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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 649-659
Copyright © 1998 by The Endocrine Society

Original Studies

Ovarian Steroid Protection against Coronary Artery Hyperreactivity in Rhesus Monkeys1

Richard D. Minshall, Frank Z. Stanczyk, Koichi Miyagawa, Barry Uchida, Michael Axthelm, Miles Novy and Kent Hermsmeyer

Oregon Regional Primate Research Center (R.D.M., K.M., M.A., M.N., K.H.), Departments of Medicine (K.H.), Cell and Developmental Biology (K.H.), and Obstetrics and Gynecology (M.N.), and The Dotter Interventional Institute (B.U.), Oregon Health Sciences University, Portland, Oregon 97201; and the Department of Obstetrics and Gynecology, Women and Children’s Hospital, University of Southern California (F.Z.S.), Los Angeles, California 90033

Address all correspondence and requests for reprints to: Dr. Kent Hermsmeyer, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our hypothesis was that estrogen and progesterone modulate coronary artery reactivity in rhesus monkeys. Adult ovariectomized (ovx) monkeys were treated for 1, 2, or 4 wk with physiological concentrations of 17ß-estradiol (E2), natural progesterone (P), and/or therapeutic levels of medroxyprogesterone acetate (MPA). Steroid concentrations in venous blood, coronary artery estrogen receptor (ER) and progesterone receptor (PR) localization, and isolated vascular muscle cell (VMC) Ca2+ and protein kinase C responses to serotonin and U46619 (a thromboxane A2 mimetic) were measured. Ovx monkey VMC responses were hyperreactive, showing prolonged increases in intracellular Ca2+ and protein kinase C that correlated with exaggerated in vivo coronary artery vasoconstrictor responses. The hyperreactive Ca2+ responses were abolished by in vivo treatment with E2 and/or P. However, VMC from ovx monkeys treated with the combination of E2 and MPA or E2, P, and MPA remained hyperreactive to vasoconstrictor stimuli, suggesting that MPA negated the protective effects of E2. ER were detected primarily in interstitial and endothelial cells and a minor fraction of the VMC. PR were localized to coronary artery VMC and interstitial cell nuclei. In vivo treatment of ovx monkeys with E2 tended to up-regulate PR in VMC, but MPA appeared to down-regulate PR expression. These results suggest that E2 and P replacement decreases coronary artery reactivity through direct interactions with ER and PR in coronary artery VMC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR disease is now recognized to be more prevalent among postmenopausal women than any other segment of the female population, a statistic that has been linked to the hypoestrogenic state (1). Among women who are current hormone users, estrogen decreases the incidence of coronary heart disease, including atherosclerosis, myocardial infarction, and coronary vasospasm, reducing the total risk by as much as 50% (2, 3, 4, 5). Although the mechanisms of estrogen’s cardioprotective effects are not completely established, the potential mechanisms include 1) a favorable impact on the circulating lipid and lipoprotein profile (4, 6); 2) inhibition of lipoprotein oxidation (7); 3) a direct antiatherosclerotic effect in arteries (8, 9); 4) a beneficial effect on endothelium-dependent responses, i.e. augmenting vasodilator and antiplatelet aggregation factors, especially nitric oxide and prostacyclin, and attenuating endothelin-1-stimulated coronary artery contraction (10); 5) vasodilation by endothelium-independent mechanisms (11); and 6) an increase in aortic compliance and a direct inotropic action on the heart (12, 13).

Considerable attention has been given to the changes in lipid and lipoprotein profiles induced by hormone replacement therapy, as this concept may account for approximately 25% of the observed reduction in relative risk of death from coronary heart disease (1, 14). However, it was also shown that 17ß-estradiol (E2), alone or in combination with natural progesterone (P), effectively reduced plaque area in monkey coronary arteries caused by an atherogenic diet by 50%, independent of changes in total plasma cholesterol, lipoprotein cholesterol, apoprotein A-1 and B concentrations, HDL sub-fraction heterogeneity, and low density lipoprotein mol wt (8). Furthermore, Bourassa and co-workers (15) discovered that estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice, implying that estrogen has additional beneficial effects directly on the arterial wall. Thus, substantial evidence has accumulated to support the hypothesis that E2 and P act directly on blood vessels (16, 17, 18).

Both endothelial cell dysfunction and vascular muscle hyperreactivity, characterized by reduced production of vasodilators and increased responsiveness to vasoconstrictors, have been shown in humans and in animal models to be associated with the loss of estrogen (19, 20, 21). Studies in nonhuman primates demonstrate that E2 modifies endothelium-dependent and -independent vascular responses. In monkeys, an abnormal coronary vasoconstrictor response to acetylcholine can be converted to a vasodilator response by chronic E2 administration (22). However, the effect of E2 was diminished when it was administered in combination with medroxyprogesterone acetate (MPA), a synthetic progestin that is commonly used in hormone replacement regimens to counter the endometrial effects of unopposed E2 (23). Clinical studies of cardiovascular diseases such as angina, syndrome X, and acute myocardial infarction (2, 3, 19, 24) also suggest that E2 may act directly on the arterial wall and modulate vascular reactivity in humans, as has been found in monkeys (20, 21, 22, 23). In women with risk factors for atherosclerosis and evidence of impaired vascular function, E2 potentiates forearm vasodilatation in response to acetylcholine and sodium nitroprusside (25) and attenuates abnormal coronary artery responses to acetylcholine (26). Although the mechanisms of estrogen’s actions are not entirely clear, it appears that physiological levels of E2 regulate vascular reactivity by augmenting endothelial cell nitric oxide release (27, 28) and modulating vascular muscle reactivity to vasoconstrictors (11, 21).

