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(Circulation. 1997;96:1970-1975.)
© 1997 American Heart Association, Inc.

Articles

Contrasting Effects of Conjugated Estrogens and Tamoxifen on Dilator Responses of Atherosclerotic Epicardial Coronary Arteries in Nonhuman Primates

J. Koudy Williams, DVM; Erika K. Honoré, DVM, MPH; ; Michael R. Adams, DVM

From the Comparative Medicine Clinical Research Center and the Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC. Dr Honoré's present address is Department of Medicine and Physiology, Southwest Foundation for Biomedical Research, San Antonio, Tex.

Correspondence to J. Koudy Williams, DVM, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1040. E-mail kwilliams{at}cpm.bgsm.edu

	Abstract

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Background Estrogens have been shown to improve dilator responses of atherosclerotic coronary arteries. Tamoxifen is a mixed estrogen agonist/antagonist with as yet unexplored effects on vascular function. Therefore, the goal of this study was to compare the effects of conjugated equine estrogens (CEEs) with those of tamoxifen on epicardial coronary artery dilator responses in atherosclerotic, ovariectomized monkeys.

Methods and Results Fifty ovariectomized cynomolgus monkeys were fed an atherogenic diet for 34 months. During this time, monkeys were assigned to one of three treatment groups: (1) control, no hormone replacement (n=15); (2) CEEs mixed in the diet at a dose of 0.043 mg · kg^-1 · d^-1 (n=14); or (3) tamoxifen mixed in the diet at a dose of 1.3 mg · kg^-1 · d^-1 (n=21). Quantitative angiography was used to measure coronary artery dilator responses to intracoronary infusions of acetylcholine (10^-8, 10^-7, and 10^-6 mol/L) and nitroglycerin (15 µg/min). Coronary arteries of the tamoxifen-treated group constricted in response to high-dose acetylcholine (-5.4± 2.3%, P<.05 versus control), whereas those of the CEE group did not (P>.05 versus control). Conversely, arteries from the CEE group dilated in response to nitroglycerin (9.1±2.1%, P<.05 versus control), whereas those from the tamoxifen group did not (P>.05 versus control). Statistical adjustments for variations in plaque extent (determined subsequently after necropsy) and plasma lipoproteins did not alter the results.

Conclusions Tamoxifen has primarily estrogen-antagonistic effects on epicardial coronary artery dilator responses in atherosclerotic monkeys. Results implicate the estrogen receptor as a modulator of coronary artery dilator responses in ovariectomized, atherosclerotic monkeys.

Key Words: atherosclerosis • arteries • hormones • tamoxifen • vasculature

	Introduction

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Tamoxifen is a mixed estrogen agonist/antagonist that is widely used as a treatment for breast cancer. It is also recommended for reduction of tumor recurrence^{1 2} and is currently being considered as a preventive therapy in women with a familial risk of breast cancer.^{3 4} Another potential benefit of tamoxifen is an apparent reduction in risk of cardiovascular disease in postmenopausal women taking tamoxifen.⁵

Cardiovascular disease remains the most common cause of death in postmenopausal women.⁶ Therefore, it is essential that the potential effect of tamoxifen therapy on cardiovascular disease risk factors be elucidated. Several studies of women with breast cancer have shown that tamoxifen has beneficial effects on plasma lipids and lipoproteins.^{7 8 9 10 11} However, the data are still inconclusive. A recent study of surgically postmenopausal female nonhuman primates showed no effect of tamoxifen on plasma cholesterol concentrations but significant reductions in arterial LDL degradation and in the progression of coronary artery atherosclerosis.¹² Investigators also report that tamoxifen prevents LDL oxidation in postmenopausal women.¹³

One of the manifestations of coronary artery disease is an impairment of normal vasodilation.^{14 15 16 17} These alterations in endothelium-mediated vascular reactivity appear very early in the progression of atherosclerosis^{18 19} and may contribute to the pathogenesis of coronary vasospasm, angina, and myocardial infarction. Estrogen replacement therapy improves dilator responses of large epicardial coronary arteries among postmenopausal women^{20 21 22 23} and nonhuman primates.^{24 25 26} Whether tamoxifen has estrogen-agonist or estrogen-antagonist effects on coronary artery reactivity is undetermined.

