Estrogens and Experimental Atherosclerosis in the Baboon (papio cynocephalus) HENRY C. MCGILL, JR., M.D., LEONARD R. AXELROD, PH.D., C. ALEX MCMAHAN, PH.D., HERMAN S. WIGODSKY, M.D., PH.D., AND GLEN E. MOTT, PH.D. triglyceride, and phospholipid in whole serum, low density lipoproteins and high density lipoproteins showed no consistent statistically significant differences among the groups. Triglyceride and phospholipid concentrations were higher in the estrogen-treated and intactovary groups than in the ovariectomized nonestrogen treated group, but not all pairwise comparisons were statistically significant. There were no consistent statistically significant differences in atherosclerotic lesions among the groups. Neither ovariectomy nor estrogen replacement influence diet-induced experimental atherosclerosis in the baboon within two years.
SUMMARY One hundred twenty-six adult female baboons (Papio cynocephalus) were hysterectomized and all except 18 were ovariectomized. The animals were fed a moderately atherogenic diet (40% calories from hydrogenated vegetable oil, 1.5 mg cholesterol/kcal) for two years. Ovariectomized-hysterectomized animals received estrone sulfate, ethynyl estradiol, or diethylstilbestrol orally in daily doses similar to those given humans. An ovariectomized-hysterectomized group and a hysterectomized group received no drug. The average total serum cholesterol concentration rose from 136 mg/dl to 223 mg/dl and declined to 186 mg/dl. Concentrations of cholesterol,
changes in arteries of rats25 28; and augmented, inhibited, and failed to affect diet-induced hypercholesterolemia and atherosclerosis in rabbits.29 32 In the hope of resolving some of these conflicting observations, this experiment was planned to examine the effects of ovariectomy and estrogen replacement therapy on serum lipids and atherogenesis in the baboon, whose reproductive physiology, estrogen and cholesterol metabolism, greater prevalence of aortic fatty streaks in the feral female compared to the feral male, and response to an atherogenic diet are more human-like than are those of chickens, rats, and rabbits.33 In order to induce moderate hyperlipidemia and experimental atherosclerosis, the animals were fed a mildly atherogenic diet for two years. The estrogens tested were estrone sulfate, a biologically active conjugate of estrone and a major circulating estrogen in humans; 17ca-ethynyl estradiol, a highly potent, orally effective synthetic estrogen structurally similar to estradiol; and diethylstilbestrol, a nonsteroidal compound differing in chemical structure from the natural estrogens but having estrogenic activity.
THERE ARE CONFLICTING OBSERVATIONS on the relationship between estrogens and atherogenesis in both humans and experimental animals. Premenopausal white women consistently have a lower incidence and prevalence of myocardial infarctionl-' and a lessened extent of advanced atherosclerotic lesions in the coronary arteries5' 6 than do white men of the same age. However, some studies show that women ovariectomized before the natural menopause lose their relative immunity to coronary heart disease,7-9 while others find no effect of ovariectomy.0' 11 One study finds atherosclerosis more severe in ovariectomized women,'2 but another finds no difference." According to observations from The Framingham Study, postmenopausal women have a higher incidence of coronary heart disease than age-matched premenopausal women.'4 Despite a prevailing impression that the menopause increases risk of coronary heart disease, age and sex specific mortality rates do not increase after the menopause more than would be expected with age.3' 15 "16 Attempts to reduce the incidence of coronary heart disease in postmenopausal women by estrogen replacement therapy also have yielded conflicting
results.'7 '9
Methods and Materials
The results of animal experiments on the relationship between estrogens and atherogenesis are as inconsistent as those from human studies. Estrogens induced hyperlipidemia in chickens20 but inhibited coronary artery lipidosis in cockerels21; reduced the size of plaques in White Carneau pigeons in high doses22-24; produced a variety of
Subjects
The subjects were 126 adult female baboons (Papio cynocephalus) ranging in estimated age from 5 to 15 years. The animals were of feral origin, but many had been in the Southwest Foundation for Research and Education (SFRE) breeding colony for several years. During this experiment, they were individually caged. All animals were hysterectomized to eliminate the uterine bleeding that occurs with continuous estrogen therapy. All but 18 also were ovariectomized at the time of hysterectomy. Eight animals died during the experiment from causes unrelated to the experimental procedure. On the average, these did not differ in age, serum lipids, or weight from the remaining 118.
From the Southwest Foundation for Research and Education, and the Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas. Supported by grants HE-14112 and HL-15914 from the National Heart, Lung and Blood Institute. Dr. Leonard R. Axelrod, who designed and initiated this experiment, left the Southwest Foundation for Research and Education on May 1, 1973, to become Director of the Criteria and Evaluation Division and Chief Scientist of the Office of Pesticide Programs, Environmental Protection Agency, Washington, D.C. The experiment was completed in his absence. Dr. Axelrod died on July 31, 1975. The analyses and interpretation of data contained in this report were completed after Dr. Axelrod's death. Address for reprints: Henry C. McGill, Jr., M.D., The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio,
Experimental Design
Although the original principal investigator (LRA) did not use a strict randomization procedure for the assignment of treatments to animals, we analyzed the results as a com-
Texas 78284. Received March 21, 1977; revision accepted May 18, 1977.
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TABLE 1. Selected Characteristics of Experimental Animals, Ovarian Status, and Estrogen Treatment by Experimental Groups
Group
1 2 3 4 5 6
7
Number of baboons Start End
18 18 18 18 18 18 18
17 17 18 17 15 16 18
Estimated age, years Mean SD
At start of experiment Weight, Serum kg oholesterol Mean SD Mean SD
Days after operation Mean SD
11.6 11.1 10.9 11.5 11.8 11.3 11.2
14.3 14.3 14.3 14.0 14.9 14.0 14.7
126.8 157.5 133.2 146.1 155.4 104.0 61.7
2.3 2.2 1.7 1.7 1.4 1.7 1.1
1.9 1.6 1.6 2.3 2.1 1.7 1.4
131.3 138.1 141.9 139.2 130.6 129.6 136.9
Estrogen treatment
19.9 26.4 24.1 29.1 34.9 21.8 19.0
Abbreviations: ES = estrone sulfate; EE = 17 a-ethynyl estradiol; DES
=
94.0 84.7 86.5 76.8 92.2 69.6 13.9
Duration,
Ovarian status
Year 1
Year 2
days Mean SD
Ablated Ablated Ablated Ablated Ablated Ablated Intact
ES None EE None DES None
ES ES EE EE DES None
625.8 11.3 374.6 8.3 624.5 12.1 374.1 7.9 623.8 11.3
None
None
-
diethylstilbestrol.
pletely randomized design with seven treatments. There was evidence of bias in the assignment process. Table 1 outlines characteristics of the treatment groups. All animals received the atherogenic diet from the beginning of the experiment. Drug treatment for groups 1, 3, and 5 began on day 133 after initiating the diet. Drug treatment for groups 2 and 4 began on day 498 to examine the possibility that estrogens might modify lesions previously established by dietinduced hyperlipidemia.
Its caloric value was about 3.8 kcal/g wet weight. Each baboon received 360 g per day, about 90 kcal/kg/day, for the first 487 days of the experiment. Because many animals gained excess weight, the amount offered each day thereafter was reduced by 20%. The final diet mixture contained 5.62 mg/g (wet weight) cholesterol, 0.12 mg/g campesterol, and 0.40 mg/g 3-sitosterol by gas-liquid chromatographic analysis. Thus, the diet contained about 1.5 mg cholesterol per kcal.