The phenomenon of sex steroid modulation of vascular reactivity has recently been characterized in a nonhuman primate nonatherosclerotic model that simulates the postmenopausal condition in women (20). In this model, focal coronary artery constrictions that appear relevant to human coronary vasospasms are reliably induced pharmacologically in hypoestrogenic monkeys with serotonin (5-HT) and U46619, a thromboxane A2 mimetic, and are prevented when E2 in combination with P is given to ovariectomized (ovx) rhesus monkeys for 6 wk (20, 29). Furthermore, we have demonstrated that MPA added to the replacement regimen with E2 consistently negated the protective effects of E2 on coronary reactivity in vivo (29) and the agonist-stimulated Ca2+ and protein kinase C (PKC) responses of isolated coronary artery vascular muscle cells (VMC) in vitro (21). It was suggested that in hyperreactive VMC, PKC activation is required to maintain the pathophysiological sustained increase in intracellular Ca2+ (21).

In the present work, we have extended our previous in vivo observations to investigate the cellular mechanisms of ovarian hormone modulation of primate coronary artery VMC reactivity. In vivo coronary artery reactivity was correlated with 1) circulating ovarian steroid hormone levels, 2) intracellular Ca2+ and PKC responses of freshly dissociated VMC from coronary arteries of treated and control monkeys, and 3) expression of receptors for E2 (ER) and P (PR) in coronary artery sections taken from the same animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Adult female rhesus monkeys (Macaca mulatta), aged 7–21 yr (mean ± SEM, 13.2 ± 0.4 yr; n = 51) and weighing 4.0–9.3 kg (mean ± SEM, 6.15 ± 0.16 kg), were used in these studies. Monkeys were ovx at least 3 months before the study. All procedures were conducted consistent with the policies in the Guide for the Care and Use of Laboratory Animals, as approved by the Oregon Regional Primate Research Center animal care and use committee. Hormone replacement therapy consisted of subdermal implants of SILASTIC brand tubing (40 mm; Dow Corning, Midland, MI) containing 200 mg E2, 400 mg P, and/or 400 mg MPA for 1–4 wk. Our goal was to achieve a reproductive hormone status at the time of angiography and tissue isolation that would closely mimic that of untreated postmenopausal women or women treated with E2 and progestin replacement regimens. Target levels of 50–100 pg/mL for E2, 4–8 ng/mL for P, and 800-1600 pg/mL for MPA were based on levels that are effective by reproductive system indexes in women (30) and also in monkeys (30, 31). Monkey treatment groups and abbreviations were 1) ovx at least 3 months before examination (ovx; n = 7); 2) treatment with E2 for 2 wk followed by withdrawal of E2 for 2 wk (n = 7), 3) E2 for 2 wk (E2; n = 12), 4) E2 for 2 wk followed by natural P alone for 4 wk (P; n = 4), 5) E2 for 1 week and then E2 plus P for 1 week (E2+P; n = 8), 6) E2 for 1 week followed by E2 plus MPA for 1 week (E2+MPA; n = 9), and 7) E2 for 1 week followed by E2, P, and MPA for 1 week (E2+P+MPA; n = 4). In addition, coronary arteries and a single blood sample taken at necropsy from intact female monkeys, aged 8–20 yr (mean ± SEM, 12.0 ± 1.3 yr; n = 11) and weighing 4.2–7.5 kg (mean ± SEM, 5.6 ± 0.3 kg), were obtained from the Tissue Distribution Program at the Primate Center. These animals were divided into luteal phase (n = 4; P > 0.7 ng/mL) and follicular phase (n = 7; P < 0.7 ng/mL) groups.

Coronary vasospasm

Coronary artery vasospasm challenges were performed as described in detail previously (20, 29). The protocol of the in vivo vasospasm provocation stimulus was applied to each study animal, and recovery to physiological conditions for at least 1 h was allowed before isolating coronary artery muscle cells. In brief, the vasospasm protocol employs a series of 1-mL intracoronary injections of 5-HT (100 µmol/L) and U46619 (1 µmol/L), first alone and then in combination, and finally in conjunction with endothelin-1 or angiotensin II. The combination challenge was repeated a total of at least five times in monkeys with no vasospasm. A positive vasospasm provocation test was determined as contractions of epicardial coronary arteries that include focal or diffuse areas of constriction to less than 25% of the control diameter, followed by adjacent downstream dilation, persisting for at least 5 min (20). Coronary artery diameters were measured from number coded angiograms and summarized along with the electrocardiogram recordings and hemodynamic measurements (20) to obtain a complete evaluation of monkey coronary artery responses to the drug challenges.

RIA

Venous blood samples (5 mL) were collected before the onset of hormone treatment and at weekly intervals until angiography and death of the animal. E2, P, and MPA circulating levels were measured by RIA as previously described (30, 33, 34).

VMC preparation

VMC from the left anterior descending, circumflex, and right coronary arteries were isolated and studied immediately as freshly dispersed single cells (35). Coronary artery pieces were dissociated by treatment for 15–30 min in Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS) containing type II collagenase (Worthington Biochemical Corp., Freehold, NJ; 0.37 mg/mL), protease type XXIV (bacterial; 0.07 mg/mL), BSA (0.67 mg/mL), and trypsin inhibitor (1.33 mg/mL). The dispersed cells were collected by centrifugation for 1 min at 200 x g, resuspended in Dulbecco’s PBS, and held at 4 C until used. These acutely dissociated VMC maintained the characteristics of the source tissue, including contraction, relaxation, and receptor integrity, similar to those of our primary cultures (36). Phenol red was excluded from all solutions because of known actions on estrogen receptors (37).

