger RNAs [25]. Harrison-Bernard et al. found that estrogen reversed elevated AT1 receptor protein expression in the kidney of Dahl salt-sensitive rats after ovariectomy and increased their systolic blood pressure. In addition, it was shown that chronic blockade of the AT1 receptor normalized the blood pressure in OVX Dah1 saltsensitive rats. This indicates that the AT1 receptor does have a role in the hypertension process and may be related to increased salt sensitivity [26]. Blood pressure is mainly determined by the balance of two antagonistic hormonal systems: the vasoconstrictor factors, such as RAS, endothelin, and the sympathetic nervous system (SNS); and vasodilatory substances, such as ANP and NO. ANP is released into the circulation in response to hypervolemia and acts in an endocrine manner to regulate blood pressure and body fluid homeostasis [27]. The present study confirmed that estrogen deficiency decreases the plasma ANP level in ovariectomized rats, and that estrogen treatment can reverse this effect. So far, only limited data are available dealing with the effects of chronic estrogen replacement on ANP. It has been reported that transdermal

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estradiol in combination with oral dihydrogesterone given for 3 months to postmenopausal women increases the plasma ANP level with no change in the blood pressure [28]. There is a dynamic reciprocal relationship between the activation of circulating RAS and ANP. An elevation of ANP levels was reported to induce renin suppression [29]. The present study also found that OVX rats had an increased renin activity accompanied by a decreased ANP level during the whole experiment. A previous study suggested that the disruption of the coronary endothelium and the inhibition of NO synthesis could reduce the relaxant effect of ANP. In fact, the coronary dilatory and hypotensive action of ANP involves the endothelium and is partly mediated by endothelial NO/ cGMP pathways [30]. The present study suggests that ovariectomy could lead to a decreased serum NO level concentration that can be reversed by estrogen treatment. A change in the serum NO level was noted before changes in the other indices measured in the present study were noted. NO, which is produced mainly by the endothelium, regulates vasodilatation, anticoagulation, smooth muscle proliferation, and the antioxidative capacity of endothelial cells. Diminished NO production or bioavailability has been implicated in the pathogenesis of systemic and pulmonary hypertension and in other vascular disorders including atherosclerosis [31]. The present study also showed that ovariectomized female rats had a markedly decreased endothelium-dependent relaxation to acetylcholine, and that chronic estrogen replacement significantly attenuated this effect. It has been shown that estrogen receptors (ER) are present in endothelial cells [31]. Studies of wild-type mouse blood vessels showed that estrogen attenuated vasoconstriction through an ER-mediated increase in nitric oxide synthase expression. As well, as they age, ERbeta-deficient mice develop sustained systolic and diastolic hypertension [32,33]. These data support the notion that ER has an essential role in the regulation of vascular function and blood pressure. Many of the vascular actions of estrogen are mediated by increases in bioavailable NO. Estrogen is capable of increasing the NO level by upregulating eNOS expression through typical transcriptional ‘genomic' regulation and nongenomic activation of eNOS mediated by cell surface estrogen receptors [34]. In order for constitutive eNOS-derived NO to be effective, NO would have to be able to diffuse readily, and all pathways would have to have no diffusion barriers [35]. The present study confirmed that an estrogen deficiency can induce endothelial cell swelling, and even irreversible necrosis. Estrogen treatment can inhibit these endothelial cell changes. This could partially explain the discrepancy between randomized trials and animal experiments. In the WHI trial, the subjects' mean age was 63 years; thus, the majority of these women were at least 10 years post-menopause at the time that the study was started [36]. The structure of the endothelium may have been damaged irreversibly because of the loss of estrogen's protective effects for so many years. Once vascular lesions occur, they can create a diffusion barrier. Thus,