Estrogen Dosage and Administration
Blood Collection and Serum Lipid Analyses
Estrogen doses approximated, on a weight basis, the usual human doses for replacement therapy. Drugs were administered by embedding tablets in fig bars. The rate of failure to take the drug ranged from 0.1% of treatment days in group 2 to 1.5% in group 3. The overall omission rate for all groups was 0.64%, and only one animal had an omission rate greater than 5%. Baboons in groups 1 and 2 received 0.625 mg/day, or about 45 Ag/kg/day, of Ogen (Abbott Laboratories) of pure crystalline estrone sulfate (ES) stabilized with piperazine. This dose in baboons may equal a higher dose in humans since plasma concentrations of estrogenic steroids are lower in baboons than in humans.34 Baboons in groups 3 and 4 received 17 ,tg/day, or about 1.2 ,tg/kg/day, of Estinyl (Schering Corporation) as the source of 17a-ethinyl estradiol (EE). Baboons in group 5 received 62.5 ,ug/day, or about 5 ,ug/kg/day, of diethylstilbestrol, USP (DES) (Eli Lilly and
Thirty ml of blood were collected at 4 month intervals in Vacutainers without anticoagulant (Becton Dickinson Co.), with the animal under Sernylan anesthesia, about 0.8 mg/kg (phencyclidine hydrochloride) (Bio-Ceutic Laboratories, Inc.) Four lipoprotein classes, separated from 5 ml serum samples by the preparative ultracentrifugal method of Lindgren and associates,37 were analyzed for cholesterol, triglyceride, and phospholipid. The Bioregional Reference Laboratory, Inc. analyzed whole serum and lipoprotein fractions for cholesterol, triglyceride, and phospholipid. Cholesterol determinations were performed early in the study on a SMA 12/60 Autoanalyzer (Technicon Instruments Corp.) with a Liebermann-Burchard reagent, and later in the study on an ABA100 (Abbott Laboratories Diagnostic Division) by an enzymatic method. Triglycerides were analyzed by extraction, silicic acid chromatography, and the colorimetric method of Foster and Dunn.38 Quality control was maintained by repeated analyses of commercial pooled serum samples. The coefficient of variation for the cholesterol analyses was 2.4% and for triglycerides, 8.7%. Both methods met the criteria established for clinical laboratories and received a satisfactory score of 3 from the Center for Disease Control. Phospholipids were determined by the method of Zilversmit and Davis,39 with a coefficient of variation of 4% for repeated analyses.
no
Co.).
Diet
The baboons had been fed SFRE baboon chow36 (Ralston Purina Co.) from the time of their arrival at SFRE until beginning the atherogenic diet for this experiment. This diet ktable 2) was prepared by mixing ground baboon chow, hydrogenated vegetable oil (Crisco, Procter and Gamble Co.), frozen egg yolk, cholesterol, and 6 ml water per 100 g.
TABLE 2. Composition and Nutrient Value of the Atherogenic Diet per 100 g Dry Weight Component
Baboon chow Crisco Egg yolk, frozen Cholesterol, USP Total
Total g
67.1 11.7 20.8 0.4 100.0
Nutrients, g
Protein
Fat
Carbohydrate
Fiber
Ash
Cholesterol mg
14.1 0 3.1 0
3.7 11.7 5.7 0 21.1
32.9 0
2.8 0 0
5.2 0 0 0 5.2
4 0 239 366 609
17.2
2.1 0
35.0
0
2.8
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ESTROGENS AND ATHEROSCLEROSIS IN BABOONS/McGill et al. Autopsy Procedures We sedated the animals with an intramuscular injection of Sernylan and performed complete autopsies, including gross and histologic examination of all major viscera. The iliac, femoral, innominate, brachial, carotid, and coronary arteries were opened longitudinally and prepared by methods used in the International Atherosclerosis Project."0 The aorta from the ligamentum arteriosus to its bifurcation was divided along anterior and posterior midlines. The right half of the aorta was frozen for chemical analysis, and the left half, after being sampled for electron microscopy, was prepared for grading like the other arteries. Grading of Atherosclerotic Lesions Two experienced pathologists (one of the authors, H.C. McGill; and a consultant, J.P. Strong) independently graded the stained arterial specimens by the method of the International Atherosclerosis Project.'1 Graders were not aware of treatment group or other data, but all the arteries from each animal (except the coronary arteries) were in the same plastic bag. Product moment correlation coefficients between grades of different arterial segments did not exceed 0.34, a value which indicated that the grades of some segments did not unduly influence the grades of other segments. The intraclass correlation coefficient42 between estimates by the two pathologists of percent intimal surface area involved with fatty streaks for the aorta ranged from 0.77 to 0.84, values indicating acceptable interobserver variability of the grading. Independent regrading of 20 sets of specimens at a later time indicated that variability within pathologist was similar to variability between pathologist. We compared aortic fatty streaks among treatment groups on the basis of extent of intimal surface involved. Because grades of fibrous plaques in all arteries and of fatty streaks in some arteries frequently were small or zero, we compared these lesions among treatment groups on the basis of prevalence rather than extent. Specimens were scored as positive only if both pathologists independently classified the specimen as positive. Aortic Intimal Cholesterol
A technician stripped the intima from the unfixed half of each aorta. Lyophilized and saponified tissue was analyzed by gas-liquid chromatography.43 A technician measured the total intimal surface area of the unfixed half of the aorta from full size photographs using an electronic digitizer (Wang 662). The coefficient of variation after retracing a randomly selected sample of 20 photographs was 2.0%. From the area and weight of the intima and the concentration of cholesterol, we estimated cholesterol per unit area of intimal surface. Statistical Methods Responses were analysed in separate univariate analyses, usually by analysis of variance (ANOVA)." For ANOVAs that resulted in a statistically significant overall F test, all pairwise contrasts between treatment group means were computed and evaluated with Tukey's t-test.4' Analyses by the Kruskal-Wallis ANOVA for ranks46 confirmed the
659
results of the parametric ANOVAs. In some instances, as noted, analysis of covariance (ANCOVA)44 was used. For data such as presence of lesions, we tested the null hypothesis of independence between treatment groups and presence of lesions by a Chi-square test.'2 Results Evidence of Estrogen Stimulation
All baboons in the group with intact ovaries (7) continued normal menstrual cycles throughout the experiment as indicated by periodic turgescence of the sex skin. The ovariectomized group (7) showed no turgescence of the sex skin. Baboons in groups which received estrogens (1-5) showed moderate sustained turgescence of the sex skin, an observation indicating that they were receiving estrogenic stimulation. Body and Organ Weights
The group receiving EE from the start of the experiment (3) gained less weight than other groups, and the group receiving EE in the second year (4) also gained less weight after hormone treatment began. The DES treated group (5) lost weight steadily after about day 300 of the experiment; the weight loss began before the daily diet ration was reduced. Mean weights of all groups fluctuated over time in a manner apparently unrelated to treatment. Mean body weights at autopsy of the two EE treated groups (3 and 4) were lower than those of all other groups, and pairwise comparisons by ANCOVA with initial body weight as the concomitant variable were statistically significant for these two groups as compared to the group treated with ES for two years.' Organ weights at autopsy, analyzed by ANCOVA with total body weight at autopsy as the concomitant variable, showed no real differences. Serum Lipids and Lipoproteins
The mean total serum cholesterol concentration of all baboons rose in the first three months of the experimental period from 136 mg/dl to 223 mg/dl, declined during the following 18 months to 186 mg/dl, and increased slightly near the end of the experiment. We compared serum lipid and lipoprotein concentrations among groups by ANOVA, using values from two blood samples drawn between 653 and 814 days after the start of the experiment. We reasoned that long term effects of the drugs should be apparent during this period, and that they would not be masked by the large changes associated with beginning the atherogenic diet. Table 3 shows results for cholesterol, triglyceride, and phospholipid in whole serum, LDL, and HDL. Chylomicrons and VLDL (not included in table 3) contained only a minor fraction of total lipids and showed no consistent differences among groups. Neither cholesterol nor phospholipid concentrations in lipoprotein fractions or in whole serum differed between the ovariectomized group (6) and the group with intact ovaries (7). Total triglyceride, LDL triglyceride, and HDL triglyceride concentrations were higher in all estrogen treated groups (1-5) and in the group with intact ovaries (7) than in the ovariectomized group (6). However, statistically significant pairwise comparisons were limited to groups 1 and 3. Phospholipid concentrations were slightly higher in the
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TABLE 3. Statistical Comparisons of Serum Lipids and Lipoproteins
Fraction of Serum
Cholesterol LDL HDL Total Triglyceride LDL HDL Total Phospholipid LDL
1
2
92.6 70.6 199.3
77.8 62.0 165.2
6
7
mean
Estimated components of variance Between Within animal animal
79.3 64.0 174.1
83.4 66.4 175.6
83.4 64.0 176.5
251.3 473.8 272.3 387.4 1114.6 379.6
Treatment group means 3 4 5
82.1 67.4 182.5
78.3 58.1 158.3
91.9 59.5 180.3
Overall
Significant* pairwise
F
comparisons
1.25 0.70 2.25*
None None 1-4 1-6,3-6 1-6X3-6 3-6
34.1
27.9
33.6
31.2
23.8
21.5
27.7
28.7
76.8
78.5
3.11**
29.9 94.4
26.9 81.6
29.9 98.6
24.5 82.7
24.4 82.2
20.9 70.4
26.9 79.1
26.3 84.4
7.9 472.3
74.9 172.7
3.63**
2.77*
96.6
76.4
93.3
64.1
74.3
66.9
78.1
78.5
113.6
636.6
5.86**
1-4,1-6,3-4, 3-6
HDL
165.5
132.2
168.0
138.4
161.5
154.3
154.7
153.1
241.0
455.6
6.55**
1-2,1-4,2-3,
3-4,2-5,4-5, 2-7
Total
292.7
259.1
292.6
244.9
266.8
256.8
265.6
268.7
954.0
508.9
1-4,3-4,3-6
4.67**
N = 118 baboons, approximately 2 values/animal between 650 and 820 days. *Statistically significant at 0.05 level. **Statistically significant at 0.01 level.