VMC Ca2+ and PKC responses

Measurements of intracellular Ca2+ and PKC fluorescence intensity and mobilization/translocation (21, 38) were made from digitized images of freshly dissociated monkey coronary artery VMC 2–8 h after vasospasm provocation in these same monkeys. Ca2+ and PKC images (total of 15 5-s shuttered exposures) were acquired simultaneously via filter changes and a staggered time course using a Zeiss Axiovert microscope with a C-Apochromat 40X/1.2 W Korr confocal-design objective (Zeiss, New York, NY). Fluorescence digital image analysis was carried out with very low light levels, using multiple (n = 4–7) layers of filtering, and an ultrahigh sensitivity (photon counting) microchannel plate (VIM, Hamamatsu Phototonics, Japan) camera to avoid fluorescence fading. Data acquired with the Hamamatsu VIM camera were controlled and processed with Image Pro software customized with Visual Basic (Microsoft, Redmond, WA) for our studies using a high speed Pentium personal computer with an Imagraph PCI local bus real-time digitizer (Micron Electronics, Nampa, IN). The fluorescent images were analyzed, corrected for background and camera noise, normalized to calibration standards, and expressed as a percentage of the control value. Data were digitally stored on magneto-optical disks, analyzed by region of interest pixel count integrations, compared by ANOVA, and color mapped for visual displays of intracellular Ca2+ and PKC distribution.

Ca2+ fluorescence

Freshly dispersed VMC were placed in a 300-µL laminar flow chamber (39). Ionic solution for mammals [ISM2; containing 100 mmol/L NaCl, 4 mmol/L NaHCO3, 0.5 mmol/L NaH2PO4, 4.7 mmol/L KCl, 1.8 mmol/L CaCl2, 0.41 mmol/L MgCl2, 0.41 mmol/L MgSO4, 50 mmol/L HEPES (pH 7.37 at 22 C), and 5.5 mmol/L dextrose] continuously suffused the cells (at 1 mL/min). Ca2+ was observed in single live VMC using the cell-permeant fluorescent Ca2+ indicator fluo3 acetoxymethyl ester (fluo3). After a 15-min equilibration period in ISM2, VMC were loaded at room temperature with 0.3–1.0 µmol/L fluo3 for 15 min, washed for 5 min, and then stimulated for 15 s with 1 µmol/L serotonin and 10 nmol/L U46619. After 15 s under no-flow conditions, continuous flow of ISM2 at 1 mL/min was reinstated to wash out unbound constrictors. Fluorescent Ca2+ images (excitation filter, 487 nm; dichroic mirror, 505 nm; emission filter, 515 nm) were acquired at the end of the 5-min washout period (time zero control) and 1, 2, 5, 10, 15, 20, and 30 min after stimulation. Illumination was limited by shutters to eight (total) exposures of 5-s duration. The fluorescence intensity of each image (whole cell thickness) was calculated relative to the predrug baseline (percentage of control value).

Measurements of intracellular Ca2+ with higher affinity indicators (e.g. fura-2, indo-1, or Ca2+-green-1) inescapably influence the intracellular Ca2+ concentration that is being measured by strongly binding Ca2+, as these indicators are also strong Ca2+-sequestering agents. For this reason, and to maximize the dynamic range (<=200 times), these experiments were designed with lower affinity fluo3 (Kd = 0.3 µmol/L). Fluo-3 has additional virtues of the greatest dynamic range, low fluorescence at basal Ca2+ concentrations (40), and thus greater reliability of localization, kinetics, and Ca2+ transient amplitudes.

PKC fluorescence

To determine relative PKC responses to vasoconstrictor stimuli, individual VMC were probed with the fluorescent PKC probe hypericin (21, 41). Detection of PKC in VMC was performed essentially as described previously for 12{alpha}-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-5-indacene-8-propionyl)-phorbol 13ß-acetate (38, 42). Live cell PKC fluorescence was determined with 30–300 nmol/L hypericin (concentrations well below the 3 µmol/L or greater levels that inhibit PKC). Hypericin was loaded for 5 min, and excess indicator was washed out for 5 min (the last 5 min of the fluo3 loading). Using the end of the 5-min washout of fluo3 and hypericin as time zero, fluorescent images were acquired immediately before and 3, 4, 9, 16, 21, and 31 min after stimulation. Hypericin fluorescent images were obtained with 5-s duration shuttered exposures with the following filters: 535-nm excitation, 560-nm dichroic mirror, and 590-nm long pass emission filter.

Immunocytochemical detection of steroid receptors

Immunocytochemical detection of nuclear ER and PR was conducted on fresh-frozen coronary artery sections using methods described previously (32). In brief, monkey coronary arteries were microwave stabilized and frozen in liquid propane. Cryostat sections (10 µm) were thaw-mounted on gelatin-coated slides, placed on ice, and microwaved for 2 s. Mounted sections were fixed in 0.2% picric acid-2% paraformaldehyde in PBS and 1.5% polyvinylpyrrolidine for 10 min at 25 C and for 2 min at 4 C in 85% ethanol and 1.5% polyvinylpyrrolidine and washed extensively. The sections were then treated for 20 min with nonspecific serum (goat), avidin-biotin blocked, and incubated overnight at 4 C with 0.5–2 µg/mL ER-21, 0.03–2 µg/mL rat anti-PR monoclonal antibody, JZB39 (43), or a 1:100 dilution of a monoclonal antibody against smooth muscle myosin heavy chain isoforms SM-1 and SM-2, clone 9A9 G4-G9 (44) (provided by Dr. Gary Owens, University of Virginia Medical Center, Charlottesville, VA). JZB39 and ER-21 were provided by Dr. Geoffrey Greene, University of Chicago (Chicago, IL). Nonspecific monoclonal antibody staining was determined using the same concentrations of an unrelated monoclonal antibody (rat or mouse anti-Timothy) as primary antibody. Specific ER-21 immunostaining was determined by preabsorbing the antibody with an equivalent amount (weight/weight) of the immunizing peptide. After the primary antibody incubation, the slides were again blocked with goat serum for 20 min, incubated with antimouse or antirat IgG biotinylated second antibody (30 min at 25 C), and detected with an avidin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA). The sections were counterstained with eosin, dehydrated, cleared with 100% xylene, and mounted in Permount (Fisher Scientific, Fairlawn, NJ). All sections shown in Figs. 4Go.1 and 4.2 were processed under identical conditions.



