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(Hypertension. 2003;42:781.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
From the Hypertension and Vascular Disease Center, Wake Forest University Health Sciences, Winston-Salem, NC.
Correspondence to Mark C. Chappell, PhD, Hypertension and Vascular Disease Center, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157-1095. E-mail mchappel{at}wfubmc.edu
| Abstract |
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, and endothelin-1 were elevated; however, renal mRNA levels of eNOS were suppressed after ovariectomy. Estrogen replacement reduced blood pressure below both the sham and OVX by 11 weeks (125±2.9 mm Hg, n=7, P<0.01 versus OVX and sham). Moreover, the AT1 receptor antagonist olmesartan (CS866; week 12 to 16) essentially normalized blood pressure to 113±5.4 mm Hg (n=6, P<0.01 versus OVX and sham). The attenuation of the hypertension was still evident 7 weeks after complete withdrawal of treatment (124±4.1 mm Hg at week 23). In summary, the OVX mRen.2.Lewis exhibited a rapid and sustained increase in blood pressure. Estrogen or olmesartan lowered pressure by a similar extent. We conclude that the ovary exerts considerable influence on the regulation of the blood pressure in the mRen2.Lewis strain, possibly by limiting activation of the renin-angiotensin system.
Key Words: angiotensin II angiotensin-converting enzyme estrogen rats, transgenic hypertension, genetic
| Introduction |
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Various hypertensive models have marked gender differences in the expression of hypertension. Although the majority of the studies have focused on the relation of androgens to the development of blood pressure,810 there clearly remains strong evidence for the contribution of estrogens or their metabolites in cardiovascular regulation.11 For example, homozygous and heterozygous (mRen2)-27 male rats have blood pressures
100 and 50 mm Hg higher, respectively, than their female littermates.12 We and other groups extensively use the (mRen2)-27 transgenics to study the mechanisms of renin overexpression and the development of hypertension. However, to eliminate the variability of the outbred SD background, (mRen2)-27 transgenics were back-crossed into the inbred Lewis rat, creating the new mRen(2).Lewis strain. After 9 generations, characterization of the mRen(2).Lewis rat revealed essentially the same degree of hypertension as the original transgenics, but the congenics lacked the malignant hypertension and the inability to concentrate urine.13 Of particular interest, the mRen(2).Lewis strain also demonstrates a significant gender difference in the development of hypertension. Therefore, in the present studies, we tested the hypothesis that early depletion of estrogen in the congenic mRen(2).Lewis rats influences the development of blood pressure and the expression of the circulating and renal RAS components, as well as related mediators, including endothelin-1 and the reactive oxygen species (ROS) metabolite 8-isoprostane F2
. In addition, we determined the influence of both estrogen replacement and selective blockade with the AT1 antagonist olmesartan on blood pressure in the ovariectomized mRen(2).Lewis strain.
| Methods |
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The concentration of Angiotensin (Ang) II was determined in the plasma, urine, and kidneys from control and treated rats, as described by Allred et al.14 Urine was collected over a 24-hour period into 4% acetic acid and BHT. Excretion of 8-isoprostane F2
(Cayman Chemicals) and endothelin-1 (ALPCO Diagnostics) were determined according to instructions for each assay kit. Serum estradiol concentrations were determined by RIA with a kit from Adaltis Italia S.P.A. Serum ACE activity was determined with the synthetic substrate Hip-His-Leu in the presence and absence of the ACE inhibitor lisinopril (10 µmol/L).14 Plasma renin concentration (PRC) was determined by addition of exogenous angiotensinogen (nephrectomized rat plasma) incubated at either pH 6.5 (rat renin) or pH 8.5 (mouse renin).
Total RNA was isolated from the renal cortex and medulla of each rat with TRIzol (Invitrogen) and then amplified with or without AMV reverse transcriptase, as described by Gallagher et al.5 Primer pairs were eNOS forward 5'-CTGCTGCCCGAGATATCTTC-3' and reverse 5'-AAGTAAGRGAGAGCCTGGCGCA-3', yielding a 435-bp fragment; ACE forward 5'-TTGACGTGAGCAACTTCCAG-3' and reverse 5'-GGCTGCAGCTCCTGGTATAG-3', yielding a 421-bp fragment; and EF1
forward 5'-GGAATGGTGACAACATGCTG-3' and reverse 5'-CGTTGAAGCCTACATTGTCC-3', yielding a 347-bp fragment. Amplification conditions were 60-second denaturation at 94°C, annealing for 60 seconds at 60°C for ACE or 62°C for eNOS, and extension at 72°C for 30 cycles followed by a final extension at 72°C for 5 minutes. EF1
primers were added after 10 cycles were completed. Products were separated on 6% polyacrylamide gels, and band intensities were quantified by phosphorimage analysis. The mRNA concentration was expressed as the ratio of eNOS or ACE to the control EF1
to account for variations in the RT-PCR assay. We use EF1
because Ang II is known to regulate other "control" genes such as GADPH.
