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(Circulation. 1997;95:1301-1307.)
© 1997 American Heart Association, Inc.

Articles

Sexually Dimorphic Response of the Balloon-Injured Rat Carotid Artery to Hormone Treatment

Suzanne Oparil, MD; Ronald L. Levine, MD; Shi-Juan Chen, MD; Joan Durand, BS; Y.F. Chen, PhD

the University of Alabama at Birmingham, Vascular Biology and Hypertension Program.

Correspondence to Suzanne Oparil, MD, 1034 Zeigler Research Bldg, 703 S 19th St, Birmingham, AL 35294-0007. E-mail card027{at}uabdpo.dpo.uab.edu

	Abstract

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Background Estrogen blunts the neointimal response to vascular injury in gonadectomized rats of both sexes; addition of a progestin blocks the estrogen effect. This study tested, in intact rats of both sexes, whether (1) exogenous estrogen has a vasoprotective effect in injured carotid arteries, (2) progestin (medroxyprogesterone acetate, MPA) blocks the vasoprotective effect of estrogen, and (3) any observed sexual dimorphism in the responses to estrogen and/or MPA can be accounted for by differences in serum 17ß-estradiol levels.

Methods and Results Intact male and female Sprague-Dawley rats were randomly divided into four subgroups treated with either (1) 17ß-estradiol, (2) MPA, (3) 17ß-estradiol+MPA, or (4) vehicle and were subjected to balloon injury of the right common carotid artery. Two weeks later, rats were killed by an overdose of pentobarbital, and the carotid arteries were evaluated for myointimal thickening. Neither estradiol nor MPA altered the neointimal response in males. In females, estradiol reduced and MPA enhanced the response, whereas addition of MPA to estradiol blocked the vasoprotective effects of estrogen.

Conclusions Intact male rats but not intact females are resistant to the vasoprotective effects of exogenous estrogen, despite attainment of physiological (for females) serum 17ß-estradiol levels. MPA enhances the neointimal response in intact females, presumably by blocking the production and thus the vasoprotective effect of endogenous estrogen.

Key Words: restenosis • hormones • sex • women • endothelium • muscle, smooth • carotid arteries

	Introduction

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Epidemiological and clinical studies have defined a sexually dimorphic pattern in the development of atherosclerotic vascular disease in humans.¹ The incidence of cardiovascular disease is much lower in premenopausal women than in men but rises steadily after the menopause, approaching but never attaining the levels observed in age-matched men.² Postmenopausal women on estrogen replacement therapy have less severe coronary artery disease and a lower risk of cardiovascular mortality than women without hormone treatment.³ Thus, estrogens inhibit the development of atherosclerotic cardiovascular disease in women.⁴ Meta-analyses of observational studies have estimated that postmenopausal estrogen replacement therapy reduces the relative risk of myocardial infarction and other forms of atherosclerotic cardiovascular disease to 0.5.^{5 6} This vasoprotective effect of estrogen involves alterations in lipid metabolism, endothelial function, smooth muscle cell proliferation and associated extracellular matrix formation, and vascular reactivity.⁷ In contrast, there is little evidence that estrogens prevent atherosclerotic disease in men. In the only controlled trial of estrogen use in men with cardiovascular end points, the Coronary Drug Project, estrogen use (albeit at high doses no longer recommended for clinical use) was associated with an increased incidence of myocardial infarction, thromboembolism, and stroke.^{8 9} These clinical data suggest the possibility of a sexual dimorphism in the vascular effects of estrogen. The mechanisms of this putative sexual dimorphism are poorly understood.

We have previously observed a sexual dimorphism in the response to balloon injury of the rat carotid artery: the intact male rat develops a more robust neointimal response to injury than the intact female.¹⁰ The sex difference in myointimal response after vascular injury is estrogen dependent, because gonadectomy of female but not male rats results in increased neointima formation, and treatment with estradiol but not testosterone inhibits neointima formation in gonadectomized rats of both sexes. We have further shown that MPA blocks the antiproliferative effects of estradiol in balloon-injured carotid arteries of gonadectomized rats of both sexes.¹¹ The present study examined the effects of estrogen and progestin, alone and in combination, against a background of endogenous sex hormones, on the vascular injury response in male and female rats with their gonads in place.