treated with ES and EE for 2 years (1 and 3) than in controls or other treatment groups, and several of the differences were statistically significant. The decrease in caloric intake after day 487 on the atherogenic diet showed no relationship to changes in cholesterol, triglyceride, or phospholipid concentrations in whole serum or in lipoprotein fractions.
groups
Characteristics of Arterial Lesions
Table 4 shows overall measures of fatty streaks and fibrous plaques by artery. Grossly, the lesions resembled those previously described in the baboon.35 Although some coronary artery lesions were elevated and resembled fibrous plaques, none produced significant stenosis. Microscopically, all lesions showed intimal thickening with smooth muscle cells. Lesions classified grossly as fibrous plaques were principally smooth muscle cells with varying amounts of lipid in the subintimal layer, and occasionally a core of necrotic debris and extracellular lipid. Electron micrographs from the thoracic and abdominal aorta and coronary arteries of several animals from each experimental group showed that most intimal lipid was within smooth muscle cells, but some was extracellular and some was in macrophages. We detected no qualitative differences among the experimental groups in histologic sections or electron micrographs. Aortic Fatty Streaks
Table 5 shows treatment group means of percent surface involvement with fatty streaks by aortic segments. The component of variance within animal (due to interobserver difference in grading) was less than the component of variance between animals. Only two pairwise comparisons were in the abdominal aorta, DES statistically significant treated (5) and ovariectomized untreated (6) groups were more extensively involved by fatty streaks than the untreated group with intact ovaries (7). In five of the other arteries, the null hypothesis of independence between treatments and presence of fatty streaks was -
rejected (P < 0.05). These five were further examined by partitioning the degrees of freedom.47 The group treated with EE for two years (3) had a significantly lower prevalence of fatty streaks in the right carotid, left carotid, left brachial, and left circumflex coronary arteries. The group treated with ES in the second year (2) and the ovariectomized group (6) had a significantly higher prevalence of fatty streaks in the left coronary circumflex artery than other groups. The group treated with ES from the beginning (1) and the group with intact ovaries (7) had a significantly lower prevalence of fatty streaks in the left anterior descending coronary artery than did all other groups. Concentration of Cholesterol in Aortic Intima
The overall means of concentrations of cholesterol in the stripped aortic intima were 25.0 mg/g and 111.9 mg/cm2, TABLE 4. Percent of Intimal Surface Involved with Fatty Streaks and Prevalence of Fatty Streaks and Fibrous Placques by Arterial Segment Intimal surface involved with fatty streaks,
percentt
Arterial segment
Aortic arch
Thoracic aorta Abdominal aorta Right carotid Left carotid Right brachial Innominate Left brachial Right iliac-femoral Left iliac-femoral Right coronary Left coronary, ant desc. Left coronary, circumflex
Mean
Range Low High
11.8 35.1 18.6 7.1 6.7 6.1 12.3 11.3 3.7
0. 1. 0. 0. 0. 0. 0. 0. 0.
3.3 0.4
Prevalence,
percent positive Fatty Fibrous streaks plaques
0. 0.
73.1 86.6 81.7 54.5 27.0 28.4 59.4 45.5 42.1 42.1 17.3
91.5 96.6* 97.5 90.7 90.7 84.7 82.2 93.2 79.7 78.0 7.6
3.4 5.1 13.6 5.9 9.3 2.5 0.8 9.3 15.3 16.1 0.8
1.2
0.
17.3
31.4
2.5
0.7
0.
17.9
18.6
0.0
N = 118 baboons. *Prevalence less than 100% although lowest percent surface involvement is not 0 because range is from the mean of independent estimates and prevalence is based on both observers scoring specimen as positive. tMean of independent estimates by 2 observers.
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TABLE 5. Percentage Intimal Surface Area Involvement of Aortic Segments with Fatty Streaks Estimated
Overall Group
Aortic
segment
Arch Thoracic Abdominal
1
15.8 42.2 15.2
mean
mean
percent surface
2
3
4
5
6
7
17.8 37.8 24.6
9.5 27.0 10.9
13.0 34.3 17.6
12.4 28.0 27.2
10.3 31.8 27.2
4.2 43.3 9.8
percent surface
11.8 35.1 18.6
components F
Significant* pairwise comparisons
1.92 1.15 3.70**
None None 5-7,6-7
of variance
Between Within
animals animals
159.3 571.5 215.0
43.0 107.5 73.2
*Statistically significant at 0.05 level. **Statistically significant at 0.01 level.
and overall estimated standard deviations were 17.8 and 60.3. No real differences among the treatment groups were apparent by either measure. Fibrous Plaques
The null hypothesis of independence between treatments and presence of fibrous plaques was rejected (P < 0.05) only for the left iliac-femoral artery. The group treated with ES for the second year (2) and the group treated with DES (5) had a significantly higher prevalence of fibrous plaques as indicated by partitioning the degrees of freedom.