In this study, we used a well-established nonhuman primate model of diet-induced atherosclerosis and surgical menopause to compare the vasodilator effects of tamoxifen with CEEs in atherosclerotic coronary arteries.

	Methods

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Experimental Design
The subjects of this study were 50 adult female cynomolgus monkeys (Macaca fascicularis) imported from Indonesia (Institut Pertanian Bogor). All animals consumed monkey chow (High Protein Monkey Chow, Ralston Purina Co) during a 3-month quarantine period. Animals were then ovariectomized and fed a moderately atherogenic diet (40% of calories from fat and 0.28 mg cholesterol/kcal)²⁷ for 4 months. They were randomly assigned to one of three experimental groups by means of a stratified randomization procedure using baseline (ie, while eating the moderately atherogenic diet) TPC and HDL-C concentrations as stratification variables. The experimental groups were (1) controls (no drug treatment, n=15); (2) CEE (treated with CEEs [Premarin, Wyeth-Ayerst]) (n=14); and (3) treated with tamoxifen (Nolvadex, ICI Pharma) (n=21). All hormones were administered in the diet continuously for 30 months with the same atherogenic diet as fed in the preexperimental period. The appropriate dose for the monkeys was calculated by use of the difference in caloric intake between monkeys and human beings adjusted for body size and metabolic rate.^{28 29} On this basis, monkeys in the hormone treatment groups received a daily dose of 0.043 mg/kg of CEEs (equivalent to a woman's dose of 0.625 mg) or 1.3 mg/kg tamoxifen (equivalent to a woman's dose of 20 mg). Plasma concentrations of estradiol and estrone were measured by radioimmunoassay of samples collected 2 hours after feeding to verify that plasma concentrations were similar to those of women taking these drugs at the equivalent doses. Plasma concentrations of the tamoxifen metabolite 4-hydroxytamoxifen in these samples were measured with high-performance liquid chromatography. Monkeys in the control group consumed the same atherogenic diet with no hormones added. Body weight was measured every 3 months to ensure that caloric intake remained constant.

Blood samples for TPC,³⁰ HDL-C,³¹ and triglycerides³² were taken from sedated monkeys (ketamine hydrochloride 10 mg/kg IM) at 3-month intervals for a concurrent study. Plasma concentrations of TPC and triglycerides reported here were taken after 27 months of treatment, because these have the most relevance to the condition of the animal at the time of coronary angiography. Plasma lipoprotein fractions (HDL, LDL, and IDL+VLDL) were obtained after 24 months of treatment (the last date of sampling).^{33 34}

Animals were housed in 4- to 6-member social groups during the experiment. All experimental procedures were conducted in compliance with the "Principles of Laboratory Animal Care" and the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Bowman Gray School of Medicine.

Measurement of Coronary Artery Vascular Responses
Monkeys were anesthetized with ketamine hydrochloride (10 to 15 mg/kg IM) and butorphanol tartrate (0.025 mg/kg IM). Doses of both agents were repeated as needed to maintain light anesthesia. Anesthetized monkeys were placed on a circulating-warm-water blanket pad and allowed to breathe spontaneously. ECG monitoring (Aloka 118, Johnson and Johnson Ultrasound) was performed throughout the period of anesthesia. Heparin was administered at the start of surgery (100 mg/kg IV). A custom-designed 3F (tapered to 1.8F) catheter was inserted into the left femoral artery and advanced into the left main coronary artery under fluoroscopic guidance. An infusion pump (Harvard Apparatus) was used to make serial 2-minute infusions of (1) 5% dextrose in water (control 1); (2) acetylcholine (10^-8, 10^-7, and 10^-6 mol/L estimated final concentration in the coronary artery); (3) 5% dextrose in water (control 2); and (4) nitroglycerin (15 µg/min). It has been shown previously that these doses of agonists, when infused into the coronary artery, have minimal effects on blood pressure and heart rate.^{24 25} Data were not used if heart rate was significantly elevated or decreased (>20%) during infusion of the agonist. Cineangiographic images were taken in the 30° right anterior oblique projection at 60 frames per second immediately after each infusion, during a hand injection of 2 mL of nonionic contrast solution (Omnipaque, Squibb) into the left main coronary artery.