	Methods

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Animals
Ten-week-old male and female S-D rats were obtained from Charles River Breeding Laboratories (Wilmington, Mass). All rats were maintained at constant humidity (60±5%), temperature (24±1°C), and light cycle (6 AM to 6 PM) and were fed a standard rat pellet diet (Ralston Purina Diet) ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No 85-23, revised 1985). Body weights were determined before and 2 weeks after balloon injury of the carotid artery.

Gonadectomy and Hormone Therapy
Male and female rats were randomly divided into four subgroups, and hormone therapy was initiated. The first treatment group (n=9 male, n=6 female) received daily injections of estrogen (17ß-estradiol 20 µg·kg^-1·d^-1 in 100 µL cottonseed oil SC); the second (n=8 male, n=8 female) received progestin (MPA, 10 mg·kg^-1·d^-1 in 100 µL saline SC); the third (n=8 male, n=6 female) received estrogen+progestin in the doses described above given daily as separate injections; and the fourth (n=20 male, n=13 female) received vehicle (100 µL/d cottonseed oil SC). The 17ß-estradiol and MPA were purchased from Sigma Chemical Co.

Balloon-Injury Procedure
After 3 days of hormone therapy, rats were anesthetized with sodium pentobarbital (50 mg/kg IP), and the right carotid artery was isolated by a middle cervical incision, suspended on ties, and stripped of adventitia. The distal right common carotid artery and region of the bifurcation were exposed. A 2F Fogarty balloon catheter (Baxter V. Mueller) was introduced through the external carotid artery and advanced into the thoracic aorta. The balloon was inflated with saline to distend the common carotid artery and was then pulled back to the external carotid artery. After six repetitions of this procedure, the endothelium was removed completely, as assessed by the Evans blue dye technique, and there was some injury to medial smooth muscle layers throughout the common carotid artery. After removal of the catheter, the external carotid artery was ligated and the wound closed. The left carotid artery was not damaged and served as a control.

Morphometric Analysis
Two weeks after balloon injury of the right carotid artery, rats were killed with an overdose of sodium pentobarbital (75 mg/kg) and perfused with 10% formalin at a pressure of 120 mm Hg. The vascular system was rinsed with 10 mL PBS before infusion of fixative solution. Both carotid arteries were isolated from adherent tissue and fixed in 10% formalin for morphometric analysis. Vessels were embedded in paraffin, and the middle fifth (0.2 cm) of the damaged right carotid artery was serially sectioned (30 µm). The left carotid artery was not damaged and served as a control. Morphometric analysis of each arterial segment was performed with a computer-based Bioquant II Morphometric system. Tissue was stained with Verhoeff's elastic-tissue stain, which demonstrated several layers of elastic laminae. At least five sections of each vessel were examined, and the measurements were averaged for statistical analysis. All morphometric analyses were carried out by a single examiner, who was blinded with respect to the experimental group to which each sample belonged. The cross-sectional surface areas of the vessel within the external elastic lamina (total area), within the internal elastic lamina (intimal area), and within the lumen (lumen area) were measured. The degree of myointimal proliferation of the injured carotid artery was expressed as the absolute area of neointima and the I/M ratio.

Gonadal Hormone Assays
At the time the animals were killed, a 1-mL blood sample was removed from the femoral arterial cannula and allowed to clot. Serum estradiol and progesterone levels were determined by radioimmunoassay with commercially available kits (Diagnostic Products Corp). Assay sensitivity was 8 pg/mL for estradiol and 30 pg/mL for progesterone. Intra-assay and interassay coefficients of variation were 5.3% and 6.4% for estradiol and 4.7% and 7.9% for progesterone, respectively. The estradiol antiserum is highly specific for 17ß-estradiol, with low cross-reactivity with 17-estradiol (0.017%), estriol (0.32%), aldosterone (0%), progesterone (0%), cortisol (0%), testosterone (0.001%), and 5-dihydrosterone (0.004%). The progesterone antiserum is highly specific for progesterone, with no cross-reactivity (0%) with MPA, estradiol, cortisol, testosterone, or pregnenolone.

Statistical Analysis
Results were expressed as mean±SEM. Data were analyzed with the CRUNCH statistical package on an IBM 486–compatible computer. Statistical comparisons of body weight, serum 17ß-estradiol and progesterone levels, neointimal area, medial area, total area, lumen area, and I/M ratio among experimental groups were performed with two-way ANOVA. Differences were reported as significant at a value of P<.05.

