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Division of Cardiology (C.D., J.M., K.B.), University of Michigan, Ann Arbor, Michigan 48109; Cardiology Section (C.D., A.B.), Veterans Affairs Medical Center, Ann Arbor, Michigan 48105; Nuclear Medicine (O.M.), Wayne State University Positron Emission Tomography Center, Detroit, Michigan 48201; and Preventive Cardiology (L.M.), Columbia University, New York, New York 10032
Address all correspondence and requests for reprints to: Dr. Claire Duvernoy, Assistant Professor of Medicine, Division of Cardiology, University of Michigan, Veterans Affairs Medical Center, 2215 Fuller Road, Box 111A, Ann Arbor, Michigan 48105-2399. E-mail: duvernoy{at}umich.edu.
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Abstract |
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We performed dynamic [13N]ammonia positron emission tomography in 15 postmenopausal women in a 7-month placebo-controlled crossover trial of continuous conjugated equine estrogen/cyclical micronized progesterone. MBF was measured at rest, after sympathetic stimulation with the cold pressor test (CPT), and after iv adenosine infusion, to determine baseline, endothelium-dependent, and maximal flows, respectively.
Response to CPT was neutral in all women at baseline (–0.51 ± 27%). Adenosine induced a marked increase in MBF (161 ± 111%). Treatment with 3 months of combined estrogen/progestin CHT did not change CPT or adenosine MBF responses. Myocardial flow reserve was unchanged as well.
In this group of postmenopausal women at higher cardiovascular risk, no association was found between CHT assignment and change in MBF. Further study is needed to clarify the effects of CHT on the endothelium of women with presumably diseased vasculature.
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Introduction |
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- and ß-adrenergic stimulation, and leads to vasodilation. Without an intact vascular endothelial layer, sympathetic stimulation leads to paradoxical vasoconstriction (2). Zeiher et al. (2) have shown that a noninvasive sympathetic stimulus such as the cold pressor test (CPT) can be used to assess the functional integrity of the coronary vascular endothelium. Exogenous adenosine exerts direct vasodilatory effects on vascular smooth muscle cells and exerts NO-mediated effects on the vascular endothelium as well (3). Adenosine effects can be characterized as a composite index of vascular reactivity; clinically, the agent is used to assess maximal myocardial blood flow (MBF) and myocardial flow reserve (MFR). Coronary heart disease (CHD) risk factors, such as hypercholesterolemia, hypertension, smoking, family history of premature CHD, and obesity, have all been associated with impaired vascular reactivity, indicating abnormal endothelial function. Because invasive coronary flow measurements are difficult to justify in healthy women, we sought to use a noninvasive technique to measure microcirculatory vascular reactivity and MBF. Dynamic positron emission tomography (PET) in combination with [13N]ammonia imaging can accurately measure MBF noninvasively. The aging process and postmenopausal status itself have been shown to adversely affect vascular endothelial reactivity (4). Estrogen has been shown to improve endothelial function in both the peripheral circulation and in coronary arteries in postmenopausal women (5). However, randomized controlled studies have shown that estrogen/progestin hormone therapy (HT) using conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) does not reduce cardiovascular events, and in fact may increase the risk of myocardial infarction and CHD events (6, 7). We sought to further delineate the effects of a different HT combination, CEE plus micronized progesterone (MP), on the myocardium in a primary prevention setting. We chose these agents based on data showing that MP does not attenuate estrogen-mediated peripheral vasodilation (8) and has more favorable effects on lipid parameters than does MPA (9). Purpose
To determine whether coronary microcirculation can be affected by short-term CHT, we performed PET in a randomized, placebo-controlled, double-blinded crossover design trial of 15 postmenopausal women with risk factors for CHD.