The catheter was removed after the last infusion, and the monkey was allowed to recover from anesthesia. Postoperative care was provided in accordance with state and federal regulations.

Angiographic Measurements
QCA was done in the Bowman Gray Cardiology Image Analysis Laboratory. A single frame after each infusion was selected for analysis on the basis of clarity of the image of the proximal 2 to 3 cm of the left circumflex coronary artery. Criteria for clarity included maximal opacification, no overlapping structures, and minimal motion artifact. Care was taken to select all frames from a single monkey at the same time in the cardiac cycle (end diastole). Each frame was optimally magnified with a cine-video projector (SME-3500, Sony Corp of America) and digitized to a 480x384x10-bit gray-scale image with a frame grabber (4 megabytes, Epix Inc) installed in a 486 personal computer. The mean diameter of the segment of interest was measured by previously validated QCA (QCA Plus, Sanders Data Systems). Specific anatomic landmarks were used to ensure that the same portion of the vessel was analyzed after each infusion. Each film was analyzed by an operator who was unaware of the subject's treatment group. Although each film was analyzed only once, previous QCA studies in our laboratory have shown a high degree of correlation between two repeated measurements of the same artery (r=.98). Each digitized analysis was examined for accuracy of measurement by personnel blinded to treatment groups. To further ensure accuracy and precision of the QCA methods used, images of a Plexiglas phantom with five precision-drilled holes ranging from 0.73 to 4.79 mm were obtained under radiographic conditions similar to those for the angiographic images. The correlation coefficient of these images was 0.99, with an SEM of 0.05 mm.

Measurement of Atherosclerotic Plaque Size
On completion of the angiographic studies, the monkeys were transported immediately to the necropsy room, where sodium pentobarbital (30 mg/kg IV) was administered to attain surgical anesthesia. An infusion of Ringer's solution was initiated via an 18-gauge needle inserted into the left ventricle. The monkeys were then euthanized with an injection of sodium pentobarbital (80 mg/kg IV). A 1-cm longitudinal incision was made in the abdominal vena cava for drainage of blood from the cardiovascular system. The heart was removed and perfusion-fixed for 1 hour at 100 mm Hg with 10% neutral buffered formalin. The left circumflex coronary artery (the artery measured angiographically) was cut en bloc at 5-mm intervals for a total of five blocks. The tissue blocks were dehydrated through increasing concentrations of ethanol and embedded in paraffin. Two 5-µm sections were cut from each block and stained with Verhoeff–van Gieson's stain. The sections then were projected onto a digitizer plate. The component parts of the artery were traced with a hand-held stylus and computer-assisted digitizer. The intimal area was used as the measurement of atherosclerosis extent and was expressed as square millimeters.

Statistical Analysis
All values shown are mean±SEM unless otherwise stated. ANOVA was used to compare body weight, lipoprotein concentrations, and vascular reactivity to acetylcholine and nitroglycerin between treatment groups. The percent change in coronary artery diameter in response to each dose of acetylcholine or nitroglycerin was calculated by comparison of arterial diameter after each drug infusion with its diameter during the preceding control infusion. Vascular reactivity data also were analyzed by ANCOVA, with plaque size or plasma lipid concentrations as the covariates. Differences were considered statistically significant when P<.05.

	Results

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Treatment groups did not differ in age (12 years) or body weight (3 kg) at the end of the experiment (Table). Among animals treated with CEEs, plasma concentrations of estradiol and estrone were 3.9 and 2.6 pmol/L, respectively. Among untreated controls, estradiol and estrone concentrations were 0.3 and 0.6 pmol/L, respectively. Animals treated with tamoxifen had undetectable concentrations of plasma estradiol and estrone but mean plasma concentrations of 17.5 nmol/L 4-hydroxytamoxifen (a metabolite with high binding affinity for the estrogen receptor³⁵ ).