	Results

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Body weights of male S-D rats (344.2±5.9 g, n=52) were significantly greater than those of females (218.0±4.1 g, n=36) before experimentation. Rats were assigned at random to treatment groups, as previously described. Two weeks after balloon injury of the right carotid artery, body weights of all groups of male rats were significantly greater than those of comparable groups of females (Table). Estradiol treatment was associated with a decrease in body weight in both male and female groups compared with controls. MPA treatment did not alter body weight compared with control groups. Treatment with the combination of MPA and estradiol resulted in decreased body weight in males but not in females.

View this table: [in this window] [in a new window]   Table 1. Effects of Administration of 17ß-Estradiol 20 µg·kg-1·d-1 and MPA 10 mg·kg-1·d-1 for 2 Weeks on Myointimal Proliferation After Balloon Injury of Carotid Arteries of Intact Male and Female S-D Rats

Serum estradiol and progesterone levels are summarized in the Table and Fig 1 (top). Serum 17ß-estradiol levels doubled in estrogen-treated male rats and increased by 50% in females compared with vehicle-treated rats of the same sex. 17ß-Estradiol levels in estrogen-treated male rats (33.4±8.2 pg/mL) were within the normal range for intact female rats reported in the literature (30 to 50 pg/mL, depending on the stage of the estrus cycle¹² ) and not significantly different from values for intact, vehicle-treated females (36.6±3.8 pg/mL) and estrogen-treated females (55.0±12.2 pg/mL) in the present study (two-way ANOVA). MPA treatment alone did not alter serum estradiol levels in male rats from values obtained during vehicle treatment, but it lowered estradiol levels significantly in female rats compared with vehicle-treated controls. Addition of MPA to estradiol treatment did not alter serum estradiol levels in males but lowered (P<.05) estradiol levels in females compared with females treated with estradiol alone.

View larger version (33K): [in this window] [in a new window]   Figure 1. Effects of administration of 17ß-estradiol (E2, 20 µg·kg-1·d-1) and MPA (10 mg·kg-1·d-1) on serum 17ß-E2 levels (top) and serum progesterone levels (bottom) in intact male (solid bars) and female (hatched bars) S-D rats at 14 days after injury of the common carotid artery. Results are mean±SEM. #P<.05 vs respective vehicle control groups of same sex; P<.05 vs respective 17ß-E2 groups of same sex; &P<.05 vs respective MPA groups of same sex; *P<.05 vs respective male groups.

Serum progesterone levels (Table and Fig 1, bottom) were significantly lower in vehicle-treated male rats than in females. Estrogen treatment was associated with significant increases in serum progesterone levels in both sexes; the magnitude of the increase was significantly less in males than in females. MPA treatment caused progesterone levels to fall to barely measurable levels in both sexes. This is probably a result of suppression of native progesterone by the artificial progestin, which does not cross-react with antibodies raised to native progesterone. Addition of MPA to estrogen treatment reduced serum progesterone concentrations to barely measurable levels in males but did not alter them in females.

Both the damaged right and undamaged left carotid arteries were examined histologically after perfusion fixation at 2 weeks after injury. In the undamaged left carotid artery, the intima was a single cell layer thick; the internal elastic lamina was intact, and the external elastic lamina was in contact with the adventitia in all rats examined. There were no differences in the total area or the medial area (wall thickness) of the undamaged left carotid artery among experimental groups, indicating that the anatomy of the intact carotid artery was not significantly different between the sexes and was not significantly altered by hormone treatment.

Two weeks after balloon injury of the right carotid artery, the neointimal area in male rats was significantly greater than in females and was unaffected by estrogen or MPA treatment (Table and Figs 2 through 4). The neointima, consisting of circumferentially uniform, multiple layers of cells, was observed in the damaged vessel of all rats but was greater in all male treatment groups than in females (Figs 2 and 3).

View larger version (121K): [in this window] [in a new window]   Figure 2. Representative light micrographs of right common carotid arteries from intact male S-D rats 14 days after balloon injury. Balloon-injured right carotid arteries from vehicle control (top left), 17ß-estradiol (E2)–treated (top right), MPA-treated (bottom left), and E2+MPA–treated (bottom right) rats. Magnification x400.

