Bi-directional actions of estrogen on the renin-angiotensin system

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogens inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7)) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the reninangiotensin vasoconstrictor/vasodilator system is to promote the antihypertensive effect. Correspondence


Introduction
Cardiovascular disease is the leading cause of female mortality, resulting in more deaths in women over 50 than all cancers (1).The incidence of cardiovascular disease in women rises steadily, approaching the incidence in men during the fifth to seventh decade of life (2).Estrogen protects women from cardiovascular disease by inhibiting atherosclerosis (3) and through effects on lipid metabolism (4)(5)(6), carbohydrate metabolism (7,8), body composition (8), and vasomotor reactivity (9)(10)(11)(12).Administration of estrogen to healthy women after menopause is associated with a lower rate of coronary disease (13)(14)(15).Barrett-Connor and Bush (13) estimated that the long-term use of postmenopausal estrogen decreased cardiovascular disease by 50%.However, changes in plasma lipids and atherosclerosis accounted for only 25-50% of the cardioprotective ef-fect (1,15), suggesting that estrogen maintains vascular health through additional processes.One potential mechanism is the effect of estrogen on the renin-angiotensin system (RAS).
Estrogen causes over-expression of the RAS by augmenting both tissue and circulating levels of Aogen (51,52) and renin (53)(54)(55)(56).Plasma renin activity (PRA) increases after estrogen treatment in nephrectomized rats (53).In addition, tissue renin is increased in the ovary, submaxillary gland, uterus, and adrenal gland after estrogen treatment (54).Ovariectomy of spontaneously hypertensive female rats is associated with a fall in kidney renin mRNA; this decrease is not observed in tissue from rats receiving estrogen supplement (53).In association with increased circulating estrogen, renin is increased in the mother during pregnancy (57).Plasma Aogen also increases in normotensive and hypertensive menopausal women placed on estrogen replacement therapy (58).However, no change in plasma Ang II was observed.The use of higher doses of estrogen in oral contraceptives increases hepatic and plasma Aogen and blood pressure (59,60), especially for the higher dose estrogens found in early contraceptives.However, lower doses of estrogen in modern contraceptives also result in an increase in plasma Aogen, without the complication of hypertension (59,60).During pregnancy, there is a large increase of plasma Aogen due to stimulation of hepatic Aogen synthesis by estrogen (57,59).During the normal menstrual cycle, plasma Aogen and prorenin, a precursor of renin, increase during the follicular phase, but active plasma renin does not change until the luteal phase when both estrogen and progesterone are elevated (61).Thus, Aogen and renin are elevated in response to increased plasma estrogen due to normal cycling events or pharmacological hormone replacement.
If estrogen treatment activates the RAS, why do the hyper-reninemia and hyperaogenemia states not result in significant increases in blood pressure?The effects of hormone replacement on blood pressure are conflicting, with findings of either no change or a decrease in blood pressure (12,(62)(63)(64).In investigating this question, we found that estrogen shifts the pattern of angiotensin peptide formation in a tissue-specific manner, reducing production of Ang II, while augmenting the production of the N-terminal Ang II fragment, Ang-(1-7).This shift in the pattern of peptides arises in part due to estrogen decreasing the activity of ACE.

Chronic hormone replacement in cynomolgus monkeys
In the first study we evaluated the effects of chronic hormone replacement in a model of post-menopausal hormone replacement, i.e., ovariectomized monkeys receiving premarin (conjugated equine estrogens; CEE) orally.At the time the study was initiated 29 feral adult female cynomolgus monkeys (Macaca fascicularis) ranging in age from 5 to 13 years had been fed a moderately atherogenic diet for 22 months to induce atherosclerosis.Bilateral ovariectomies were performed to initiate surgical menopause on all animals 4 months after the diet started.At the end of 22 months of continuous feeding of the atherosclerotic diet, all animals received a lipid-lowering diet and were randomly assigned to a replacement therapy protocol lasting 30 months as indicated: group 1 (placebo, N = 14) and group 2 (CEE, N = 15).Groups 2 received 7.2 µg/day of CEE (Premarin ® , Wyeth-Ayerst, Radnor, PA, USA) for the first eight months and 166 µg/ day of CEE for the remaining 22 months.The latter dose of CEE was used to raise the levels of circulating 17 ß-estradiol to approximately 150 pg/ml, a concentration equivalent to the therapeutic concentrations achieved in women (~0.625 mg/day) (65).Hormones were administered twice daily in the diet.Blood samples characterizing the levels of sex hormones were taken 4 h after administration of the oral drug (peak level) under ketamine hydrochloride sedation (10 to 15 mg/kg body weight, im) (Fort Dodge Laboratories, Inc., Fort Dodge, IA, USA) 5 months before necropsy.
There was no significant effect of hormone replacement on mean arterial blood pressure or body weight.As expected, plasma 17-ß-estradiol levels were near the minimum detectable level of the assay in untreated control animals (2.4 ± 1.1 pg/ml) but rose significantly (148.4 ± 13.4 pg/ml, P<0.05) in CEE-treated animals.Figure 1 shows that long-term replacement therapy with CEE produced significant increases in PRA.These changes were accompanied by a significant reduction in plasma ACE activity in CEE-treated animals.Replacement of estrogen resulted in a significant, three-fold increase in plasma Ang I in animals given CEE (Figure 2A).On the other hand, there