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid Concentrations and Coronary Vascular Responses in Study Animals at End of Treatment Period

Baseline TPC and HDL-C concentrations in groups of animals before treatment were 11.0 and 1.4 mmol/L, respectively. Treatment effects on plasma lipids and coronary vascular reactivity are presented in the Table. Treatment with CEEs or tamoxifen increased plasma triglyceride concentrations and reduced LDL molecular weight (P<.05, Table). Tamoxifen treatment had no effect on TPC, HDL, IDL+VLDL, or LDL compared with control animals (P>.05, Table).

Arteries from control animals and tamoxifen-treated animals constricted in response to the two higher doses of acetylcholine, whereas arteries from animals treated with CEEs dilated in response to all concentrations of acetylcholine (P<.05, CEE versus control or tamoxifen, Table). Although plaque extent was less in the CEE group (P<.05 versus control) and somewhat less (P=.57 versus control) in tamoxifen-treated monkeys (Table), statistical adjustments for variation in atherosclerotic plaque size or plasma lipids had no influence on this outcome. The effect of treatment on coronary vascular responses to the highest concentration of acetylcholine (10^-6 mol/L) is shown graphically in the Figure.

View larger version (0K): [in this window] [in a new window]   Figure 1. Mean±SEM responses of left circumflex coronary arteries (% change in diameter vs infusion of vehicle only) to intracoronary infusion of 10-6 mol/L acetylcholine in ovariectomized cynomolgus monkeys with diet-induced atherosclerosis. a, P<.05 vs control. b, P<.05 vs CEEs.

Arteries from animals in all groups dilated in response to nitroglycerin, although the magnitude of response was significantly higher in the CEE group (P<.05 versus control and tamoxifen; Table). The response to nitroglycerin of the tamoxifen group was increased only marginally over that of the control group (P=.08, Table). Statistical adjustment for variation in atherosclerotic plaque size or plasma lipids did not affect these results.

	Discussion

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The major finding of this study is that, unlike estradiol, the mixed estrogen agonist/antagonist tamoxifen does not enhance the endothelium-dependent vasodilator response of atherosclerotic coronary arteries in ovariectomized female monkeys.

Vascular Reactivity
The finding that CEEs improved large epicardial coronary artery responses to acetylcholine is consistent with our previous results.²⁶ The current findings confirm that, like short-term CEE treatment (1 month),²⁶ longer treatment (2 years) resulted in the amelioration of constrictor responses of atherosclerotic coronary arteries to acetylcholine. Interestingly, in the present study CEEs also improved dilator responses to nitroglycerin, suggesting that CEEs may have affected both endothelium- and smooth muscle–mediated dilation of these atherosclerotic coronary arteries. These results are consistent with those of other studies, which suggest an effect of estrogens on endothelium-independent rather than endothelium-dependent dilation.^{36 37} Unfortunately, our data do not resolve the issue of whether estrogens affect vascular reactivity through endothelium- or smooth muscle–mediated mechanisms, but this study was not designed to do so. It does seem, however, that both cell types may be affected, resulting in overall improvement in coronary dilator capability.

We report in this study that unlike estrogens, tamoxifen does not improve epicardial coronary dilator responses to acetylcholine (Figure). Furthermore, tamoxifen does not improve dilator responses to nitroglycerin (Table). To our knowledge, this is the first report of the effects of tamoxifen on epicardial coronary artery reactivity in atherosclerotic subjects. Since tamoxifen is both an estrogen receptor blocker and an estrogen agonist, our results suggest that under these experimental conditions, tamoxifen acts as an estrogen antagonist on the vessel wall. There is also the suggestion that estrogen receptors (which are blocked by tamoxifen) may play a role in the improved vascular reactivity reported with pure estrogens. Estrogen receptors have been implicated previously as important modulators of vascular physiological and pathological events,^{38 39 40 41} although no studies to date have directly examined the role of the estrogen receptor in modulating the pathophysiological characteristics of the vessel wall in vivo. Although suggestive, the results presented here also do not directly implicate the estrogen receptor in modulating vascular reactivity. Additional groups, such as a combined estrogen+ tamoxifen group, and careful examination of estrogen-receptor binding and activity would be required to test that hypothesis. Such measurements were beyond the scope of the present study. Use of tamoxifen has been associated with increased hot flushes in women, suggesting an effect of tamoxifen on small-artery reactivity. However, the current experiment was not designed to examine resistance-size vessel reactivity.