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Subjects and Methods |
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Fifteen postmenopausal women with elevated total cholesterol and at least one other risk factor for CHD were recruited for this study. All subjects were without HT for at least 3 months before enrollment. Screening dobutamine stress echocardiograms were performed to evaluate for significant CHD, and were normal in all study subjects enrolled. Postmenopausal status was confirmed by lack of menses for at least 12 months as well as baseline estradiol levels. Baseline patient characteristics, including preexisting medications, are shown in Table 1
. Exclusion criteria included prior recent (
3 month) HT or initiation of lipid-lowering therapy, personal or family history of breast cancer, and history of gynecologic malignancy. After enrollment, patients underwent dynamic [13N]ammonia PET studies to measure MBF at rest, in response to sympathetic stimulation with the CPT, and after maximal coronary vasodilation using iv adenosine. The subjects were then randomized to 3 months of continuous CEE (0.625 mg/d) plus MP (200 mg/d) on d 1–12 of each month, or placebo. At the end of the initial 3-month period, the subjects returned for repeat PET studies (97 ± 17 d later). They then had a 1-month washout phase, followed by crossover to the opposite therapy (placebo or CHT) for another 3-month period, and a final set of PET studies at the end of the second 3-month period (125 ± 8 d later). PET studies were always scheduled during the last phase of the month when patients were taking estrogen only, to standardize hormone effects throughout the study. The study protocol was approved by the institutional review boards of the University of Michigan and of the Ann Arbor Veterans Affairs Medical Center. Each patient gave informed consent before being enrolled in the study.
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Subjects came to the PET suite after an overnight fast, and were without caffeine for 12 h and without vasoactive medications for 24 h before PET. [13N]Ammonia was synthesized by the 16O(p,)13N reaction as described by Gelbard et al. (10). Dynamic PET measurements were performed using a whole-body PET scanner (ECAT 931 or Exact HR+; Siemens, New York, NY), which allows simultaneous acquisition of 15 contiguous transaxial images. After placement in the PET scanner, a scout scan was obtained to align the heart within the field of view using 2 mCi [13N]ammonia. The patient’s position with respect to the camera was checked by a cross-shaped laser beam that was aligned with pen marks on the patient’s skin. A 15-min transmission scan was then acquired to correct for photon attenuation using retractable germanium-68 ring sources. After completion of the transmission scan, 20 mCi [13N]ammonia diluted in 10 cc normal saline was administered as a slow bolus over 30 sec using a volumetric pump (Pump 33; Harvard Apparatus, Holliston, MA) via an iv line. At study onset, dynamic scan acquisition was initiated with varying frame duration (12 x 10 sec/4 x 15 sec/4 x 30 sec/3 x 300 sec). Total scanning time was 20 min.
After acquisition of the baseline [13N]ammonia scan, at least 50 min was allowed for [13N]ammonia decay (physical half-life, 9.9 min). During this time, [13N]ammonia activity decayed to <3% of its initial activity.
For the CPT, the subject’s hand was placed in an ice-water slurry for 2.5 min. [13N]Ammonia (20 mCi) was administered after 90 sec in the ice bath, and images were obtained in a fashion identical with that of the baseline study. Heart rate and blood pressure were monitored at 1-min intervals during CPT.
Fifty minutes later, adenosine was infused iv at 0.14 mg/kg·min over 6 min using a syringe pump (Graseby 3400; Watford, Herts, UK). Heart rate and blood pressure were monitored at 1-min intervals during the first 10 min of the study and at the conclusion of scanning. Rate-pressure product (RPP) for the scans was calculated as heart rate multiplied by systolic blood pressure divided by 100. Twelve-lead electrocardiograms were continuously monitored. Three minutes after onset of the adenosine infusion, 20 mCi [13N]ammonia was administered. The PET data acquisition protocol was identical with that of the baseline study.
Image analysis and quantification of blood flow
PET data analysis was performed as previously reported (11, 12) by one or two experienced observers, each of whom evaluated all rest/stress studies. The validity of this method has been assessed by members of the PET study group; interobserver variability has been shown to be excellent, with an r value of 0.96 (12). Interstudy variability is good when resting MBF is corrected for changes in the RPP (r = 0.69), and is good as well for maximal MBF during adenosine stress (r = 0.65) (12).
