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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:562-570

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:562-570.)
© 1995 American Heart Association, Inc.

Articles

Effects of Androgens on Coronary Artery Atherosclerosis and Atherosclerosis-Related Impairment of Vascular Responsiveness

Michael R. Adams; J. Koudy Williams; Jay R. Kaplan

From the Department of Comparative Medicine and Comparative Medicine Clinical Research Center, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC.

Correspondence to Michael R. Adams, DVM, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040.


*    Abstract
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 *Abstract
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Abstract The factors responsible for the marked gender differences in risk of coronary heart disease and atherosclerosis severity remain largely undetermined. While some clinical and experimental evidence supports a protective effect of endogenous estrogen on the initiation and progression of atherosclerosis and incidence of coronary heart disease, much of the epidemiological data do not support this conclusion. The possibility that endogenous androgens may have adverse effects on atherosclerosis progression and coronary risk has received little attention. We investigated the effects of experimentally induced hyperandrogenism in female cynomolgus monkeys with diet-induced atherosclerosis. Animals were assigned randomly to one of four treatment groups: (1) untreated controls, (2) ovariectomized (sex hormone-deficient) controls, (3) treated with androstenedione and estrone (mild hyperandrogenism), or (4) treated with testosterone (male plasma androgen pattern). At necropsy, coronary atherosclerosis was approximately twice as extensive (P<.05) in testosterone-treated animals relative to untreated controls, while treatment with androstenedione and estrone had no effect on atherosclerosis extent. Coronary plaque size was positively correlated with lumen size in intact and ovariectomized controls; however, there was no evidence of a similar relation between animals in either androgen treatment group. The atherogenic effects of testosterone were independent of variations in plasma lipoprotein and nonlipoprotein risk variables. Although chronic hyperandrogenism had adverse effects on atherosclerosis progression, it reversed (P<.03) atherosclerosis-related impairment of endothelium-dependent vasodilator responses. We conclude that an experimentally induced male plasma androgen pattern results in exacerbation of diet-induced atherosclerosis and has potentially adverse effects on atherosclerosis-related arterial remodeling in female monkeys. The results indicate that testosterone may have a direct role in the increased rate of atherosclerosis progression and increased risk of coronary heart disease seen in men, relative to women, in most Western societies.


Key Words: atherosclerosis • coronary heart disease • cynomolgus monkeys • vasomotion • androgens


*    Introduction
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 *Introduction
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In most Western societies, male gender is a powerful risk factor for coronary heart disease (CHD). Men of all ages are at higher risk than similarly aged women. Although this disparity is widely thought to originate in greater levels of endogenous estrogen in women, direct evidence supporting this hypothesis is lacking. The possibility that endogenous androgens have adverse effects on cardiovascular risk in men has received little attention, and the existing evidence conflicts. Morbidity and mortality data indirectly support an adverse effect of androgen. For example, women are at lower risk of CHD at all ages, even after menopause, when endogenous estrogen production decreases markedly.1 Furthermore, women show a linear increase in coronary mortality with age, with no apparent upward inflection in rate of increase at the age of menopause, while the rate of increase in men is greater prior to age 50 years and declines thereafter.1 While this may represent selective mortality, ie, many susceptible men die prior to 50 years of age, it also seems possible that the decline in androgen production that occurs in many men after 50 years of age2 3 may be responsible.