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  		Figure 4. ER and PR immunostaining of coronary artery sections. Coronary artery serial sections from an intact female rhesus monkey (4.1) were stained with hematoxylin and eosin (4.1a) and a 1:100 dilution of monoclonal antibody 9A9 that specifically labels SM-1 and SM-2 smooth muscle myosin heavy chain isoforms (4.1b) to characterize the localization of nuclei and specific cell types within the vessel wall. With 0.5 µg/mL ER-21 (4.1c), positive ER immunostaining of endothelial and interstitial cells and some VMC was observed. No staining was observed after preabsorbing ER-21 with an equivalent amount (wt/wt) of the immunizing peptide (4.1d). Sections from the intact monkey stained with 1 µg/mL JZB39 (4.1e) showed abundant PR immunostaining of VMC and some interstitial cells. By comparison, using 1 µg/mL rat anti-Timothy (4.1f), a nonspecific monoclonal antibody, no nuclear staining was observed.

(continued). Coronary artery PR immunostaining of ovx, E2+P, and E2+MPA monkeys is shown in 4.2. Fresh-frozen ovx (4.2a–c), E2+P (4.2d–f), and E2+MPA (4.2g–i) monkey coronary artery sections were stained with the rat antihuman PR monoclonal antibody JZB39 at three concentrations (0.3, 0.1, and 0.03 µg/mL), followed by secondary antibody labeling and visualization by avidin-biotin complex and diaminobenzidine. The dose of anti-PR antibody at which specific PR staining disappears was used as a relative index of PR expression. At 0.1 µg/mL, PR staining was no longer observed in the ovx monkey coronary artery (4.2b), but was still present in the E2+P (4.2e) and E2+MPA (4.2h) monkey coronary artery sections. At 0.03 µg/mL, the lowest concentration studied, slightly less PR immunostaining in MPA-treated monkeys (4.2i) was observed compared to that in E2+P monkeys, although additional studies are required to quantitate any real differences. Note the specific localization of nuclear PR staining in the muscle cells in the E2+P and E2+MPA sections and the relative paucity of immunostaining in sections from an ovx monkey. The pictures shown are representative of several experiments performed. Original magnification, x400.

 
Each slide was graded for ER or PR staining by the first author and a laboratory technician. A score of 0–3 was assigned based on the percentage of endothelial, vascular muscle, and interstitial cell nuclei that showed discrete brown nuclear staining. The four categories corresponded to 0%, 1–25%, 26–75%, and more than 75% of the nuclei labeled in each cell group. The two independent evaluations of each section were compared, and differences were resolved during joint examination.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The experiments described were designed to correlate in vivo steroid hormone concentrations with indexes of vascular reactivity (intracellular Ca2+ and PKC responses of freshly isolated coronary artery VMC in vitro) to vasoconstrictor stimuli and with localization of ER and PR expression in the coronary arteries. The primary findings in female rhesus macaque epicardial coronary arteries are 1) in vivo treatments resulting in physiological levels of E2 and P (both alone and in combination) restored the reactivity of isolated VMC from ovx monkeys to the normal state observed in VMC from intact female monkeys; 2) E2 and P treatment in combination with MPA were associated with hyperreactive VMC Ca2+ and PKC responses; 3) ER were localized in endothelial and interstitial cell nuclei and were less abundant in vascular muscle cells; and 4) PR were found in VMC, but were relatively absent from the endothelium and adventitia.

Serum steroid levels

E2 and P target levels achieved with subdermal SILASTIC brand implants are shown in Table 1Go. E2 levels significantly increased from approximately 5 pg/mL in E2 withdrawal and ovx monkeys to 61–112 pg/mL in all E2-treated animals (P < 0.05 vs. ovx). P levels were significantly increased from approximately 0.1 ng/mL in ovx monkeys to 5.9–7.4 ng/mL in E2+P or P animals (P < 0.05 vs. ovx). The levels of E2 and P achieved via SILASTIC brand implants were similar to the measured levels in monkeys in the follicular and luteal phases of the menstrual cycle, respectively (Table 1Go). MPA levels achieved in monkeys ranged from 0.25–1.70 ng/mL. The average levels (1.05 ± 0.13 ng/mL; n = 13) were in the target range of 0.80–1.60 ng/mL. This amount of MPA is 5–10 times less than the peak levels observed in women taking Provera (MPA), but is similar to the steady state levels achieved (see Discussion).


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  		Table 1. Rhesus monkey serum steroid hormone levels and vasospasm incidence

 
Incidence of coronary vasospasm

Table 1Go shows the striking difference in the incidence of vasospasm (as defined in Materials and Methods) in untreated ovx monkeys compared to that in monkeys treated with SILASTIC brand implants containing E2 and/or P. In 83% (5 of 6) of the untreated ovx monkeys and 83% (5 of 6) of the monkeys treated for 2 wk and then withdrawn from E2 for 2 wk, vasospasms could be drug induced with intracoronary injections of the pathophysiological vasoconstrictor combination of 5-HT and U46619. Physiological blood levels of E2 and P, both alone and in combination, that were typical of the levels observed in monkeys in the follicular and luteal phases of the menstrual cycle were protective (Table 1Go). Of 11 monkeys challenged that were treated with E2 alone, 10 were protected from developing coronary vasospasm, whereas 3 of 4 monkeys treated with P alone and 7 of 8 monkeys treated with the combination of E2+P were protected. However, treatment of monkeys with MPA in place of natural P, although in the presence of E2, did not protect and resulted in 9 of 9 animals that developed coronary vasospasm with less repetitions of vasoconstrictor challenge. In addition, 4 of 4 monkeys pretreated with all 3 steroids, E2, P, and MPA, also showed coronary vasospasm in response to 5-HT+U46619, indicating that 1.2 ± 0.2 ng/mL MPA negated the protective effect of 61 ± 23 pg/mL E2 even in the presence of 7.4 ± 0.8 ng/mL P.