Discussion Serum Lipids and Lipoproteins
The slight and consistent, but not always statistically significant, elevations in serum triglyceride and phospholipid concentrations in the estrogen treated groups and in the group with intact ovaries compared to ovariectomized untreated controls are consistent with the reported effects of estrogenic compounds in humans.48s60 The difference we observed, however, is small, and slight elevations of triglyceride and phospholipid concentrations in the absence of increased cholesterol levels would not be expected to augment atherogenesis. Furthermore, no major shifts in distribution of lipids among the lipoproteins appear to result either from ovariectomy or estrogen replacement. The failure to find a higher serum cholesterol concentration in ovariectomized untreated baboons than in those with intact ovaries or in those receiving estrogens is not consistent with the elevation in serum cholesterol associated with surgical or natural menopause in humans.7' 8, 14, 51, 52 Detailed comparison of these results with observations on other animal specieS20-25, 29-32, 53 shows some similarities but many differences. Atherosclerosis
The comparisons of arterial lesions in this experiment indicate no consistent effect of estrogen deficiency or estrogen replacement on extent or quality of atherosclerotic lesions. The results, therefore, are consistent with reports that ovariectomized women have no greater atherosclerosis at autopsy'3 and experience no higher incidence or prevalence of atherosclerotic disease than women with intact ovaries.10-1' They are consistent with reports that estrogen replacement therapy does not influence coronary heart disease prevalence in postmenopausal women.'9 They also are consistent with reports that physiological doses of estrogens do not reduce spontaneous atherosclerosis in pigeons23' 24 or experimental atherosclerosis in rabbits.2' 31 32 We have
found no reports of the effects of estrogens on diet-induced atherosclerosis in nonhuman primates. The extent of fatty streaks exceeded that produced in other baboons by similar moderate levels of hyperlipidemia.35 '4-57 This difference may be due to age or sex, since most experiments with atherogenic diets in baboons have used young adult males rather than females. The abundant smooth muscle and fibrous tissue in fatty streaks may be typical of the reaction of nonhuman primates to moderate prolonged hyperlipidemia, as observed in rhesus monkeys.58 These lesions differ from the foam cell lesions typical of experimental animals with higher serum cholesterol levels. Multiple pregnancies also may have caused smooth muscle proliferation, since Wexler59 has described nonlipid containing intimal thickening in the arteries of breeder female rats. Most of the baboons were multiparous, but because they were feral, accurate assessment of parity was not possible. Lesions classified grossly as fibrous plaques resembled fibromuscular cushions described in the coronary arteries and aortas of normal rhesus monkeys80' 61 and feral baboons autopsied immediately after capture.82 We believe that our baboons probably had developed many of these plaques prior to the experiment. The relatively high frequency of fibrous plaques in the femoral arteries may have been due to trauma to the arteries during venipuncture. Hyperlipidemia may have augmented fibrous plaques, and probably was responsible for lipid deposition within them as reported for hyperlipidemic rhesus monkeys.83 Whatever their origin, they do not appear to have been affected either by ovariectomy or estrogen replacement. Conclusions
In the female baboon fed a moderately atherogenic diet for two years, neither ovariectomy nor estrogen replacement after ovariectomy produces consistent and statistically significant differences in serum lipid or lipoprotein concentrations or in atherosclerosis. There is a trend (not statistically significant) for baboons with intact ovaries or ovariectomized baboons receiving exogenous estrogens to have slightly higher serum triglyceride and phospholipid concentrations than ovariectomized baboons. The results are consistent with epidemiologic studies of humans which show no effects of either estrogen deficiency or estrogen replacement therapy on serum lipid or lipoprotein concentrations or on atherosclerosis.
Acknowledgments The authors gratefully acknowledge the assistance of Dr. Joseph W. Goldzieher in completing the experiment after Dr. Axelrod's death and in reviewing the manuscript; of Dr. Thomas W. Culp in supervising the serum lipid analyses; and of Dr. Jack P. Strong in grading the arterial specimens. Cornelio Celaya and Ignacio Gomez cared for the baboons throughout the experi-
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ment, Mary Jo Haas performed the lipoprotein fractionations, and Cynthia Gaudot performed the aortic lipid analyses.
References
31.
1. Levy H, Boas EP: Coronary artery disease in women. JAMA 107: 97,
1936 2. Gertler MM, White PD: Coronary Heart Disease in Young Adults: A Multidisciplinary Study. Cambridge, Massachusetts, Harvard University Press, 1954 3. Moriyama IM, Krueger DE, Stamler J: Cardiovascular Diseases in the United States. Cambridge, Massachusetts, Harvard University Press, 1971 4. Shurtleff D: Some characteristics related to the incidence of cardiovascular disease and death: Framingham Study, 18-year follow-up. In The Framingham Study. An Epidemiological Investigation of Cardiovascular Disease, section 30, edited by Kannel WB, Gordon T. Department of Health, Education and Welfare Publication (National Institutes of Health) No. 74-599, 1974 5. Tejada C, Strong JP, Montenegro MR, Restrepo C, Solberg LA: Distribution of coronary and aortic atherosclerosis by geographic location, race, and sex. Lab Invest 18: 509, 1968 6. Sternby NH: Atherosclerosis in a defined population. An autopsy survey in Malmo, Sweden. Acta Pathol Microbiol Scand (suppl 194): 1-216, 1968 7. Robinson RW, Higano N, Cohen WD: Increased incidence of coronary heart disease in women castrated prior to the menopause. Arch Intern Med 104: 908, 1959 8. Oliver MF, Boyd GS: Effect of bilateral ovariectomy on coronary-artery disease and serum-lipid levels. Lancet 2: 690, 1959 9. Parrish HM, Carr CA, Hall DG, King TM: Time interval from castration in premenopausal women to development of excessive coronary atherosclerosis. Am J Obstet Gynecol 99: 155, 1967 10. Ritterband AB, Jaffe IA, Densen PM, Magagna JF, Reed E: Gonadal function and the development of coronary heart disease. Circulation 27: 237, 1963 11. Manchester JH, Herman MV, Gorlin R: Premenopausal castration and documented coronary atherosclerosis. Am J Cardiol 28: 33, 1971 12. Wuest JH Jr, Dry TJ, Edwards JE: The degree of coronary atherosclerosis in bilaterally oophorectomized women. Circulation 7: 801, 1953 13. Novak ER, Williams TJ: Autopsy comparison of cardiovascular changes in castrated and normal women. Am J Obstet Gynecol 80: 863, 1960 14. Kannel WB, Hjortland M, McNamara PM, Gordon T: Menopause and risk of cardiovascular disease: The Framingham Study. Ann Intern Med 85: 447, 1976 15. Winkelstein W Jr, Rekate AC: Age trend of mortality from coronary artery disease in women and observations on the reproductive patterns of those affected. Am Heart J 67: 481, 1964 16. Tracy RE: Sex difference in coronary disease: Two opposing views. J Chron Dis 19: 1245, 1966 17. Rivin AU, Dimitroff SP: The incidence and severity of atherosclerosis in estrogen-treated males, and in females with a hypoestrogenic or a hyperestrogenic state. Circulation 9: 533, 1954 18. Higano N, Robinson RW, Cohen WD: Increased incidence of cardiovascular disease in castrated women. Two-year follow-up studies. N Engl J Med 268: 1123, 1963 19. Rosenberg L, Armstrong B, Jick H: Myocardial infarction and estrogen therapy in post-menopausal women. N Engl J Med 294: 1256, 1976 20. Lorenz FW, Chaikoff IL, Entenman C: The endocrine control of lipid metabolism in the bird. II. The effects of estrin on the blood lipids of the immature domestic fowl. J Biol Chem 126: 763, 1938 21. Pick R, Stamler J, Rodbard S, Katz LN: The inhibition of coronary atherosclerosis by estrogens in cholesterol-fed chicks. Circulation 6: 276, 1952 22. Prichard RW, Clarkson TB, Lofland HB: Estrogen in pigeon atherosclerosis. Arch Pathol 82: 15, 1966 23. Souadjian JV, Kottke BA, Titus JL: Estrogen effect on spontaneous atherosclerosis. Experimental studies in white Carneau pigeon. Arch Pathol 85: 463, 1968 24. Hanash KA, Kottke BA, Greene LF, Titus JL: Effects of conju'gated estrogens on spontaneous atherosclerosis in pigeons. Arch Pathol 93: 184, 1972 25. Moskowitz MS, Moskowitz AA, Bradford WL, Jr., Wissler RW: Changes in serum lipids and coronary arteries of the rat in response to estrogens. Arch Pathol 61: 245, 1956 26. Gammal EB: Intimal thickening in arteries of rats treated with synthetic sex hormones. Br J Exp Pathol 57: 248, 1976 27. Wolinsky H: Effects of estrogen treatment on the oophorectomized female rat aorta. Proc Soc Exp Biol Med 144: 864, 1973 28. Cutts JH: Vascular lesions resembling polyarteritis nodosa in rats undergoing prolonged stimulation with oestrogen. Br J Exp Pathol 47: 401, 1966 29. Ludden JB, Bruger M, Wright IS: Effect of testosterone propionate and estradiol dipropionate on the cholesterol content of the blood and aorta of rabbits. Endocrinol 28: 999, 1941 30. Ludden JB, Bruger Ml Wright IS: Experimental atherosclerosis. IV.
32.
33. 34. 35. 36. 37.
38.
39. 40.
41. 42.
43. 44. 45.
46. 47. 48.
49. 50. 51.
52. 53.
54. 55. 56.