MBF was measured at rest, during CPT, and after adenosine. To account for interindividual differences in cardiac work, MBF at rest and during CPT was normalized to cardiac work by dividing MBF by the RPP and multiplying by 10,000. Because adenosine uncouples flow from cardiac work, maximal hyperemic MBF was not normalized to the RPP. MFR was defined as the ratio between MBF in response to adenosine and MBF at rest.
Statistical analysis
No good published data exist for the comparison of MBF changes in response to HT compared with placebo. However, a study comparing MBF rate during CPT before and after L-arginine reported the flow rates of 0.80 ml/g·min (SD = 0.16) for before treatment and 1.06 ml/g·min (SD = 0.16) after treatment (13). Our own pilot data in four women using CEE/MP gave mean MBF normalized to RPP during CPT of 0.85 ml/g·min (SD = 0.16) before treatment and 1.07 ml/g·min (SD = 0.16) after 3 months of CEE/MP treatment. Therefore, using a conservative absolute change in MBF of 0.22, with the previously reported interstudy variability, we calculated a study sample size of 13 subjects required to achieve 90% power to detect an increase in MBF during CPT of 0.22 ml/g·min with CEE/MP treatment. Study results are presented as summary statistics of groups, given as mean ± SD. A two-tailed Student t test with equal variances was used to compare mean values between study phases. A value of P < 0.05 was considered significant. Microsoft Excel (Microsoft, Redmond, WA) was used for initial comparisons. Correlations were assessed for using SPSS for Windows, version 11.0.
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Results |
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gives baseline characteristics of the study participants; these women were a relatively overweight group with multiple risk factors. All of the women had suboptimal cholesterol levels; two thirds had a family history of heart disease, and one third were smokers and/or had elevated blood pressure. Diabetes was not common in the study group; only two women in the group were being treated for it. Each woman had between two and four traditional risk factors for CHD, giving them a 10-yr CHD hard-event risk between 15 and 30%, according to Framingham risk scores (14). Laboratory, hemodynamic, and blood flow results
Lipid and hormone values are given in Table 2. Baseline total cholesterol levels were 226 ± 45 mg/dl, and did not change significantly during the study. Baseline low-density lipoprotein cholesterol levels (140 ± 27 mg/dl), triglycerides (185 ± 136 mg/dl), and high-density lipoprotein cholesterol levels (48 ± 15 mg/dl) also showed no significant changes during the two phases of the study. Estrogen levels changed appropriately during the two phases of the study, from 15 ± 7 pg/ml at baseline, to 56 ± 33 pg/ml during the hormone phase, and down to 20 ± 15 pg/ml during the placebo phase. Progesterone levels rose during the hormone phase of the study, although the changes did not reach statistical significance, likely reflecting the fact that women were tested during the estrogen-only phase of CHT. We found no significant correlation between cholesterol or cholesterol subfractions and MBF values during any phase of the study. Furthermore, no associations were detected between baseline MBF values and smoking status, history of hypertension, diabetes, or family history of premature coronary artery disease.
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gives peak heart rate, systolic blood pressure, RPP, and raw and normalized MBF values during each study phase. RPP rose significantly from rest during cold pressor testing (reflecting primarily a rise in peak systolic blood pressure) and during adenosine infusion in each study phase. At baseline, resting RPP was 9,794 ± 2,391; this value increased to 11,115 ± 2,487 during cold pressor testing (P = 0.0003), and to 14,230 ± 2,823 during adenosine infusion (P < 0.00001). Although MBF rose significantly during adenosine infusion (1.1 ± 0.2 ml/g·min to 2.6 ± 0.9 ml/g·min; P = 0.00002), reflecting maximal hyperemic blood flow, there was no significant change in MBF during CPT vs. MBF at rest (1.1 ± 0.2 ml/g·min to 1.1 ± 0.3 ml/g·min; P = 0.85). Overall, there were no significant differences in resting MBF, MBF during cold pressor testing, MBF after adenosine infusion, or MFR between baseline and CHT, placebo and CHT, or baseline and placebo study periods (Fig. 1).