VMC reactivity

Coronary artery VMC derived from E2 withdrawal/ovx and MPA-treated monkey groups showed hyperreactive intracellular Ca2+ (Fig. 1Go) and PKC responses (Fig. 2Go) to the combined stimuli of 1 µmol/L 5-HT and 10 nmol/L U46619 compared to VMC from intact monkeys, which showed only small, transient responses. In VMC from E2 withdrawal/ovx monkeys (pooled because the serum E2 and P levels, incidence of vasospasm, and reactivity of the isolated cells were identical), Ca2+ levels (whole cell averages) increased to 139% of their prestimulation levels by 30 min after stimulation (P < 0.05 vs. intact monkey VMC; Table 2Go). VMC from E2+P monkeys minimally responded to 5-HT+U46619 and were not statistically different from intact (control) monkey VMC responses. The initial Ca2+ transients that occurred before 1 min are not shown. Also, in VMC from the P alone and E2 alone monkeys, Ca2+ levels only transiently increased at the early time points (1, 2, and 5 min; Fig. 1Go). VMC from animals treated with E2 and MPA in both the presence and absence of natural P showed hyperreactive intracellular Ca2+ levels of prolonged duration in response to the brief 15-s 5-HT+U46619 stimulus. Ca2+ responses from MPA-treated monkeys increased to 159 ± 23% and 191 ± 31% of their respective control levels, persisted for the criterion duration of 15 min after stimulation (Fig. 1Go), and then declined to 139 ± 10% and 115 ± 10% of the prestimulation levels by 30 min. E2 or E2+P treatment in vivo did not result in Ca2+ increases by the vasospasm stimulus at 10 or 15 min or any later time point.



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  		Figure 1. Ca2+ fluorescence in freshly dispersed coronary artery VMC as a function of steroid hormone treatment. Whole cell Ca2+, as indicated by fluo3 fluorescence intensity, was measured from images recorded immediately before (time zero) and 1, 2, 5, 10, 15, 20, and 30 min after 15-s stimulation with 1 µmol/L serotonin and 10 nmol/L U46619. The data are the mean ± SEM of the fluo3 fluorescence intensity from experiments on four to seven VMC from each group, which were calculated as the percent change from the control (time zero) fluorescence image of each individual VMC. Fluo3 fluorescence in VMC from E2 withdrawal/ovx (E2 WD/ovx; n = 7 cells), E2+MPA (n = 5), and E2+P+MPA (n = 5) monkeys showed amplified Ca2+ responses with sustained time courses (with increases persisting beyond 30 min) that differed significantly (P < 0.05) from those in intact monkey VMC (n = 6). Ca2+ responses in VMC form (n = 4), P (n = 4), and E2+P (n = 5) groups did not significantly differ from those in intact monkey VMC.

 


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  		Figure 2. PKC fluorescence in freshly dispersed coronary artery VMC as a function of steroid hormone treatment. Whole cell PKC, as indicated by hypericin fluorescence intensity, was measured in freshly isolated VMC (concurrently with Ca2+) immediately before (time zero) and 3, 4, 9, 16, 21, and 31 min after stimulation with 1 µmol/L serotonin and 10 nmol/L U46619. The averaged data represent experiments from three to seven VMC from each monkey treatment group. Note the sustained increase in PKC in VMC from monkeys treated with E2+MPA (n = 5), E2+P+MPA (n = 5), and E2 withdrawal/ovx monkeys (E2 WD/ovx; n = 7) compared to that in VMC from intact monkeys (n = 6) and those treated with E2 (n = 3), P (n = 5), or E2+P (n = 5) replacement regimens.

 

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  		Table 2. Maximum Ca2+ and PKC responses in untreated and treated monkey coronary artery VMC

 
PKC levels in VMC from E2 withdrawal/ovx monkeys, as indicated by hypericin fluorescence intensity, increased in response to vasoconstrictor stimulation to 122 ± 7% of the prestimulated baseline. As with Ca2+, the increase in fluorescence intensity was maintained for up to 31 min, whereas VMC from intact monkey coronary arteries showed no change or a slight decrease in PKC fluorescence intensity through the 31-min time course (Fig. 2Go). PKC fluorescence intensity in VMC from E2+MPA and E2+P+MPA monkeys increased to 131 ± 14% and 119 ± 10% of the control value at the criterion time point 16 min after stimulation (Fig. 2Go).

Treatment of ovx monkeys in vivo with E2, P, or both, but not with E2+MPA, restored normal Ca2+ and PKC reactivity of VMC, similar to that observed in intact female monkey coronary artery VMC. As shown in Table 2Go, which summarizes the peak Ca2+ and PKC fluorescence responses to 5-HT+U46619 stimulation, hyperreactive Ca2+ and PKC responses reached a maximum of 139 ± 7% and 122 ± 7% of baseline in the E2 withdrawal monkeys (P < 0.05 vs. intact monkey VMC peak responses). Significant PKC increases were not present in VMC from intact monkeys or ovx monkeys treated with E2 and/or P. E2+MPA or E2+P+MPA ovx monkey VMC Ca2+ and PKC peak responses were significantly increased compared to those of VMC from intact animals (P < 0.05).