Estrogen replacement in transgenic hypertensive (mRen-2)27 rats
In the next study, we evaluated hormone replacement in hypertensive and normotensive rats.Fifty-three female transgenic negative Tg(-) and heterozygous hypertensive Tg(+) rats (body weight: 220~250 g) from the Hypertension Center Transgenic Rat Colony of Wake Forest University School of Medicine underwent bilateral ovariectomy at age 12 weeks under general anesthesia with ketamine (30 mg/kg, im) and xylazine (5 mg/kg, im).Pellets containing either 17 ßestradiol (E 2 ) (1.5 mg/rat, for 3-week release; Innovative Research of America, Toledo, OH, USA) or vehicle (VEH) were implanted into the subcutaneous tissue.
The magnitude of the pressor response produced by the injection of three doses of Ang II was similar in VEH-treated Tg(+) and Tg(-) rats.In contrast, chronic 17 ß-estradiol treatment significantly attenuated the pressor responses to intravenous injection of Ang II in both strains at all doses tested (Figure 3A and B).Intravenous injections of Ang-(1-7) in normotensive and transgenic rats elicited a biphasic response consisting of a rapid (55.6 s) pressor component followed by a longer lasting (138.9 s) depressor component.Estrogen replacement therapy had a small but significant effect on the pressor but not the depressor component of the response to intravenous injections of Ang-(1-7) in normotensive rats (Figure 4A  and C).In Tg(+) rats treated with estrogen, the blunting of the pressor component of the Ang-(1-7) response was comparable to that obtained in Tg(-) rats (Figure 4A and B).In hypertensive rats, estrogen potentiated the magnitude of the fall in blood pressure produced by Ang-(1-7) (Figure 4D).Replacement with estrogen resulted in a nearly 2-fold reduction in the circulating levels of Ang II in Tg(+) rats while it had no effect on plasma Ang II in Tg(-) rats (Figure 5A).In contrast, estrogen replacement significantly increased the levels of plasma Ang-(1-7) in Tg(+) animals (Figure 5B).In accordance with the reduction in plasma Ang II levels in Tg(+) rats, estrogen significantly reduced plasma ACE activity in Tg(+) animals (Figure 6A).A similar reduction in plasma ACE was observed in Tg(-) rats.There was no difference in plasma, kidney, or aorta ACE activity levels between Tg(-) and Tg(+) animals on similar treatment (Figure 6A-C), whereas chronic estrogen replacement therapy significantly decreased both kidney and aorta ACE activity in Tg(+) but not in Tg(-) rats.
In summary, this study combined a monogenetic model of renin-dependent hypertension with a surgically induced postmenopausal model.Hormone replacement in this model attenuates hypertension in association with reduced generation of Ang II and increased production of Ang-(1-7).In spite of the overall impression in the literature that estrogen activates the RAS (51,52,67) by increasing the levels of angiotensinogen and renin, estrogen acts downstream in relation  to these two proteins by reducing ACE activity and shifting the profile of the circulating angiotensin peptides.Thus, estrogen acts as a fulcrum reducing the magnitude of the response to and levels of Ang II, while increasing the formation and vasodilator effect of Ang-(1-7).These studies provide new information on the potential mechanisms that may contribute to the therapeutic action of estrogen replacement therapy in postmenopausal women who are at an increased risk of cardiovascular morbidity.

Estrous cycle influence on angiotensin peptides
A third study was conducted to evaluate if local regional angiotensin peptide levels are modulated by the estrous cycle.Tenweek-old normotensive (N = 15) and hypertensive (mRen2)27 (N = 15) rats were sacrificed by decapitation without prior anesthesia.Vaginal smears were obtained from each rat to determine the stage of the estrous cycle at sacrifice, as described by Long and Evans (68).Immunoreactive (Ir) peptide levels were measured using three different radioimmunoassays, as previously described (69).Table 1A shows the influence of the estrous cycle on angiotensin peptides in the hypothalamus.There was no influence of the estrous cycle on hypothalamic peptide levels of normotensive rats.Ir-Ang I was significantly elevated during proestrus as compared to diestrus in hypertensive rats.Ir-Ang II levels were unchanged with the estrous cycle.Ir-Ang-(1-7) levels were significantly elevated during both proestrus and estrus as compared to diestrus.Table 1B illustrates that the peptide levels in the lower brain stem of normotensive or hypertensive rats did not change with the estrous cycle.In summary, the estrous cycle exerts an influence on hypothalamic angiotensin peptides, enhancing the expression of Ang I and Ang-(1-7) in hypertensive rats.
Gender differences in cardiovascular dis-   7).By reducing ACE activity, estrogen would decrease the breakdown of Ang-(1-7) and add further to the increased levels of Ang-(1-7), potentially resulting in an enhancement of vasodilation.Because ACE reduction also leads to less degradation of bradykinin, another aspect of estrogens action would be to enhance
Ang-(1-7) and BK.Our studies enhance the understanding of the role of estrogen in the regulation of the RAS and may provide a new rationale for the use of estrogen to prevent cardiovascular disease in postmenopausal women who are at increased cardiovascular risk.