57.
58. 59. 60. 61. 62.
63.
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Effect of testosterone propionate and estradiol dipropionate on experimental atherosclerosis in rabbits. Arch Pathol 33: 58, 1942 Bruger M, Wright IS, Wiland J: Experimental atherosclerosis. V. Effect of testosterone propionate and estradiol dipropionate on the cholesterol content of the blood and the aorta in castrate female rabbits. Arch Pathol 36: 612, 1943 Fillios LC, Mann GV: The importance of sex in the variability of the cholesteremic response of rabbits fed cholesterol. Circ Res 4: 406, 1956 Goldzieher JW, Joshi S, Kraemer DC: Non-human primates in contraceptive research. Acta Endocrinol (Copenhagen) (suppl 185): 90, 1974 Goncharov N, Aso T, Cekan Z, Pachalia N, Diczfalusy E: Hormonal changes during the menstrual cycle of the baboon (Papio hamadryas). Acta Endocrinol (Copenhagen) 82: 396, 1976 McGill HC Jr, Mott GE, Bramblett CA: Experimental atherosclerosis in the baboon. Primates Med 9: 41, 1976 Hummer RL: Observations of the feeding of baboons. In Feeding and Nutrition of Nonhuman Primates, edited by Harris RS. New York, Academic Press, 1970, pp 183-203 Lindgren FT, Elliott HA, Gofman JW: The ultracentrifugal characterization and isolation of human blood lipids and lipoproteins, with applications to the study of atherosclerosis. J Phys Colloid Chem 55: 80, 1951 Foster LB, Dunn RT: Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin Chem 19: 338, 1973 Zilversmit DB, Davis AK: Microdetermination of plasma phospholipids by trichloroacetic acid precipitation. J Lab Clin Med 35: 155, 1950 Guzman MA, McMahan CA, McGill HC Jr, Strong JP, Tejada C, Restrepo C, Eggen DA, Robertson WB, Solberg LA: Selected methodologic aspects of the International Atherosclerosis Project. Lab Invest 18: 479, 1968 Guzman MA, McMahan CA, Strong JP: Unaided visual estimation of atherosclerotic lesions. Lab Invest 31: 398, 1974 Snedecor GW, Cochran WG: S+atistical Methods, ed 6. Ames, Iowa, Iowa State University Press, 1967 Miettinen TA, Ahrens EH Jr, Grundy SM: Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 6: 411, 1965 Ostle B: Statistics in Research, ed 3. Ames, Iowa, Iowa State University Press, 1963 Li CC: Introduction to Experimental Statistics. New York, McGrawHill, 1964 Siegel S: Nonparametric Statistics for the Behavioral Sciences. New York, McGraw-Hill, 1956 Maxwell AE: Analysing Qualitative Data. London, Methuen, 1961. Glueck CJ, Scheel D, Fishback J, Steiner P: Progestagens, anabolicandrogenic compounds, estrogens: effects on triglycerides and postheparin lipolytic enzymes. Lipids 7: 110, 1972 Beck P: Contraceptive steroids: Modifications of carbohydrate and lipid metabolism. Metabolism 22: 841, 1973 Warren MP: Metabolic effects of contraceptive steroids. Am J Med Sci 265: 4, 1973 Weiss NS: Relationship of menopause to serum cholesterol and arterial blood pressure: The United States Health Examination Survey of Adults. Am J Epidemiol 96: 237, 1972 Hjortland MC, McNamara PM, Kannel WB: Some atherogenic concomitants of menopause: The Framingham Study. Am J Epidemiol 103: 304, 1976 de Alvarez RR, Jahed FM, Spitalny KJ: The influence of oral contraceptive steroids on serum lipids. Am J Obstet Gynecol 116: 727, 1973 Strong JP, McGill HC Jr: Diet and experimental atherosclerosis in baboons. Am J Pathol 50: 669, 1967 Howard AN, Gresham GA, Bowyer DE, Lindgren FT: Aortic and coronary atherosclerosis in baboons. Prog Biochem Pharmacol 4: 438, 1968 Kritchevsky D, Davidson LM, Shapiro IL, Kim HK, Kitagawa M, Malhotra S, Nair PP, Clarkson TB, Bersohn I, Winter PAD: Lipid metabolism and experimental atherosclerosis in baboons: influence of cholesterol-free, semi-synthetic diets. Am J Clin Nutr 27: 29, 1974 Strong JP, Eggen DA, Jirge SK: Atherosclerotic lesions produced in baboons by feeding an atherogenic diet for four years. Exp Mol Pathol 24: 320, 1976 Armstrong ML, Megan MB, Warner ED: Intimal thickening in normocholesterolemic rhesus monkeys fed low supplements of dietary cholesterol. Circ Res 34: 447, 1974 Wexler BC: Spontaneous arteriosclerosis in repeatedly bred male and female rats. J Atheroscler Res 4: 57, 1964 Cockrell BY, Valerio MG, Loeb WF: Fibrous plaques in the aortas of adult female rhesus monkeys (Macaca mulatta). J Med Primatol 4: 120, 1975 Stary HC, Strong JP: Coronary artery fine structure in Rhesus monkeys: Nonatherosclerotic intimal thickening. Primates Med 9: 321, 1976 McGill HC Jr, Strong JP, Holman RL, Werthessen NT: Arterial lesions in the Kenya baboon. Circ Res 7: 670, 1960 Stary HC: Coronary artery fine structure in Rhesus monkeys: The early atherosclerotic lesion and its progression. Primates Med 9: 359, 1976
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Estrogens and experimental atherosclerosis in the baboon (Papio cynocephalus). H C McGill, Jr, L R Axelrod, C A McMahan, H S Wigodsky and G E Mott Circulation. 1977;56:657-662 doi: 10.1161/01.CIR.56.4.657 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1977 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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SUMMARY One hundred twenty-six adult female baboons (Papio cynocephalus) were hysterectomized and all except 18 were ovariectomized. The animals were fed a moderately atherogenic diet (40% calories from hydrogenated vegetable oil, 1.5 mg cholesterol/kcal) for two years. Ovariectomized-hysterectomized animals received estrone sulfate, ethynyl estradiol, or diethylstilbestrol orally in daily doses similar to those given humans. An ovariectomized-hysterectomized group and a hysterectomized group received no drug. The average total serum cholesterol concentration rose from 136 mg/dl to 223 mg/dl and declined to 186 mg/dl. Concentrations of cholesterol,
changes in arteries of rats25 28; and augmented, inhibited, and failed to affect diet-induced hypercholesterolemia and atherosclerosis in rabbits.29 32 In the hope of resolving some of these conflicting observations, this experiment was planned to examine the effects of ovariectomy and estrogen replacement therapy on serum lipids and atherogenesis in the baboon, whose reproductive physiology, estrogen and cholesterol metabolism, greater prevalence of aortic fatty streaks in the feral female compared to the feral male, and response to an atherogenic diet are more human-like than are those of chickens, rats, and rabbits.33 In order to induce moderate hyperlipidemia and experimental atherosclerosis, the animals were fed a mildly atherogenic diet for two years. The estrogens tested were estrone sulfate, a biologically active conjugate of estrone and a major circulating estrogen in humans; 17ca-ethynyl estradiol, a highly potent, orally effective synthetic estrogen structurally similar to estradiol; and diethylstilbestrol, a nonsteroidal compound differing in chemical structure from the natural estrogens but having estrogenic activity.