Comparisons of the digitally recorded and corrected example images from four of the groups are shown in Fig. 3Go. Pseudocolor maps of Ca2+ (second and third columns) and PKC (fourth and fifth columns) in freshly dispersed VMC from monkeys treated for 2 wk with E2 followed by withdrawal of E2 for 2 wk (Fig. 3AGo), continued treatment with E2 for 2 wk (Fig. 3BGo), treatment with E2+P for 2 wk (Fig. 3CGo), or treatment with E2+MPA for 2 wk (Fig. 3DGo), contrasting protected and hyperreactive states of VMC, are shown. Time zero Nomarski nonfluorescent images are shown in the first column, followed by fluorescent images of Ca2+ (0 and 15 min after 15-s stimulation with 1 µmol/L 5-HT and 10 nmol/L U46619) and PKC (0 and 16 min after stimulation). VMC from E2 withdrawal animals (Fig. 3AGo) showed prolonged increases in Ca2+ and PKC that were not found after E2 treatment (Fig. 3BGo). Treatment of monkeys with E2+P (Fig. 3CGo) also produced less reactive VMC Ca2+ and PKC responses. However, monkeys treated with E2+MPA (Fig. 3DGo) were not protected, as evidenced by the abnormally large increases in Ca2+ that were sustained for more than 20 min and the elevated PKC (hypericin fluorescence intensity) coincident with the late phase increase in Ca2+.



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  		Figure 3. Representative Ca2+ and PKC fluorescent images from freshly isolated monkey coronary artery VMC. Whole cell Ca2+ (fluo3 fluorescence) and PKC images (hypericin fluorescence) from monkey coronary artery VMC, contrasting the hyperreactive state (E2 withdrawal and E2+MPA treated) with the protected state (after E2 or E2+P replacement), are shown. In the left column, the brightfield Nomarski nonfluorescent image of each cell is shown, followed by the 0 and 15 min Ca2+ images (second and third columns) and 0 and 16 min PKC images (fourth and fifth columns). The color bar shows exponentially increasing Ca2+ and PKC levels from violet (lowest) to red (highest). Note the dramatically increased Ca2+ signals and activation of PKC 15 and 16 min after stimulation by 5-HT+U46619 in VMC from E2 withdrawal (A) and E2+MPA (D) monkeys compared to the little or no elevation in VMC from E2 (B) or E2+P monkeys (C).

 
In response to vasoconstrictor stimulation, PKC fluorescence intensity not only increased, but also shifted from central cytoplasmic locations before stimulation to peripheral sites near the plasma membrane 4–16 min after stimulation, and then back to central locations by 31 min. Ca2+ in hyperreactive cells (from ovx or MPA categories) increased centrally and was sustained for more than 15 min, whereas Ca2+ in protected VMC (from E2 or P categories) transiently increased in perinuclear regions between 1–5 min.

Coronary artery steroid receptor expression

Figure 4Go.1 shows the distribution, specificity, and relative degree of ER and PR immunostaining in a representative coronary artery section from an intact rhesus monkey. In Fig. 4Go.1a, a hematoxylin- and eosin-stained section of the left anterior descending coronary artery from an intact female monkey is shown, accompanied by specific staining of the vascular muscle layer with a smooth muscle myosin heavy chain monoclonal antibody (Fig. 4Go.1b). ER immunostaining of fresh-frozen coronary artery sections with polyclonal antiserum raised against the human ER (ER-21; 0.5 µg/mL) showed uniform positive labeling of endothelial and interstitial cell nuclei and relatively less labeling of VMC nuclei (Fig. 4Go.1c). The coronary artery endothelial cell, interstitial cell, and VMC ER staining using the rabbit polyclonal antibody ER-21 (0.5–2 µg/mL) did not differ among the intact, ovx, and MPA-treated monkeys (not shown). With ER-21, we found ER localized primarily to the nucleus, although some cytosolic staining was also detected. Both nuclear and cytosolic stainings were completely inhibited by preabsorbtion of the antibody with an equivalent amount (weight/weight) of the immunizing peptide (Fig. 4Go.1d).

With the monoclonal antibody JZB39 (1 µg/mL; Fig. 4Go.1e), we detected positive staining for PR specifically in VMC nuclei. A limited number of PR-positive nuclei were also detected in the adventitia surrounding the coronary vessels, in endothelial cells, and in ventricular muscle. Arterial muscle cell nuclei in fresh-frozen sections exposed to our negative control, a nonspecific rat monoclonal antibody (anti-Timothy; Fig. 4Go.1f), showed no staining.

Table 3Go shows PR immunostaining of coronary artery sections from four to seven animals in each of the ovx (including E2 withdrawal), E2+P, E2+MPA, and intact monkey groups. With 1 µg/mL JZB39, our impression was that there was relatively less PR expression in ovx (average score, 1.5 ± 0.29) and MPA-treated animals (0.86 ± 0.40) than in intact (2.67 ± 0.33) or E2+P monkey coronary artery muscle cells (3 ± 0). To further clarify this point, an additional series of experiments was performed that compared the staining of JZB39 at three concentrations in the ovx, E2+P, and E2+MPA groups. As shown in Fig. 4Go.2, PR expression in ovx monkey coronary artery sections (Fig. 4Go.2a–c) was detectable with 0.3 µg/mL, but was essentially absent at concentrations of 0.1 or 0.03 µg/mL. In contrast, PR expression in E2+P monkeys (Fig. 4Go.2d–f) was detectable even at 0.03 µg/mL JZB39 (Fig. 4Go.2f). Slightly less JZB39 staining was observed at 0.1 and 0.03 µg/mL in E2+MPA monkeys (Fig. 4Go.2g–i and Table 3Go) than in E2+P monkeys. Additional studies are required to accurately quantitate the level of PR expression in the coronary artery during various steroid treatment regimens to determine whether MPA modulates the expression of PR. However, ER expression appeared relatively stable in the absence of E2 or the presence of MPA, whereas E2 appeared to stabilize or enhance the expression of PR in arterial muscle cells.