Statistical Analysis
All measurements are expressed as mean±SEM. Comparisons between the sham, ovariectomized, and treated rats were evaluated by ANOVA and the Dunnett post hoc analysis (StatMate). All other data were analyzed by the Student unpaired t test, and figures were constructed with GraphPad Prism plotting and statistical software. A probability value of <0.05 was required for statistical significance.
| Results |
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As shown in Figure 2A, estrogen depletion in the mRen(2).Lewis increased PRC approximately 2-fold compared with either the sham or estrogen-treated groups. Measurement of mouse PRC revealed a similar level to that of rat renin after ovariectomy and estrogen replacement (Figure 2B). Serum ACE activity was also significantly higher in the OVX-mRen(2).Lewis as compared with the other two groups, although the estrogen-treated animals tended to have the lowest ACE activity (Figure 2C), consistent with the lower blood pressure in this group (see Figure 1). Also consistent with an increased activity of both renin and ACE, the circulating levels of Ang II were elevated to a similar degree compared with intact and estrogen-treated groups (Figure 2D). We also determined the free plasma concentration of 8-isoprostane F2
as an index of ROS but did not observe a difference between the OVX and estrogen-treated groups (26.8±4.6 fmol/mL versus 36.5±8.2 fmol/mL, respectively).
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In terms of the renal RAS, urinary excretion and cortical tissue levels of Ang II were significantly increased in the OVX-mRen(2).Lewis rats (Figures 3A and 3B). The urinary excretion of both endothelin-1 (ET-1) and 8-isoprostane F2
were also elevated in the OVX group in comparison to either the sham or estrogen-treated rats (Figures 3C and 3D). Consistent with the increase in serum ACE activity and renal Ang II, both cortical and medullary levels of ACE mRNA were significantly higher in the OVX group as compared with the estrogen group (Figure 3E). Finally, we assessed mRNA levels of eNOS in the OVX-mRen(2).Lewis and the estrogen replacement group. The mRNA levels for eNOS (expressed as a ratio to EF1
) were reduced 42% [0.273±0.016 U versus 0.466±0.073 U, P<0.01, n=6 to 7] in the renal medulla of the OVX-mRen(2).Lewis as compared with the estrogen-treated group. The overall cortical expression of eNOS was significantly lower than that in the medulla; however, estrogen depletion had no effect on cortical eNOS levels [OVX: 0.123±0.013 U versus OVX+E2: 0.103±0.011 U, P>0.05, n=6 to 7].
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As shown in Figure 4, oral administration of olmesartan (1 mg/kg per day) for 2 weeks to 12-week OVX-mRen(2).Lewis rats substantially reduced blood pressure and, by 4 weeks, blood pressure was not different than the untreated normotensive female Lewis rats of similar age (113±5.4 mm Hg, n=6 versus 118±7 mm Hg for Lewis, n=5). In addition, we determined the blood pressure in this group of rats after the cessation of olmesartan treatment (week 16) for 7 weeks. By the end of week 23, the systolic blood pressures in the olmesartan-treated OVX-mRen(2).Lewis were still substantially reduced as compared with either that before treatment at 12 weeks (124±4.1 mm Hg versus 201±11 mm Hg, P<0.01) or a separate group of age-matched untreated OVX-mRen(2).Lewis at 16 weeks (190±9 mm Hg, n=5) and 23 weeks (208±12 mm Hg) but not different from that of the 16-week OVX-mRen(2).Lewis group receiving olmesartan (Figure 4).
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| Discussion |
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Previous studies in female (mRen2)-27 rats, the founder strain for the congenic mRen(2).Lewis rats, revealed differences in the blood pressure response after ovariectomy. Bachman et al18 reported that ovariectomy in 16-week heterozygous (mRen2)-27 reduced blood pressure by
20 mm Hg. The reduction in blood pressure was associated with a fall in both PRA and Ang II, as well as reduced expression of rat and mouse renin mRNA levels in the kidney.18 Although the effects of estrogen replacement were not determined in this study, the lower PRA after ovariectomy is consistent with a stimulatory effect of estrogen on renin. In contrast, Brosnihan et al19 found that ovariectomy had little effect on blood pressure in 12-week (mRen2)-27, but estrogen replacement reduced blood pressure as well as decreased ACE activity and mRNA levels of the enzyme in the lung, aorta, and kidney. In addition, the reduction in ACE was associated with a differential expression of plasma Ang peptides, reduced Ang II, and increased Ang-(1-7) that most likely reflects the ability of ACE to form Ang II and degrade Ang-(1-7).20 Similar to the latter study, the present results revealed increased plasma Ang II and serum ACE activity as well as increased rat PRC after ovariectomy. Moreover, estrogen replacement reversed the changes in these parameters as well as attenuated the development of hypertension. Apart from the background of the (mRen2)-27 transgenics (Sprague-Dawley versus Lewis), the major difference in the present study was that ovariectomy was performed at an earlier age before the establishment of hypertension. Interestingly, ovariectomy in the SHR as early as 3 weeks does not alter the development of blood pressure or exacerbate the hypertension.8 Indeed, androgens are thought to mediate the expression of hypertension in this model.10 In this regard, Baltatu et al21 recently reported that the androgen antagonist flutamide reduced blood pressure as well as attenuated the development of cardiac and renal injury in female (mRen2)-27 transgenics. The influence of androgen blockade in the mRen(2).Lewis rat is currently not known. It is possible that estrogen may well have a modulatory influence on the actions of androgens in this model, although studies on the effects of the testosterone antagonist in intact or estrogen depleted mRen(2).Lewis are necessary to address this issue.