View larger version (125K): [in this window] [in a new window]   Figure 3. Representative light micrographs of right common carotid arteries from intact female S-D rats 14 days after balloon injury. Balloon-injured right carotid arteries from vehicle control (top left), 17ß-estradiol (E2)–treated (top right), MPA-treated (bottom left), and E2+MPA–treated (bottom right) rats. Magnification x400.

In estradiol-treated male rats, the extent of neointima formation in the damaged carotid artery was not significantly different from that in vehicle-treated males (Table and Figs 2 through 4). In contrast, estradiol-treated females had significantly less (>70% less) neointima formation than vehicle-treated females.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effects of administration of 17ß-estradiol (E2, 20 µg·kg-1·d-1) and MPA (10 mg·kg-1·d-1) on neointima formation of balloon-injured right common carotid artery of intact male (solid bars) and female (hatched bars) S-D rats at 14 days after injury. Top, Cross-sectional areas of neointima, and bottom, ratios of neointimal area to medial area presented as mean±SEM. #P<.05 vs respective vehicle control groups of same sex; P<.05 vs respective 17ß-E2 groups of same sex; *P<.05 vs respective male groups; #P<.05 vs respective vehicle control groups; *P<.05 vs respective intact male groups.

MPA treatment alone did not alter the myointimal response (I/M ratio) to balloon injury in male rats compared with vehicle controls but greatly increased the I/M ratio in females compared with vehicle-treated controls (Table and Figs 2 through 4). The neointimal area in the damaged carotid artery of MPA-treated male rats was not significantly different from that in vehicle-treated or estradiol-treated males. In contrast, neointima formation (I/M ratio) in MPA-treated females was significantly greater than in either vehicle- or estradiol-treated females, approaching male levels.

In male rats treated with MPA+estradiol, neointima formation in the damaged carotid artery was significantly less than in the other three treatment groups, whereas in females, addition of MPA to estradiol treatment resulted in an increase (P<.05) in neointima formation (I/M ratio) compared with the estradiol group (Table and Figs 2 through 4). The combined MPA+estradiol group was not significantly different from the MPA-treatment group of female rats.

Morphometric analysis showed that the neointimal area and I/M ratio were significantly less in female rats treated with estradiol alone than in any other treatment group (Table and Figs 2 through 4). The intimal area was reduced by >70% in estrogen-treated females compared with vehicle-treated controls. In contrast, estradiol treatment had no effect on the intimal area or the I/M ratio in male rats.

MPA alone did not alter the myointimal response to balloon injury in male rats compared with vehicle-treated males (Table and Figs 2 through 4). Administration of estradiol significantly suppressed neointima formation in female rats compared with other treatments; addition of MPA to estradiol blocked this effect and restored the myointimal response to levels seen in females treated with vehicle (Table and Figs 2 through 4). In contrast, addition of MPA to estradiol treatment in males was associated with a decrease in the neointimal response, which was not altered by estradiol treatment alone. Serum estradiol levels were not significantly different in males treated with estradiol versus estradiol+MPA. Administration of MPA alone was associated with a large increase in the neointimal response in females compared with both the vehicle-treated and estradiol-treated groups, bringing the I/M ratio into the male range. This effect was associated with a significant reduction in serum estradiol levels compared with vehicle-treated females: serum estradiol levels in MPA-treated females were not significantly different from levels measured in vehicle-treated males.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our results demonstrated that (1) estradiol treatment alone had no effect on neointima formation after balloon injury of the carotid artery in intact male S-D rats but reduced the intimal area in female rats by >70%; (2) MPA treatment alone did not alter the myointimal response to balloon injury in male rats but attenuated the vasoprotective effects of endogenous estrogen in females, so that the neointimal response in the MPA-treated female attained male levels; (3) combined MPA+estradiol treatment blocked the vasoprotective effects of estrogen and enhanced the neointimal response in females but tended to reduce the neointimal response in males; and (4) serum estradiol levels in estrogen-treated males were in the normal female range and were not altered by concomitant administration of MPA; serum estradiol levels in females tended to be higher in the estrogen-treatment group than in vehicle controls but were reduced to male levels by MPA treatment.