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  		Table 3. Effect of steroid hormone treatment on the relative level of PR immunostaining in fresh-frozen monkey coronary artery sections

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although coronary heart disease is rare in premenopausal women, it is the leading cause of death in postmenopausal women (14). The increase in prevalence of coronary heart disease after menopause has been attributed to the loss of estrogen. Meta analysis of case control and prospective studies indicates that estrogen exerts an unambiguous protective effect on coronary heart disease (45). The putative mechanism of estrogen’s benefit includes, among other factors, a lowering of lipid levels (1), attenuation of atherosclerotic lesion development (8, 9), reduction in exercise-induced myocardial ischemia (3), and prevention of coronary vasospasms by restoring normal coronary artery reactivity (20, 22, 29).

Coronary vasospasms (abnormal focal contractions associated with downstream dilation forming an hourglass pattern) (20) are considered to be an underlying cause of variant angina in patients without obstructive coronary atherosclerosis (46, 47). The mechanism of coronary vasospasm is not known, but vascular hyperreactivity has been hypothesized (20, 46, 48). The premise is that localized sites of coronary muscle exhibit hyperreactivity to multiple vasoconstrictor substances, including platelet-derived thromboxane A2 and 5-HT (20, 49, 50), which can initiate severe and sustained occlusion of a coronary artery. This, in turn, can lead to myocardial infarction, ventricular arrhythmia, and even sudden cardiac death (48). E2 and progestins appear to modulate coronary reactivity by changing the sensitivity of VMC to naturally occurring platelet release products (20, 21, 29). As E2 and P replacement therapy in our primate animal model, which is independent of atherosclerosis, provides protection against this increase in vascular reactivity, there are compelling reasons to explore clinical treatment protocols used in hormone replacement therapy with regard to their cardiovascular effects. Drug-induced coronary vasospasms recorded in rhesus monkeys were augmented after ovariectomy and were inhibited by E2 and/or P replacement therapy. Monkeys treated with the synthetic progestin MPA had a higher incidence of coronary vasospasm. The vasospasms in monkeys induced by 5-HT and U46619 were characteristically similar to the pattern of focal constrictions and downstream dilation (20, 29) observed in diseased human coronary arteries after intracoronary injection of acetylcholine (19) or serotonergic stimuli such as ergonovine (46). Similarities in human and nonhuman primate coronary vasospasm appearance and time course suggest that coronary artery reactivity modulation by ovarian steroids may be an important pathophysiological mechanism.

The steroid hormone-sensitive changes in vascular reactivity observed in vivo were preserved in the freshly isolated coronary artery VMC studied in vitro, as shown in Figs. 1–3GoGoGo. Hyperreactive coronary artery VMC Ca2+ and PKC responses to 5-HT and U46619 were correlated with low circulating E2 and P levels in ovariectomized monkeys, as first described by Miyagawa and associates (21) and expanded here. When physiological levels of E2 and P were restored, small, transient VMC Ca2+ and PKC responses, similar to those in VMC from intact female monkeys, were observed. The in vitro Ca2+ and PKC responses to 5-HT and U46619 in VMC from MPA-treated monkeys were also dramatically increased and sustained for prolonged periods of time. In fact, the incidence of coronary vasospasm and the amplitude and duration of coronary artery VMC Ca2+ and PKC signals were the highest in MPA-treated monkeys. In contrast, monkeys treated with E2 for 2 wk followed by P alone for 4 wk were protected from coronary vasospasm. Also, the Ca2+ and PKC responses of their coronary VMC were small and transient, similar to those observed in VMC from intact monkeys. These data indicate that MPA behaves virtually opposite to P, negating the protective effects of E2 on coronary reactivity both in vivo and in vitro.

Blood levels of E2 and P achieved via SILASTIC brand implants in ovx monkeys were typical of the follicular and luteal phases of the primate menstrual cycle (51). The levels of MPA achieved (range, 0.25–1.6 ng/mL) were somewhat less than the peak levels observed (2.5–12.3 ng/mL) 1–4 h after ingestion of a 10-mg tablet of Provera in women (30). However, the levels achieved in monkeys were similar to the 24-h trough levels observed after oral Provera (0.3–0.6 ng/mL) as well as to those in women 1–7 days after insertion of intravaginal rings impregnated with 100–200 mg Provera (0.9 to 1.6 ng/mL) (30).

Freshly dispersed VMC from ovx monkeys retained the hyperreactive responsiveness to 5-HT and U46619 and maintained hyperactivity even after several days in culture (21). In addition, freshly isolated or primary cultured VMC obtained from monkeys treated with E2 and P in vivo were not hyperreactive, suggesting that the protective effect of E2 and natural P on coronary artery hyperreactivity occurs at the level of the single VMC. The protective effects of E2 on VMC reactivity were negated when E2 replacement was combined with MPA for 1 week before VMC isolation. Similarly, hyperreactive VMC from MPA-treated monkeys were maintained for up to 3 wk after isolation in primary culture (21). These findings, taken together, suggest that the reactivity state of the coronary artery is modulated in vivo by steroid hormone treatment and is retained during dispersion, isolation, and primary culture of coronary artery VMC. The fact that the reactivity state of the VMC persists suggests a genomic mechanism that is regulated by E2 and P. Precedence for the phenomenon of single cells retaining the characteristics of the whole organ has previously been established in animal models of genetic hypertension. For example, intracellular Ca2+ signals in VMC from spontaneously hypertensive rats were elevated compared to those in normotensive Wistar-Kyoto rats (36, 52), thus implicating a Ca2+ hypothesis for hypertension (36, 52, 53).