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Discussion |
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Other authors have shown improvements in both peripheral and MBF responses to estrogen as well as CHT. Gilligan et al. (15, 16) showed a potentiating effect of 17ß-estradiol on endothelium-dependent coronary and brachial artery vasodilation in both healthy postmenopausal women, and those with risk factors for atherosclerosis. Riedel and Mugge (17) described both a dose-dependent estrogen-induced vasorelaxation in human coronary arteries in vitro, and significant coronary vasodilation in vivo in response to high-dose estradiol. Two more recently published studies documenting beneficial estrogen effects in animal models included the study by Rouleau et al. (18) of estrogen effects on MBF in ovariectomized rabbits, and the work of Clark et al. (19) showing increased coronary artery blood flow after 17ß-estradiol and CEE infusions in oophorectomized sheep. In postmenopausal women without cardiovascular risk factors, oral conjugated estrogen administration induced increases in coronary flow velocity reserve, as measured by transthoracic color Doppler echocardiography (20). Gerhard et al. (8) studied brachial artery responses to transdermal estradiol with vaginal MP in mildly hypercholesterolemic postmenopausal women without other risk factors, and found significant improvements in hyperemic flow during the CHT phase of their study. Hodis et al. (21) looked at carotid intima-media thickness as a surrogate marker for subclinical atherosclerosis, and reported that unopposed estradiol slowed intima-media thickness progression in hypercholesterolemic postmenopausal women, but only if they were not concomitantly taking lipid-lowering agents.
In contrast, Herrington et al. (22) analyzed brachial artery reactivity as a surrogate for coronary vascular reactivity in a longitudinal study of postmenopausal women with and without CHD risk factors. These authors found that endothelial dysfunction was unaltered by estrogen in women with risk factors, and unchanged in progressively older women. Only younger postmenopausal women without cardiovascular risk factors seemed to derive benefit (measured as improvement in reactive hyperemic response) from estrogen therapy. Several years earlier, Losordo et al. (23) showed that histopathological specimens of women with atherosclerosis were less likely to stain positively for estrogen receptors than arteries of women without atherosclerotic disease. These authors concluded that, especially in premenopausal women, atherosclerotic arteries showed distinctly diminished expression of estrogen receptors compared with normal coronary arteries. These findings were underscored by PET researchers at the University of California, Los Angeles, who looked at MBF responses to estrogen as measured by PET in several groups of women (24). The authors studied endothelium-dependent and -independent MBF in young, healthy women (who served as controls) as well as postmenopausal women taking hormone replacement therapy (HRT) and those not on HRT. The postmenopausal group was further subdivided into women with and without coronary risk factors. They found that the endothelium-dependent MBF response to cold pressor testing was abnormal in postmenopausal women with CHD risk factors with or without HRT, and that only in postmenopausal women without CHD risk factors did HRT seem to normalize MBF response to CPT. The authors concluded that irreversible endothelial dysfunction was present in postmenopausal women with risk factors for CHD, and that this impaired vascular reactivity could not be improved by estrogen therapy. Thus, these results correspond with our findings that estrogen administration did not improve endothelium-dependent MBF. Most recently, Di Carli et al. (25) compared MBF responses in healthy premenopausal women, premenopausal women with diabetes, and postmenopausal women. The authors found that premenopausal diabetics had impaired MBF responses to cold, similar in degree to postmenopausal women. They concluded that diabetes abolished the protective vascular effects normally noted in young women, and in effect induced premature aging of coronary vasculature.