The most dramatic finding of the present study was that administration of exogenous estrogen to male rats had no effect on the carotid injury response, whereas the same dose (adjusted for body weight of the recipient) of the same estrogen preparation (17ß-estradiol) reduced the injury response in female rats by >70%. This unresponsiveness to exogenous estrogen in the intact male was very different from the response we previously elicited in gonadectomized male S-D rats with an identical dose schedule of estradiol administration: administration of exogenous estrogen to the gonadectomized male converted the response to one resembling that of the intact female, ie, reduced neointimal proliferation.¹⁰ This result confirms the previous observations of Foegh et al¹³ that 17ß-estradiol inhibits neointima formation (VSMC proliferation and extracellular matrix formation) after balloon injury of the common and external iliac arteries of rabbits. Furthermore, preliminary studies in our laboratory suggest that the sexual dimorphism in the vascular injury response and its modulation by estrogen may not be an intrinsic property of the VSMCs.¹⁴ We have shown that VSMCs derived from aortas of male and female SHRs grow and proliferate at similar rates when cultured in medium free of sex hormones, manifest similar hypoproliferative responses to estrogen, and appear to be unaffected by dihydrotestosterone. Thus, resistance to the vasoprotective effects of estrogen in the male appears to be dependent on the hormonal environment, ie, the presence of testosterone, rather than being an intrinsic property of the male phenotype. It should be noted that our previous cell culture studies were performed on VSMCs from aortas of SHRs, whereas the present study used S-D rats. Although the growth properties of VSMCs from the two strains undoubtedly differ in some respects, the sexual dimorphism of the vascular injury response is similar.¹⁵ Therefore, we believe that data on the sex hormone dependence of VSMC proliferation obtained from VSMCs of SHRs are applicable to the present study.

Many of the vasoprotective effects of estrogen are mediated by the endothelium. These include enhancement of endothelial degradation of LDL cholesterol, suppression of collagen and elastin synthesis, and restoration of endothelium-dependent vasodilator mechanisms after injury.^{16 17 18 19 20 21 22 23 24 25 26 27 28} Estrogens also interfere with the oxidation of deposited LDL particles, thus limiting the progression of atherosclerosis.²⁹ The endothelium plays a central role in the control of vascular tone and the growth of the blood vessel wall, mainly through synthesis and release of endothelium-derived relaxing factor, or NO.³⁰ NO is both a vasorelaxant and an antimitogen for VSMCs. The vasodilator response to infused ACh, the standard test for assessing endothelium-dependent vascular tone, is responsive to acute and chronic estrogen administration. Vasoconstrictor responses to ACh infusion resulting from endothelial dysfunction have been demonstrated in the coronary arteries of ovariectomized monkeys fed an atherogenic diet; normal vasodilator responses were restored after either long-term (3-month)¹⁸ or short-term (20-minute)¹⁹ estrogen administration. Similar restitution of normal coronary vasomotor responses to ACh have been reported in postmenopausal women with coronary artery disease after short-term administration of either ethinyl estradiol²⁰ or 17ß-estradiol.^{22 23}

Whether abnormal coronary vasomotor responses can also be corrected by short-term estrogen administration in male subjects with coronary artery disease is controversial.^{23 24 25 26 27} Coronary vasodilator responses to ACh were not restored in men with coronary artery disease after short-term 17ß-estradiol administration in one published study.²³ However, preliminary evidence from several laboratories suggests that acute intravenous administration of conjugated estrogen improves coronary vascular reactivity in response to ACh challenge or cold pressor testing in men with coronary artery disease and male cardiac transplant recipients.^{24 25 26} Furthermore, 17-dihydroequilenin sulfate, a nonfeminizing conjugated equine estrogen, has been shown to reduce coronary artery LDL degradation and improve coronary artery vascular reactivity in atherosclerotic male rhesus monkeys.²⁷ The lack of effect of the naturally occurring estrogen 17ß-estradiol on coronary arteries in men and male animals supports the concept of a sex-related action of estrogen on the vasculature that is greater in females than in males. Whether the positive effect on endothelium-dependent coronary vascular tone in males that was recently observed with conjugated estrogens is dependent on the dose of estrogen delivered to the vessel or to some other unknown property of the conjugated estrogen that is not shared by estradiol is unknown, and this underscores the need for further research on the vascular effects of estrogen in males. The recent observation that intracoronary injection of 17ß-estradiol effects similar increases in coronary artery cross-sectional area and coronary blood flow in male and female dogs adds complexity to this interpretation.²⁸ However, acute estrogen-induced dilation of canine coronary arteries is endothelium independent and not estrogen receptor mediated and may be unrelated to the vasoprotective effect of estrogen in our model.