Additional evidence that estrogen may influence the RAS and the development of hypertension in the mRen(2).Lewis are the results that blood pressure was normalized after 4-week treatment with the AT1 receptor antagonist olmesartan. Indeed, systolic blood pressures in the olmesartan-treated OVX-mRen(2).Lewis group were not different from that of either the intact or OVX-Lewis rat. Estrogen reduces the expression of the AT1 receptor in the vasculature, adrenal gland, and kidney, as well as inhibits the AT1-dependent actions of Ang II in vascular smooth muscle cells.4,6 The effects of increased expression of Ang II and the AT1 receptor after estrogen depletion may be further enhanced by the reduced capacity of eNOS in the kidney. Cowley and colleagues22 showed that reduced NO production in the renal medulla significantly influences blood pressure in the Dahl S rats. The influence of NOS may also explain the delayed effects of exogenous estrogen to attenuate the development of hypertension in the OVX-mRen(2).Lewis rats. Rahimian et al23 observed that 3 weeks of estrogen treatment was required to restore NO activity after ovariectomy. This treatment period corresponds to the time point for the development of blood pressure to diverge in the present study. Studies are in progress to determine whether the treatment of ovariectomized mRen(2).Lewis with olmesartan restores the renal expression of eNOS. Regarding the influence of other mediators, we do not know whether alterations in the renal excretion of ET-1 and isoprostanes are directly influenced by estrogen or an activated Ang IIAT1 axis. Ang IIdependent hypertension is associated with both increased ET-1 and activated ROS.2428 Furthermore, RAS blockade and estrogen attenuate expression of both systems.2832 However, the issue of enhanced renal ET-1 is complicated by the presence of both ET-A and ET-B receptors in the kidney that mediate opposing actions.33 Thus, the balance of these two receptors may dictate whether increased ET-1 contributes to or counters the increase in blood pressure after estrogen depletion. In addition, the effects of estrogen depletion may encompass increased ROS in the kidney that may further diminish the bioavailability of NO.32,34 Additional studies to assess whether the blockade of these two systems contributes to the increase in blood pressure or alters renal status in the mRen(2).Lewis rat are also in progress.
Finally, an unanticipated finding of the present study was the persistent effect of olmesartan treatment on blood pressure once the antagonist was withdrawn. In the ovariectomized mRen(2).Lewis, the systolic pressures were still markedly reduced (-36%) at 7 weeks after cessation of olmesartan administration and were not different than the blood pressures determined at the end of the 4-week treatment period (-42%). Previous studies, all performed in the SHR, clearly showed that RAS blockade with either an ACE inhibitor or ARB exhibits similar effects, provided that treatment is initiated at an early age (3 to 4 weeks) before the development of hypertension.3538 Furthermore, the long-lasting effects are generally not shared by other anti-hypertensive drugs despite a similar reduction in pressure during the treatment period.3941 In contrast, studies in the Milan and Lyon hypertensive rats revealed that RAS blockade had no persistent effect on blood pressure despite marked improvements in indexes of vascular and renal injury.4243 To our knowledge, the present study is the first to demonstrate a substantial effect on blood pressure after cessation of AT1 receptor blockade in adult females with established hypertension and in a hypertensive model other than the SHR.38 Future studies are necessary to ascertain the mechanism and the duration of the persistent effects of olmesartan in the mRen(2).Lewis rat. In this regard, it will also be of interest to determine whether cessation of estrogen treatment in the ovariectomized mRen(2).Lewis rat also exhibits a persistent effect on blood pressure similar to that of the AT1 antagonist.
Perspectives
The role of estrogen to influence the cardiovascular system, particularly in a hypertensive setting, is complex. The mRen(2).Lewis strain represents a model of monogenetic Ang IIdependent hypertension in which estrogen depletion has a profound influence on the development of blood pressure, most likely through attenuating activation of the RAS and other downstream mediators. The early influence of estrogen in the current studies may be more relevant to the status of estrogen in the premenopausal versus postmenopausal period. Kaplan and colleagues44 have demonstrated that reduced levels of estrogen arising from stress in the premenopausal period are associated with increased incidence of cardiovascular disease. Thus, the early loss or reduction of estrogen may play a more significant role in the setting and progression of cardiovascular disease than estrogen loss in the postmenopausal state.45
| Acknowledgments |
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Received May 12, 2003; first decision May 30, 2003; accepted June 25, 2003.
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