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estrogen administration would not be able to reverse the disease process due to the loss of estrogen's interaction with its receptors, which would subsequently diminish the amount of bioavailable NO. Previous studies have confirmed that estrogen is of no benefit in animals that have artery damage caused either by balloon injury or an atherosclerotic diet prior to the initiation of hormone therapy [37]. The effect of estrogen on the cardiovascular system is a complex process that involves signal integration of multiple pathways [38]. Therefore, it may well be that the protective effect of estrogen on the cardiovascular system is mediated by the modulation of not one but several of these pathways. To the best of our knowledge, this is the first systematic animal study that assessed the effects of ERT on blood pressure, ultrastructural characteristics, and endotheliumdependent relaxation of the thoracic aorta, as well as circulating RAS, ANP, and NO, in the same study. In addition, the longer duration of the study offers an advantage over previous short-term studies, and it allows the assessment of the dynamic changes that occur in the indices of the same subjects. Overall, our study suggests that, in OVX + E2 rats, estrogen may have a protective effect on the endothelium, and may increase plasma ANP and NO levels while decreasing renin activity; all of these effects may contribute to a blood pressure reduction. However, there was a discordance between the effects that estradiol had on angiotensin II compared to its effect on blood pressure. Future studies are required to elucidate the mechanism by which estradiol caused these discordant effects. Better understanding of the complex interactions between estrogen, menopause, the RAS, and vascular biology will provide a proper insight into the pathogenesis of cardiovascular diseases in women and facilitate the development of optimal, gender-specific, cardiovascular disease prevention and therapeutic strategies. References [1] Mendelsohn ME. Protective effects of estrogen on the cardiovascular system. Am J Cardiol 2002;89(suppl):12E–8E. [2] Chappell MC, Gallagher PE, Averill DB, et al. Estrogen or the AT1 antagonist olmesartan reverses the development of profound hypertension in the congenic mRen2.Lewis rat. Hypertension 2003;42:781–6. [3] Fisher M, Baessler A, Schunkert H, et al. Renin angiotensin system and gender differences in the cardiovascular system. Cardiovasc Res 2002;53:672–7. [4] Bader M, Ganten D. Regulation of renin: new evidence from cultured cells and genetically modified mice. J Mol Med 2000;78:130–9. [5] Roesch DM, Tian YW, Zheng W, et al. Estradiol attenuates angiotensin-induced aldosterone secretion in ovariectomized rats. Endocrinology 2000;141:4629–36. [6] Wu Z, Maric C, Roesch DM, et al. Estrogen regulates adrenal angiotensin AT(1) receptors by modulating AT(1) receptor translation. Endocrinology 2003;144:3251–61. [7] Sanada M, Higashi Y, Nakagawa K, et al. Estrogen replacement therapy in postmenopausal women augments reactive hyperemia in the forearm by reducing angiotensin converting enzyme activity. Atherosclerosis 2001;158:391–7.