All of these studies were limited by their nonrandomized nature; the studies of Campisi et al. (24) and Herrington et al. (22) included women taking multiple different formulations and combinations of HT for variable time periods. In our study, we sought to control for many of these variables to standardize our results as much as possible. We used one HT combination and studied all subjects at the same phase in their HT regimen. MP was chosen as the progestin agent because of previous studies showing more favorable effects on lipid profiles with this agent vs. MPA (9). Our study subjects were relatively obese postmenopausal women with multiple CHD risk factors, chosen to maximize benefit in a high-risk primary prevention model, if a benefit could be found. Our negative results indicate that, in this diverse group of women with subclinical, yet presumably established vascular disease, CHT is not associated with a vasodilatory effect, much as the earlier noninvasive studies have indicated. Furthermore, our results corroborate the negative outcomes seen in both the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI) (6, 7). These two landmark trials found no CHD risk reduction with combined HRT in either a primary or secondary prevention model. Previously demonstrated estrogen benefits did not translate to reduction in cardiovascular events despite favorable estrogen effects on lipid profiles and other biochemical markers of risk. Because of these initially inexplicable findings, recognition has emerged that estrogen effects are far more complex than previously thought, and that one potentially favorable effect may be counterbalanced or outweighed by other deleterious effects. Stabilization or reversal of age- and menopause-associated vascular endothelial dysfunction had been one estrogen-mediated mechanism through which CHD benefit was thought to occur. Our study indicates that this effect may not occur in a high-risk population, in whom other factors such as genetic predisposition, cigarette smoking, hypertension, and hypercholesterolemia may already have irreversibly impaired vascular function.
Study limitations
The women enrolled in this trial had multiple cardiovascular risk factors which may well have affected their MBF responses. However, we were not able to detect significant correlations between traditional risk factors and MBF at baseline. Although carefully designed and controlled, the current study enrolled a small number of patients and thus may have had insufficient power to detect an estrogen effect. We used a crossover design to increase the study’s power, and we saw no trends whatsoever, indicating that enrollment of more patients likely would not have altered the outcome. Another potential limiting factor was the 3-month duration of therapy, which may have been too short to effect any substantial change in vascular endothelial function. However, other studies showing estrogen-mediated improvement in vascular reactivity have been comparable or even shorter in duration. We did not assess MBF in a control population without risk factors because of limited resources, but normal volunteers have been tested using PET with CPT and PET with adenosine infusion (12, 26). In a recently published analysis of short- and long-term effects of vitamin C administration on CPT-mediated MBF as assessed by PET, Schindler et al. (26) showed that patients with risk factors such as smoking and hypertension had neutral or even negative responses to cold, whereas normal volunteers showed a marked increase in flow. We used a noninvasive technique to measure MBF along with a sympathetic stimulus that may have heterogeneous effects. Others have demonstrated that this technique is valid and reproducible (12, 27), however, and have shown that the CPT selectively targets NO release from the vascular endothelium (13), thus making it the best noninvasive tool to assess functional integrity of the vascular endothelium. Finally, we chose CEE with MP as our CHT regimen. This study does not permit conclusions about other CHT regimens—a synthetic estrogen such as estradiol may provide more consistent therapeutic levels and, by extension, greater vascular benefit. Our rationale for choosing CEE was that this agent is the most frequently used estrogen in the United States; we used MP for its more favorable effect on the lipid profile, as documented in the Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial (9), which compared multiple CHT strategies including CEE with cyclical or continuous MPA, and CEE with cyclical MP. Others (28, 29) have also shown that MPA attenuates the favorable effects seen with estrogen alone, and thus we hoped to avoid a potential loss in estrogen vasoprotective effects.
Conclusions
Our study showed that postmenopausal CHT using CEE and MP does not improve endothelium-dependent microcirculatory vascular reactivity or maximal MBF. These results are compatible with recent findings that estrogen/progestin HT does not reduce CHD risk, and with recent mechanistic studies indicating that endothelial function may be irreversibly impaired in postmenopausal women with risk factors for CHD.
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Acknowledgments |
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Footnotes |
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Abbreviations: CEE, Conjugated equine estrogen; CHD, coronary heart disease; CHT, combination HT; CPT, cold pressor test; HRT, hormone replacement therapy; HT, hormone therapy; MBF, myocardial blood flow; MFR, myocardial flow reserve; MP, micronized progesterone; MPA, medroxyprogesterone acetate; NO, nitric oxide; PET, positron emission tomography; RPP, rate-pressure product.
Received September 25, 2003.
Accepted March 1, 2004.
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