THERE ARE CONFLICTING OBSERVATIONS on the relationship between estrogens and atherogenesis in both humans and experimental animals. Premenopausal white women consistently have a lower incidence and prevalence of myocardial infarctionl-' and a lessened extent of advanced atherosclerotic lesions in the coronary arteries5' 6 than do white men of the same age. However, some studies show that women ovariectomized before the natural menopause lose their relative immunity to coronary heart disease,7-9 while others find no effect of ovariectomy.0' 11 One study finds atherosclerosis more severe in ovariectomized women,'2 but another finds no difference." According to observations from The Framingham Study, postmenopausal women have a higher incidence of coronary heart disease than age-matched premenopausal women.'4 Despite a prevailing impression that the menopause increases risk of coronary heart disease, age and sex specific mortality rates do not increase after the menopause more than would be expected with age.3' 15 "16 Attempts to reduce the incidence of coronary heart disease in postmenopausal women by estrogen replacement therapy also have yielded conflicting
results.'7 '9
Methods and Materials
The results of animal experiments on the relationship between estrogens and atherogenesis are as inconsistent as those from human studies. Estrogens induced hyperlipidemia in chickens20 but inhibited coronary artery lipidosis in cockerels21; reduced the size of plaques in White Carneau pigeons in high doses22-24; produced a variety of
Subjects
The subjects were 126 adult female baboons (Papio cynocephalus) ranging in estimated age from 5 to 15 years. The animals were of feral origin, but many had been in the Southwest Foundation for Research and Education (SFRE) breeding colony for several years. During this experiment, they were individually caged. All animals were hysterectomized to eliminate the uterine bleeding that occurs with continuous estrogen therapy. All but 18 also were ovariectomized at the time of hysterectomy. Eight animals died during the experiment from causes unrelated to the experimental procedure. On the average, these did not differ in age, serum lipids, or weight from the remaining 118.
From the Southwest Foundation for Research and Education, and the Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas. Supported by grants HE-14112 and HL-15914 from the National Heart, Lung and Blood Institute. Dr. Leonard R. Axelrod, who designed and initiated this experiment, left the Southwest Foundation for Research and Education on May 1, 1973, to become Director of the Criteria and Evaluation Division and Chief Scientist of the Office of Pesticide Programs, Environmental Protection Agency, Washington, D.C. The experiment was completed in his absence. Dr. Axelrod died on July 31, 1975. The analyses and interpretation of data contained in this report were completed after Dr. Axelrod's death. Address for reprints: Henry C. McGill, Jr., M.D., The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio,
Experimental Design
Although the original principal investigator (LRA) did not use a strict randomization procedure for the assignment of treatments to animals, we analyzed the results as a com-
Texas 78284. Received March 21, 1977; revision accepted May 18, 1977.
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CIRCULATION
TABLE 1. Selected Characteristics of Experimental Animals, Ovarian Status, and Estrogen Treatment by Experimental Groups
Group
1 2 3 4 5 6
7
Number of baboons Start End
18 18 18 18 18 18 18
17 17 18 17 15 16 18
Estimated age, years Mean SD
At start of experiment Weight, Serum kg oholesterol Mean SD Mean SD
Days after operation Mean SD
11.6 11.1 10.9 11.5 11.8 11.3 11.2
14.3 14.3 14.3 14.0 14.9 14.0 14.7
126.8 157.5 133.2 146.1 155.4 104.0 61.7
2.3 2.2 1.7 1.7 1.4 1.7 1.1
1.9 1.6 1.6 2.3 2.1 1.7 1.4
131.3 138.1 141.9 139.2 130.6 129.6 136.9
Estrogen treatment
19.9 26.4 24.1 29.1 34.9 21.8 19.0
Abbreviations: ES = estrone sulfate; EE = 17 a-ethynyl estradiol; DES
=
94.0 84.7 86.5 76.8 92.2 69.6 13.9
Duration,
Ovarian status
Year 1
Year 2
days Mean SD
Ablated Ablated Ablated Ablated Ablated Ablated Intact
ES None EE None DES None
ES ES EE EE DES None
625.8 11.3 374.6 8.3 624.5 12.1 374.1 7.9 623.8 11.3
None
None
-
diethylstilbestrol.
pletely randomized design with seven treatments. There was evidence of bias in the assignment process. Table 1 outlines characteristics of the treatment groups. All animals received the atherogenic diet from the beginning of the experiment. Drug treatment for groups 1, 3, and 5 began on day 133 after initiating the diet. Drug treatment for groups 2 and 4 began on day 498 to examine the possibility that estrogens might modify lesions previously established by dietinduced hyperlipidemia.
Its caloric value was about 3.8 kcal/g wet weight. Each baboon received 360 g per day, about 90 kcal/kg/day, for the first 487 days of the experiment. Because many animals gained excess weight, the amount offered each day thereafter was reduced by 20%. The final diet mixture contained 5.62 mg/g (wet weight) cholesterol, 0.12 mg/g campesterol, and 0.40 mg/g 3-sitosterol by gas-liquid chromatographic analysis. Thus, the diet contained about 1.5 mg cholesterol per kcal.
Estrogen Dosage and Administration
Blood Collection and Serum Lipid Analyses
Estrogen doses approximated, on a weight basis, the usual human doses for replacement therapy. Drugs were administered by embedding tablets in fig bars. The rate of failure to take the drug ranged from 0.1% of treatment days in group 2 to 1.5% in group 3. The overall omission rate for all groups was 0.64%, and only one animal had an omission rate greater than 5%. Baboons in groups 1 and 2 received 0.625 mg/day, or about 45 Ag/kg/day, of Ogen (Abbott Laboratories) of pure crystalline estrone sulfate (ES) stabilized with piperazine. This dose in baboons may equal a higher dose in humans since plasma concentrations of estrogenic steroids are lower in baboons than in humans.34 Baboons in groups 3 and 4 received 17 ,tg/day, or about 1.2 ,tg/kg/day, of Estinyl (Schering Corporation) as the source of 17a-ethinyl estradiol (EE). Baboons in group 5 received 62.5 ,ug/day, or about 5 ,ug/kg/day, of diethylstilbestrol, USP (DES) (Eli Lilly and
Thirty ml of blood were collected at 4 month intervals in Vacutainers without anticoagulant (Becton Dickinson Co.), with the animal under Sernylan anesthesia, about 0.8 mg/kg (phencyclidine hydrochloride) (Bio-Ceutic Laboratories, Inc.) Four lipoprotein classes, separated from 5 ml serum samples by the preparative ultracentrifugal method of Lindgren and associates,37 were analyzed for cholesterol, triglyceride, and phospholipid. The Bioregional Reference Laboratory, Inc. analyzed whole serum and lipoprotein fractions for cholesterol, triglyceride, and phospholipid. Cholesterol determinations were performed early in the study on a SMA 12/60 Autoanalyzer (Technicon Instruments Corp.) with a Liebermann-Burchard reagent, and later in the study on an ABA100 (Abbott Laboratories Diagnostic Division) by an enzymatic method. Triglycerides were analyzed by extraction, silicic acid chromatography, and the colorimetric method of Foster and Dunn.38 Quality control was maintained by repeated analyses of commercial pooled serum samples. The coefficient of variation for the cholesterol analyses was 2.4% and for triglycerides, 8.7%. Both methods met the criteria established for clinical laboratories and received a satisfactory score of 3 from the Center for Disease Control. Phospholipids were determined by the method of Zilversmit and Davis,39 with a coefficient of variation of 4% for repeated analyses.
no
Co.).
Diet
The baboons had been fed SFRE baboon chow36 (Ralston Purina Co.) from the time of their arrival at SFRE until beginning the atherogenic diet for this experiment. This diet ktable 2) was prepared by mixing ground baboon chow, hydrogenated vegetable oil (Crisco, Procter and Gamble Co.), frozen egg yolk, cholesterol, and 6 ml water per 100 g.