In the present study, addition of exogenous 17ß-estradiol to endogenous estrogen in intact female rats further reduced the already blunted (compared with males or ovariectomized females) neointimal response to vascular injury. The dose dependency of the vasoprotective effect of estrogen in this model is consistent with previous observations that restoration of ACh-mediated vasodilation in coronary arteries of ovariectomized monkeys by estrogen replacement treatment is dependent on circulating estradiol levels.¹⁹ The effect of postmenopausal estrogen replacement on circulating lipoprotein concentrations in women is also dependent on the estrogen dose (and presumably on circulating estradiol levels),³¹ as are estrogen effects on ACh-induced vasodilation in the forearm vasculature of postmenopausal women.³² ACh-stimulated forearm blood flow in postmenopausal women has been shown to be enhanced by acute intra-arterial infusion but not by chronic transdermal administration of 17ß-estradiol.³² Furthermore, acute intra-arterial infusion of 17ß-estradiol enhanced the ACh response even in women who were already receiving transdermal estradiol, suggesting that the response is related to circulating levels of hormone. Thus, the magnitude of the vasoprotective effect of estrogen in women and female animals appears to be directly related to circulating estradiol levels. However, the lack of estrogen-induced vasoprotection in males in the present study cannot be attributed to alterations in absorption, metabolism, and/or excretion of estradiol, since serum estradiol levels in estrogen-treated male rats were not different from values in intact females or in estrogen-treated gonadectomized male rats in our laboratory.¹⁰ In both of the latter treatment groups, estrogen treatment significantly reduced the vascular injury response.

MPA treatment of intact female rats reduced serum estradiol concentrations to male levels and increased the neointimal response to injury into the male range. Accordingly, the vascular effect of MPA in the intact female can be attributed, at least in part, to suppression of endogenous ovarian estrogen production and circulating estradiol levels. This contrasts with our previous observation in ovariectomized rats that addition of MPA blocked the antiproliferative effects of estrogen treatment without altering serum estradiol levels, suggesting that the progestin also antagonizes the effects of estrogen on the injured blood vessel.¹¹ This may be related to progestin-estrogen interactions at the receptor level^{33 34} and/or to opposing effects on growth factors/mitogens in damaged vascular tissue.^{11 35} Whatever the cellular mechanism(s) involved, these results are consistent with previous observations that addition of progestin to estrogen treatment reduces the vasoprotective effects of estrogen. Mechanisms by which progestins may antagonize the favorable effects of concomitant estrogen treatment on cardiovascular risk in postmenopausal women include lowering HDL cholesterol levels³⁶ ; reducing NO production, as reflected in serum nitrite and nitrate levels³⁷ ; and increasing plasminogen activator inhibitor-1 levels.³⁸ Whether adding a progestin attenuates the favorable effects of postmenopausal estrogen replacement therapy on cardiovascular disease risk is less well understood, although a recent report of data from the Nurses' Health Study suggests that the addition of progestin does not attenuate the cardioprotective effects of postmenopausal estrogen therapy.³⁹ Furthermore, nonrandomized observational studies have reported that the risk of myocardial infarction was reduced as much (or more) by combined estrogen-progestin therapy as by estrogen alone,^{40 41 42} but the interpretation of such studies is severely limited by indication bias. Since combined estrogen-progestin therapy has not been used routinely until recent years, data on cardiovascular events and mortality from observational population-based studies are minimal.⁴³ Further study in controlled trials is needed to resolve this issue.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine I/M = intima/media MPA = medroxyprogesterone acetate S-D = Sprague-Dawley SHR = spontaneously hypertensive rat VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported in part by grants HL-44195, HL-47081, HL-48848, HL-07457, and HL-50147 from the National Heart, Lung, and Blood Institute and a Grant-in-Aid (93014269) from the American Heart Association. The authors thank Nancy Penney and Rochelle Hunter for their assistance in the preparation of the manuscript.

Received August 16, 1996; revision received October 15, 1996; accepted October 27, 1996.

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