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[8] Liu ML, Xu X, Rang WQ, et al. Influence of ovariectomy and 17βestradiol treatment on insulin sensitivity, lipid metabolism and postischemic cardiac function. Int J Cardiol 2004;97:485–93. [9] Burry KA. Risks and benefits of estrogen plus progestin in healthy postmenopausal women. Principal results from the Women's Health Initiative randomized controlled trial. Curr Womens Health Rep 2002;2:331–2. [10] Powledge T. NIH terminates WHI oestrogen-only study. Lancet 2004;363:870. [11] Speroff L. WHI estrogen-only arm is conceled. Maturitas 2004;48:3–4. [12] Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovascular gender differences. Science 2005;308:1583–7. [13] Castro LR, Verde I, Cooper DM, et al. Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation 2006;113:2221–8. [14] Jiang JL, Li NS, Li YJ, et al. Probucol preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level. Br J Pharma 2002;135:1175–82. [15] Harrison-Bernard LM, Schulman IH, Raij L, et al. Postovariectomy hypertension is linked to increased renal AT1 receptor and salt sensitivity. Hypertension 2003;42:1157–63. [16] Tremollieres FA, Pouilles JM, Cauneille C, et al. Coronary heart disease risk factors and menopause: a study in 1684 French women. Atherosclerosis 1999;142:415–23. [17] Seely EW, Walsh BW, Gerhard MD, et al. Estradiol with or without progesterone and ambulatory blood pressure in postmenopausal women. Hypertension 1999;276:H1355–60. [18] Karjalainen AH, Ruskoaho H, Vuolteenaho O, et al. Effects of estrogen replacement therapy on natriuretic peptides and blood pressure. Maturitas 2004;47:201–8. [19] De Lusignan S, Hague N, Van Vlymen J, et al. A study of cardiovascular risk in overweight and obese people in England. Eur J Gen Pract 2006;12:19–29. [20] Reckelhoff JF, Fortepiani LA. Novel mechanisms responsible for postmenopausal hypertension. Hypertension 2004;43:918–23. [21] Oelkers WK. Effects of estrogens and progestogens on the renin– aldosterone system and blood pressure. Steroids 1996;61:166–71. [22] Franco-Saenz R, Atarashi K, Takagi M, et al. Effect of atrial natriuretic factor on renin and aldosterone. J Cardiovasc Pharmacol 1989;13 (Suppl 6):S31–5. [23] Harvey PJ, Morris BL, Miller JA, et al. Estradiol induces discordant angiotensin and blood pressure responses to orthostasis in healthy postmenopausal women. Hypertension 2005;45:399–405.

[24] Lumbers ER. Angiotensin and aldosterone. Regul Pept 1999;80:91–100. [25] Ouali R, Berthelon MC, Bégeot M, et al. Angiotensin II receptor subtypes AT1 and AT2 are down-regulated by angiotensin II through AT1 receptor by different mechanisms. Endocrinology 1997;138:725–33. [26] Harrison-Bernard LM, Hernadez SI, Raij L. Postovariectomy hypertension is linked to upregulation of renal angiotensin II type 1 receptor and increased salt-sensitivity. Hypertension 2003;42:1157–63. [27] Maack T. Role of atrial natriuretic factor in volume control. Kidney Int 1996;49:1732–7. [28] Maffei S, Del RS, Prontera C, et al. Increase in circulating levels of cardiac natriuretic peptides after hormone replacement therapy in postmenopausal women. Clin Sci 2001;101:447–53. [29] Franco-Saenz R, Atarashi K, Takagi M, et al. Effect of atrial natriuretic factor on renin and aldosterone. J Cardiovasc Pharmacol 1989;13 (Suppl6):S31–5. [30] Brunner F, Wölkart G. Endothelial NO/cGMP system contributes to natriuretic peptide-mediated coronary and peripheral vasodilation. Microvasc Res 2001;61:102–10. [31] Mineo C, Shaul PW. Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae. Cardiovasc Res 2006;70:31–41. [32] Guo X, Razandi M, Pedram A, et al. Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors α and β. J Biol Chem 2005;280:19704–10. [33] Zhu Y, Bian Z, Lu P, et al. Abnormal vascular function and hypertension in mice deficient in estrogen receptor β. Science 2002;295:505–8. [34] Khalil RA. Sex hormones as potential modulators of vascular function in hypertension. Hypertension 2005;46:249–54. [35] Dai Z, Zhu HQ, Jiang DJ, et al. 17β-Estradiol preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level. Inter J Cardiol 2004;96:223–7. [36] Ouyang P, Michos ED, Karas RH, et al. Hormone replacement therapy and the cardiovascular system: lessons learned and unanswered questions. J Am Coll Cardiol 2006;47(9):1741–53. [37] Wagner JD, Clarkson TB. The applicability of hormonal effects on atherosclerosis in animals to heart disease in postmenopausal women. Semin Reprod Med 2005;23:149–56. [38] Babiker FA, De Windt LJ, van Eickels M, et al. Estrogenic hormone action in heart: regulatory network and function. Cardiovasc Res 2002;53:709–19.