Animals in the CEE group showed a reduction in the amount of coronary artery atherosclerosis as well as an improvement in coronary vasodilation. This comparison might suggest that hormone effects on acetylcholine responses and extent of atherosclerosis may be related. We do not believe this to be the case, however. We have shown previously that the effects of estrogen (both CEEs and ethinyl estradiol) on vascular reactivity are independent of extent of atherosclerosis and plasma lipids.^{24 25} Furthermore, statistical adjustments for variation in extent of atherosclerosis did not alter the results. The lack of correlation between plaque size and vascular response is further emphasized in the tamoxifen-treated group, which showed a reduction in atherosclerosis progression but no improvement in dilator responses.

These data suggest that tamoxifen may act as both an estrogen agonist (ie, to inhibit atherogenesis) and an estrogen antagonist (ie, to inhibit dilator responses) in the same artery. Unfortunately, it was beyond the scope of this study to examine this apparent paradox. However, it could be speculated that the mechanisms by which estrogen agonists and antagonists modulate atherogenesis and vascular reactivity are different, with the classic estrogen receptor being a more important modulator of vascular reactivity.

It also seems unlikely that vascular reactivity was significantly related to treatment effects on plasma lipids or lipoproteins. Statistical adjustment for variations in LDL molecular weight and plasma triglycerides did not alter the results. Furthermore, short-term treatment with CEEs does not alter plasma lipoprotein concentrations but does affect vascular reactivity.²⁶ Resistance of LDL to oxidation was not measured in this study but has been identified as a factor that may affect vascular reactivity.¹⁶

Tamoxifen and Coronary Heart Disease
It is estimated that several hundred thousand women in this country take tamoxifen for treatment and/or prevention of estrogen receptor–positive breast cancer.⁴² However, tamoxifen has been reported to increase the risk of endometrial cancer,^{43 44 45} suggesting that it has estrogenic activity in some tissues. Since cardiovascular disease represents a serious health threat to women, the impact of tamoxifen on cardiovascular disease risk has been of concern. The results of studies exploring the effects of tamoxifen on coronary heart disease have been varied and are confounded in large part by the fact that the studies have been done on women with breast cancer.

Most of the current data from human and animal studies suggest that tamoxifen either has no deleterious effects or may even be protective against cardiovascular disease.^{7 8 9 10 11} This conclusion is based primarily on the association of tamoxifen with reductions in known risk factors such as plasma LDL concentrations and on the results of the Scottish trial of adjuvant tamoxifen therapy, which show a 67% reduced risk of myocardial infarction.⁵ Rutqvist and Mattson,⁴⁶ for the Stockholm Breast Cancer Study Group, report a 32% reduction in risk of all cardiac disease. There is also evidence that tamoxifen may have antioxidant properties.¹³ In our study, tamoxifen had modest effects on plasma lipid concentrations. There was a trend toward lower TPC, higher HDL-C, and lower LDL-C concentrations, as well as lower LDL molecular weights, in the tamoxifen-treated animals.¹² These results are roughly comparable to those reported in women^{7 8 9 10 11} and might have been amplified if more subjects had been included in each experimental group. However, our results in monkeys will not necessarily translate directly to those in women.

To summarize, tamoxifen appears to have both estrogenic (atherosclerosis extent, plasma lipids) and antiestrogenic (vascular reactivity) effects on the cardiovascular system in nonhuman primates. However, if one considers the results of all studies (both human and nonhuman primates), it appears that the net effect of tamoxifen is a lowering of the risk of cardiovascular disease, despite a lack of improvement in coronary vascular reactivity.