TABLE 2. Composition and Nutrient Value of the Atherogenic Diet per 100 g Dry Weight Component
Baboon chow Crisco Egg yolk, frozen Cholesterol, USP Total
Total g
67.1 11.7 20.8 0.4 100.0
Nutrients, g
Protein
Fat
Carbohydrate
Fiber
Ash
Cholesterol mg
14.1 0 3.1 0
3.7 11.7 5.7 0 21.1
32.9 0
2.8 0 0
5.2 0 0 0 5.2
4 0 239 366 609
17.2
2.1 0
35.0
0
2.8
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ESTROGENS AND ATHEROSCLEROSIS IN BABOONS/McGill et al. Autopsy Procedures We sedated the animals with an intramuscular injection of Sernylan and performed complete autopsies, including gross and histologic examination of all major viscera. The iliac, femoral, innominate, brachial, carotid, and coronary arteries were opened longitudinally and prepared by methods used in the International Atherosclerosis Project."0 The aorta from the ligamentum arteriosus to its bifurcation was divided along anterior and posterior midlines. The right half of the aorta was frozen for chemical analysis, and the left half, after being sampled for electron microscopy, was prepared for grading like the other arteries. Grading of Atherosclerotic Lesions Two experienced pathologists (one of the authors, H.C. McGill; and a consultant, J.P. Strong) independently graded the stained arterial specimens by the method of the International Atherosclerosis Project.'1 Graders were not aware of treatment group or other data, but all the arteries from each animal (except the coronary arteries) were in the same plastic bag. Product moment correlation coefficients between grades of different arterial segments did not exceed 0.34, a value which indicated that the grades of some segments did not unduly influence the grades of other segments. The intraclass correlation coefficient42 between estimates by the two pathologists of percent intimal surface area involved with fatty streaks for the aorta ranged from 0.77 to 0.84, values indicating acceptable interobserver variability of the grading. Independent regrading of 20 sets of specimens at a later time indicated that variability within pathologist was similar to variability between pathologist. We compared aortic fatty streaks among treatment groups on the basis of extent of intimal surface involved. Because grades of fibrous plaques in all arteries and of fatty streaks in some arteries frequently were small or zero, we compared these lesions among treatment groups on the basis of prevalence rather than extent. Specimens were scored as positive only if both pathologists independently classified the specimen as positive. Aortic Intimal Cholesterol
A technician stripped the intima from the unfixed half of each aorta. Lyophilized and saponified tissue was analyzed by gas-liquid chromatography.43 A technician measured the total intimal surface area of the unfixed half of the aorta from full size photographs using an electronic digitizer (Wang 662). The coefficient of variation after retracing a randomly selected sample of 20 photographs was 2.0%. From the area and weight of the intima and the concentration of cholesterol, we estimated cholesterol per unit area of intimal surface. Statistical Methods Responses were analysed in separate univariate analyses, usually by analysis of variance (ANOVA)." For ANOVAs that resulted in a statistically significant overall F test, all pairwise contrasts between treatment group means were computed and evaluated with Tukey's t-test.4' Analyses by the Kruskal-Wallis ANOVA for ranks46 confirmed the
659
results of the parametric ANOVAs. In some instances, as noted, analysis of covariance (ANCOVA)44 was used. For data such as presence of lesions, we tested the null hypothesis of independence between treatment groups and presence of lesions by a Chi-square test.'2 Results Evidence of Estrogen Stimulation
All baboons in the group with intact ovaries (7) continued normal menstrual cycles throughout the experiment as indicated by periodic turgescence of the sex skin. The ovariectomized group (7) showed no turgescence of the sex skin. Baboons in groups which received estrogens (1-5) showed moderate sustained turgescence of the sex skin, an observation indicating that they were receiving estrogenic stimulation. Body and Organ Weights
The group receiving EE from the start of the experiment (3) gained less weight than other groups, and the group receiving EE in the second year (4) also gained less weight after hormone treatment began. The DES treated group (5) lost weight steadily after about day 300 of the experiment; the weight loss began before the daily diet ration was reduced. Mean weights of all groups fluctuated over time in a manner apparently unrelated to treatment. Mean body weights at autopsy of the two EE treated groups (3 and 4) were lower than those of all other groups, and pairwise comparisons by ANCOVA with initial body weight as the concomitant variable were statistically significant for these two groups as compared to the group treated with ES for two years.' Organ weights at autopsy, analyzed by ANCOVA with total body weight at autopsy as the concomitant variable, showed no real differences. Serum Lipids and Lipoproteins
The mean total serum cholesterol concentration of all baboons rose in the first three months of the experimental period from 136 mg/dl to 223 mg/dl, declined during the following 18 months to 186 mg/dl, and increased slightly near the end of the experiment. We compared serum lipid and lipoprotein concentrations among groups by ANOVA, using values from two blood samples drawn between 653 and 814 days after the start of the experiment. We reasoned that long term effects of the drugs should be apparent during this period, and that they would not be masked by the large changes associated with beginning the atherogenic diet. Table 3 shows results for cholesterol, triglyceride, and phospholipid in whole serum, LDL, and HDL. Chylomicrons and VLDL (not included in table 3) contained only a minor fraction of total lipids and showed no consistent differences among groups. Neither cholesterol nor phospholipid concentrations in lipoprotein fractions or in whole serum differed between the ovariectomized group (6) and the group with intact ovaries (7). Total triglyceride, LDL triglyceride, and HDL triglyceride concentrations were higher in all estrogen treated groups (1-5) and in the group with intact ovaries (7) than in the ovariectomized group (6). However, statistically significant pairwise comparisons were limited to groups 1 and 3. Phospholipid concentrations were slightly higher in the
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VOL 56, No 4, OCTOBER 1977
TABLE 3. Statistical Comparisons of Serum Lipids and Lipoproteins
Fraction of Serum
Cholesterol LDL HDL Total Triglyceride LDL HDL Total Phospholipid LDL
1
2
92.6 70.6 199.3
77.8 62.0 165.2
6
7
mean
Estimated components of variance Between Within animal animal
79.3 64.0 174.1
83.4 66.4 175.6
83.4 64.0 176.5
251.3 473.8 272.3 387.4 1114.6 379.6
Treatment group means 3 4 5
82.1 67.4 182.5
78.3 58.1 158.3
91.9 59.5 180.3
Overall
Significant* pairwise
F
comparisons
1.25 0.70 2.25*
None None 1-4 1-6,3-6 1-6X3-6 3-6
34.1
27.9
33.6
31.2
23.8
21.5
27.7
28.7
76.8
78.5
3.11**
29.9 94.4
26.9 81.6
29.9 98.6
24.5 82.7
24.4 82.2
20.9 70.4
26.9 79.1
26.3 84.4
7.9 472.3
74.9 172.7
3.63**
2.77*
96.6
76.4
93.3
64.1
74.3
66.9
78.1
78.5
113.6
636.6
5.86**
1-4,1-6,3-4, 3-6
HDL
165.5
132.2
168.0
138.4
161.5
154.3
154.7
153.1
241.0
455.6
6.55**
1-2,1-4,2-3,
3-4,2-5,4-5, 2-7
Total
292.7
259.1
292.6
244.9
266.8
256.8
265.6
268.7
954.0
508.9
1-4,3-4,3-6
4.67**
N = 118 baboons, approximately 2 values/animal between 650 and 820 days. *Statistically significant at 0.05 level. **Statistically significant at 0.01 level.
treated with ES and EE for 2 years (1 and 3) than in controls or other treatment groups, and several of the differences were statistically significant. The decrease in caloric intake after day 487 on the atherogenic diet showed no relationship to changes in cholesterol, triglyceride, or phospholipid concentrations in whole serum or in lipoprotein fractions.