The combination of 5-HT and U46619 provokes coronary vasospasm in monkeys and causes exaggerated and prolonged Ca2+ and PKC responses in freshly dissociated coronary artery VMC, presumably by activating phospholipase C via 5-HT2 and thromboxane A2 receptor stimulation (54). Vasospasms can be reversed by Ca2+ channel antagonists, such as mibefradil (29). Similarly, myocardial infarction or death is rare in patients treated for coronary artery spasm with Ca2+ channel blockers (47). Thus, membrane Ca2+ signals as well as phospholipase C-induced intracellular Ca2+ release mechanisms are likely to be involved in the development of spasm (21, 55). The mechanism of the sustained increase in intracellular Ca2+ needed to maintain the contractile state is the key fundamental question that has yet to be addressed. The long duration may be due to a number of factors based on deficient relaxation or on amplified activation mechanisms involving Ca2+ signal proteins. Our results suggest that PKC is involved, as suggested by previous studies involving hyperreactive VMC (21, 42, 56).

The mechanisms by which E2, P, and MPA modulate VMC reactivity may involve regulation of endothelial cell production of constrictor and dilator substances or direct inhibitory effects on the excitation-contraction coupling pathway in VMC via ER and PR. Our data suggest that ER in the monkey coronary artery is primarily expressed in endothelial cells and fibroblasts, whereas PR is mainly expressed in VMC. PR expression in the coronary artery appeared to be regulated in vivo by E2, similar to E2 regulation of PR expression in the reproductive tract (31). A similar mechanism has been suggested to occur in the cardiovascular system of the dog (57) and baboon (58).

A correlation between E2 status and ER expression could not be drawn, but perhaps more sensitive and specific quantitative techniques can detect differences. We found ER localized mainly in endothelial and interstitial cell nuclei and to a much lesser extent in VMC nuclei. This would either suggest that only a limited number of VMC ER maybe required to modulate PR expression or that an ER-activated paracrine factor is involved. E2 is known to potentiate the release of endothelium-derived vasoactive factors such as prostacyclin and nitric oxide and inhibit the production of thromboxane A2, endothelin, and superoxide ions (for review, see Ref.59). Although additional studies are required to test the idea, the hypothesis that the loss of E2 alters a delicate balance of vasoactive factors and thereby modulates the regulation of vascular tone and reactivity would be supported (60). More directly, E2 may modulate PR expression through other ER forms, such as ERß, which may act differently from the classical nuclear ER receptor, ER{alpha} (61, 62, 63). PR was localized primarily in the tunica media of coronary arteries; therefore, P may directly modulate coronary artery VMC reactivity. The mechanism by which E2 and P reduce VMC reactivity may be through modulation of Ca2+ influx, extrusion, and storage pathways (11, 21, 18, 20, 26, 55, 60).

In the human, immunostaining with the anti-ER rabbit polyclonal antibody, ER-21, and the rat monoclonal antibody, D547, was shown in coronary artery, internal mammary artery, and saphenous vein sections from premenopausal women (64, 65). The small amount of ER immunostaining detected in vascular muscle in the present study with ER-21 is consistent with the results of McGill and Sheridan (66). They showed in the baboon that the nuclear uptake of [3H]estrogen was detected mainly in interstitial and adventitial cells and to a much lesser extent in atrial and ventricular myocardial fibers, arterial endothelial cells, and vascular muscle cells of the arterial media. The variability in ER expression may reflect species differences (rhesus macaque vs. human), differences in the sensitivity of anti-ER antibodies used (1D5, H222sp{gamma}, D547, and ER-21), differences in the specificity of radioligand binding studies, or differences in the stability of the ER in vascular muscle and endothelial cells. Similar to the results of our studies in monkeys, PR was previously identified in sections of fresh and frozen human aorta, internal carotid and coronary arteries, atria (67), and saphenous vein (68). In the carotid and coronary arteries and saphenous vein, PR staining localized to endothelial nuclei in the intima and VMC nuclei in the tunica media and neointima was also reported (67).

The mechanism by which MPA increases vascular reactivity is not known, but these studies lend support to the idea that steroid hormone effects on vascular function should be considered when assessing the usefulness of synthetic estrogens and progestins. MPA is known to possess significant androgenic activity (for review, see Ref.59) and thus may modulate vascular reactivity through an androgenic component. In support of this hypothesis, testosterone treatment in vivo was shown to enhance thromboxane A2-induced coronary artery vasoconstriction in guinea pigs (69) and aortic and platelet responses to U46619 in rats by increasing thromboxane A2 receptor density in VMC and platelets (70, 71). Whether MPA stimulates androgen receptors in the vascular wall or circulating blood cells in monkeys is not yet known, but the direct modulation of thromboxane A2 receptor expression via androgen receptor activation represents a possible mechanism by which MPA induces vascular hyperreactivity and thus should be explored further.

In conclusion, our data suggest that E2 and P reverse ovariectomy-induced coronary hyperreactivity, and that this mechanism may contribute to a cardioprotective benefit of estrogens and natural P. The incidence of coronary artery vasospasm is increased in ovx monkeys and in monkeys treated with MPA, both of which appear to have reduced PR expression in vascular muscle. Additional studies currently in progress in our laboratory will determine whether in vitro application of E2, P, and MPA directly modulates VMC reactivity via steroid receptors, and whether the time course of direct effects of ovarian steroids on coronary arteries in vivo and on isolated VMC in vitro might suggest additional membrane effects.


   Footnotes
 
1 This work was supported by NIH Grants HL-51723 and HD-18185. Back

Received August 18, 1997.

Revised October 17, 1997.

Accepted October 27, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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