Experimental Considerations
Several experimental procedures must be considered in the interpretation of these data. Acetylcholine and nitroglycerin are test substances only, and any alteration of dilation in response to either agent can be interpreted only as an index of epicardial coronary artery function. Acetylcholine stimulates the release of vasodilator substances from normally functioning endothelial cells.^{15 23} Disruption of the endothelium causes acetylcholine to act directly on the smooth muscle cells, resulting in vasoconstriction. Therefore, acetylcholine is widely used as an indicator of endothelial function. Coronary artery responses to acetylcholine are similar to those reported in test subjects in response to normal daily stimuli such as anxiety or exercise.²³ Nitroglycerin causes vasodilation by acting directly on smooth muscle cells and thus is a good measure of endothelium-independent dilator capacity. The control group of animals in this study did not show a strong vasodilator response to nitroglycerin, but the arteries did recover from constriction and returned to slightly greater than their initial control diameter. The greatest degree of vasodilation was seen in the CEE group. It is possible that the arteries from animals in all other treatment groups, which had constricted during the acetylcholine infusions, had not fully recovered their ability to dilate.

The anesthetics used in this experiment (ketamine and butorphanol) were chosen because they are relatively short-lived and do not significantly reduce autonomic reflexes. We cannot rule out the possibility that these agents may have had anticholinergic actions; however, they did not increase heart rate.

We chose not to preconstrict arteries in this experiment. Rather, baseline (control) measurements were made before each drug infusion. Baseline diameters were similar among groups. Therefore, we believe that any effects of anesthesia on coronary artery diameter were similar among treatment groups.

The doses of CEEs and tamoxifen used were calculated to mimic the doses taken by women. A major assumption is that the monkeys received the correct dose and metabolized the drugs in a manner similar to women. Plasma samples were taken 4 hours after consumption of the drugs. Certainly, the plasma concentrations of these three compounds were similar to those of women taking these compounds. However, it cannot be ruled out that the overall kinetics of metabolism are different between species. It is unclear whether or to what extent different kinetics would alter the experimental results.

In female cynomolgus monkeys, diameters of the left circumflex coronary artery average 1 mm. This small diameter pushes the limits of QCA sensitivity. We have previously published²⁶ data on the reproducibility and sensitivity of QCA in monkeys and believe that we can reliably measure the percent differences in diameter presented in this report. Furthermore, we cannot rule out that small changes in heart rate and blood pressure during infusion of agonist may have affected results.

A larger question is whether the modest percent changes in diameter are significant physiologically. In the context of risk of vasospasm, they probably are not. However, one could interpret these changes as a barometer of overall vascular (endothelial?) function. If so, there are numerous ways in which vascular function, especially as it relates to endothelial function, may contribute to the pathogenesis of atherosclerosis and plaque rupture.

Results of the present study indicate that, unlike estrogen, tamoxifen (a mixed estrogen agonist/antagonist) does not improve impaired dilator responses of large epicardial coronary arteries to acetylcholine in atherosclerotic primates. Therefore, under these conditions, tamoxifen has estrogen-antagonist properties. This contrasts with the estrogen-agonist effects of tamoxifen on atherogenesis. Thus, the effect of tamoxifen on vascular reactivity appears to be unrelated to its effects on atherogenesis and may suggest two modes of action (both agonist and antagonist) on the vessel wall. Although not conclusive, these data imply that the estrogen receptor may play a role in modulating the effect of estrogen agonists on vascular reactivity.

	Selected Abbreviations and Acronyms

 

CEE = conjugated equine estrogen HDL-C = HDL cholesterol QCA = quantitative coronary angiography TPC = total plasma cholesterol

	Acknowledgments

 
This study was supported in part by grants P01-HL-45666 (Dr Adams) and HL-49085 (Dr Williams) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. The authors thank Jamie Fox for technical assistance and Karen Potvin Klein for editorial assistance.

Received July 15, 1996; revision received November 25, 1996; accepted December 16, 1996.

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