groups
Characteristics of Arterial Lesions
Table 4 shows overall measures of fatty streaks and fibrous plaques by artery. Grossly, the lesions resembled those previously described in the baboon.35 Although some coronary artery lesions were elevated and resembled fibrous plaques, none produced significant stenosis. Microscopically, all lesions showed intimal thickening with smooth muscle cells. Lesions classified grossly as fibrous plaques were principally smooth muscle cells with varying amounts of lipid in the subintimal layer, and occasionally a core of necrotic debris and extracellular lipid. Electron micrographs from the thoracic and abdominal aorta and coronary arteries of several animals from each experimental group showed that most intimal lipid was within smooth muscle cells, but some was extracellular and some was in macrophages. We detected no qualitative differences among the experimental groups in histologic sections or electron micrographs. Aortic Fatty Streaks
Table 5 shows treatment group means of percent surface involvement with fatty streaks by aortic segments. The component of variance within animal (due to interobserver difference in grading) was less than the component of variance between animals. Only two pairwise comparisons were in the abdominal aorta, DES statistically significant treated (5) and ovariectomized untreated (6) groups were more extensively involved by fatty streaks than the untreated group with intact ovaries (7). In five of the other arteries, the null hypothesis of independence between treatments and presence of fatty streaks was -
rejected (P < 0.05). These five were further examined by partitioning the degrees of freedom.47 The group treated with EE for two years (3) had a significantly lower prevalence of fatty streaks in the right carotid, left carotid, left brachial, and left circumflex coronary arteries. The group treated with ES in the second year (2) and the ovariectomized group (6) had a significantly higher prevalence of fatty streaks in the left coronary circumflex artery than other groups. The group treated with ES from the beginning (1) and the group with intact ovaries (7) had a significantly lower prevalence of fatty streaks in the left anterior descending coronary artery than did all other groups. Concentration of Cholesterol in Aortic Intima
The overall means of concentrations of cholesterol in the stripped aortic intima were 25.0 mg/g and 111.9 mg/cm2, TABLE 4. Percent of Intimal Surface Involved with Fatty Streaks and Prevalence of Fatty Streaks and Fibrous Placques by Arterial Segment Intimal surface involved with fatty streaks,
percentt
Arterial segment
Aortic arch
Thoracic aorta Abdominal aorta Right carotid Left carotid Right brachial Innominate Left brachial Right iliac-femoral Left iliac-femoral Right coronary Left coronary, ant desc. Left coronary, circumflex
Mean
Range Low High
11.8 35.1 18.6 7.1 6.7 6.1 12.3 11.3 3.7
0. 1. 0. 0. 0. 0. 0. 0. 0.
3.3 0.4
Prevalence,
percent positive Fatty Fibrous streaks plaques
0. 0.
73.1 86.6 81.7 54.5 27.0 28.4 59.4 45.5 42.1 42.1 17.3
91.5 96.6* 97.5 90.7 90.7 84.7 82.2 93.2 79.7 78.0 7.6
3.4 5.1 13.6 5.9 9.3 2.5 0.8 9.3 15.3 16.1 0.8
1.2
0.
17.3
31.4
2.5
0.7
0.
17.9
18.6
0.0
N = 118 baboons. *Prevalence less than 100% although lowest percent surface involvement is not 0 because range is from the mean of independent estimates and prevalence is based on both observers scoring specimen as positive. tMean of independent estimates by 2 observers.
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ESTROGENS AND ATHEROSCLEROSIS IN BABOONS/McGill et al.
661
TABLE 5. Percentage Intimal Surface Area Involvement of Aortic Segments with Fatty Streaks Estimated
Overall Group
Aortic
segment
Arch Thoracic Abdominal
1
15.8 42.2 15.2
mean
mean
percent surface
2
3
4
5
6
7
17.8 37.8 24.6
9.5 27.0 10.9
13.0 34.3 17.6
12.4 28.0 27.2
10.3 31.8 27.2
4.2 43.3 9.8
percent surface
11.8 35.1 18.6
components F
Significant* pairwise comparisons
1.92 1.15 3.70**
None None 5-7,6-7
of variance
Between Within
animals animals
159.3 571.5 215.0
43.0 107.5 73.2
*Statistically significant at 0.05 level. **Statistically significant at 0.01 level.
and overall estimated standard deviations were 17.8 and 60.3. No real differences among the treatment groups were apparent by either measure. Fibrous Plaques
The null hypothesis of independence between treatments and presence of fibrous plaques was rejected (P < 0.05) only for the left iliac-femoral artery. The group treated with ES for the second year (2) and the group treated with DES (5) had a significantly higher prevalence of fibrous plaques as indicated by partitioning the degrees of freedom.
Discussion Serum Lipids and Lipoproteins
The slight and consistent, but not always statistically significant, elevations in serum triglyceride and phospholipid concentrations in the estrogen treated groups and in the group with intact ovaries compared to ovariectomized untreated controls are consistent with the reported effects of estrogenic compounds in humans.48s60 The difference we observed, however, is small, and slight elevations of triglyceride and phospholipid concentrations in the absence of increased cholesterol levels would not be expected to augment atherogenesis. Furthermore, no major shifts in distribution of lipids among the lipoproteins appear to result either from ovariectomy or estrogen replacement. The failure to find a higher serum cholesterol concentration in ovariectomized untreated baboons than in those with intact ovaries or in those receiving estrogens is not consistent with the elevation in serum cholesterol associated with surgical or natural menopause in humans.7' 8, 14, 51, 52 Detailed comparison of these results with observations on other animal specieS20-25, 29-32, 53 shows some similarities but many differences. Atherosclerosis
The comparisons of arterial lesions in this experiment indicate no consistent effect of estrogen deficiency or estrogen replacement on extent or quality of atherosclerotic lesions. The results, therefore, are consistent with reports that ovariectomized women have no greater atherosclerosis at autopsy'3 and experience no higher incidence or prevalence of atherosclerotic disease than women with intact ovaries.10-1' They are consistent with reports that estrogen replacement therapy does not influence coronary heart disease prevalence in postmenopausal women.'9 They also are consistent with reports that physiological doses of estrogens do not reduce spontaneous atherosclerosis in pigeons23' 24 or experimental atherosclerosis in rabbits.2' 31 32 We have
found no reports of the effects of estrogens on diet-induced atherosclerosis in nonhuman primates. The extent of fatty streaks exceeded that produced in other baboons by similar moderate levels of hyperlipidemia.35 '4-57 This difference may be due to age or sex, since most experiments with atherogenic diets in baboons have used young adult males rather than females. The abundant smooth muscle and fibrous tissue in fatty streaks may be typical of the reaction of nonhuman primates to moderate prolonged hyperlipidemia, as observed in rhesus monkeys.58 These lesions differ from the foam cell lesions typical of experimental animals with higher serum cholesterol levels. Multiple pregnancies also may have caused smooth muscle proliferation, since Wexler59 has described nonlipid containing intimal thickening in the arteries of breeder female rats. Most of the baboons were multiparous, but because they were feral, accurate assessment of parity was not possible. Lesions classified grossly as fibrous plaques resembled fibromuscular cushions described in the coronary arteries and aortas of normal rhesus monkeys80' 61 and feral baboons autopsied immediately after capture.82 We believe that our baboons probably had developed many of these plaques prior to the experiment. The relatively high frequency of fibrous plaques in the femoral arteries may have been due to trauma to the arteries during venipuncture. Hyperlipidemia may have augmented fibrous plaques, and probably was responsible for lipid deposition within them as reported for hyperlipidemic rhesus monkeys.83 Whatever their origin, they do not appear to have been affected either by ovariectomy or estrogen replacement. Conclusions
In the female baboon fed a moderately atherogenic diet for two years, neither ovariectomy nor estrogen replacement after ovariectomy produces consistent and statistically significant differences in serum lipid or lipoprotein concentrations or in atherosclerosis. There is a trend (not statistically significant) for baboons with intact ovaries or ovariectomized baboons receiving exogenous estrogens to have slightly higher serum triglyceride and phospholipid concentrations than ovariectomized baboons. The results are consistent with epidemiologic studies of humans which show no effects of either estrogen deficiency or estrogen replacement therapy on serum lipid or lipoprotein concentrations or on atherosclerosis.
Acknowledgments The authors gratefully acknowledge the assistance of Dr. Joseph W. Goldzieher in completing the experiment after Dr. Axelrod's death and in reviewing the manuscript; of Dr. Thomas W. Culp in supervising the serum lipid analyses; and of Dr. Jack P. Strong in grading the arterial specimens. Cornelio Celaya and Ignacio Gomez cared for the baboons throughout the experi-
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662
CIRCULATION
ment, Mary Jo Haas performed the lipoprotein fractionations, and Cynthia Gaudot performed the aortic lipid analyses.
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Estrogens and experimental atherosclerosis in the baboon (Papio cynocephalus). H C McGill, Jr, L R Axelrod, C A McMahan, H S Wigodsky and G E Mott Circulation. 1977;56:657-662 doi: 10.1161/01.CIR.56.4.657 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1977 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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