Since the effects of androgens on CHD incidence could be mediated by effects on lipoprotein metabolism, some investigators have studied relations between exogenous or endogenous androgens and plasma lipoproteins. The results conflict. While plasma concentrations of HDL cholesterol (HDL-C) fall during sexual maturation in males,4 5 in most6 7 8 9 10 11 though not all12 13 14 studies of adult men there is a positive correlation between plasma testosterone and plasma HDL-C concentrations. Furthermore, in males with hypogonadism, exogenous testosterone causes an increase in plasma HDL-C concentrations.15 16 17 In contrast, supraphysiological doses of nonaromatizable androgens result in decreased plasma HDL-C concentrations,18 19 20 and pharmacological doses of testosterone have little effect.18 19 Further confusing the issue are studies of hyperandrogenic conditions of women. Polycystic ovary syndrome (PCO) is perhaps the most common of several causes of hyperandrogenism in women. Women with PCO have irregular or absent menstrual cycles, often beginning with the onset of menstrual function.21 They frequently develop acne, hirsutism, and other manifestations of increased peripheral androgenic activity. PCO patients also have "male-like" plasma lipoprotein profiles, eg, decreased plasma HDL-C concentrations and increased ratios of LDL to HDL-C.21 22 23

Some investigators have sought to determine if an association exists between circulating androgen concentrations and risk of CHD. The results of these studies have also conflicted.15 24 25 26 27 28 Most have been retrospective case-control studies involving relatively small numbers of patients and subject to inherent design limitations. Also, most relied on a single determination of plasma sex hormone concentrations, which probably do not accurately represent cumulative exposure and which may have been determined more as sequelae of a clinical event that led to the subject's inclusion in the study.

Thus, the relations among endogenous androgens, plasma lipoproteins, atherosclerosis progression, and risk of CHD remain unclear, and to date no studies have addressed this question directly.

Also unaddressed are the effects of androgens on endothelium-dependent coronary vasomotor responses. It is well established that atherosclerotic coronary arteries have impaired endothelial function, resulting in decreased dilator and augmented constrictor responses to a variety of neurohumoral stimuli.29 30 Impaired vasomotor responses of atherosclerotic coronary arteries may contribute to the pathogenesis of coronary vasospasm, which may result in transient myocardial ischemia or promote plaque rupture, thrombosis, and myocardial infarction.31 32 33 The greater incidence of variant angina due to coronary spasm in women suggests a sex difference in vasomotor function,34 but the role of sex hormones in modulating vasomotor function is unclear.

Progression of coronary artery atherosclerosis, impaired vasomotor responsiveness, and their associations with atherosclerosis risk variables are exceedingly difficult to study prospectively in human subjects. For this reason, we have studied the effects of experimentally induced hyperandrogenism on the extent of diet-induced coronary artery atherosclerosis and the resultant impairment of coronary vasomotor responses in female cynomolgus monkeys (Macaca fascicularis). We report here that relatively mild hyperandrogenism, similar to that associated with PCO, had no effect on plasma lipoproteins and atherosclerosis, while a male-like plasma androgen pattern, though associated with minimal changes in plasma lipoproteins, resulted in a doubling of atherosclerosis extent relative to untreated controls. Furthermore, the expected positive association between atherosclerosis plaque size and arterial lumen size35 was not seen in androgen-treated animals. Somewhat surprisingly, a male-like plasma androgen pattern was associated with an improvement of coronary vasomotor responsiveness to the endothelium-dependent dilator acetylcholine.


*    Methods
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 *Methods
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Experimental Design and Procedures
Sixty-four adult female cynomolgus monkeys were imported from Indonesia. For 32 months, all animals were fed a moderately atherogenic diet (40% of calories as fat and 0.28 mg cholesterol/kilocalorie). Monkeys lived in social groups consisting of four to five individuals. All procedures involving animals were conducted in compliance with Institutional Animal Care and Use Committee policies. For surgical procedures, animals were anesthetized with ketamine hydrochloride (10 mg/kg IM) and xylazine (0.6 mg/kg IM). Data from 61 of the 64 animals are reported here. Two animals died during the treatment period of causes unrelated to the experiment. A third was removed from the experiment due to its exceedingly aggressive behavior.

Hormone Manipulations
Monkeys consumed the atherogenic diet for an 8-month pretreatment period. They were then assigned, using a stratified randomization design, to one of four experimental groups: untreated controls (n=16), ovariectomy (n=14), treated with androstenedione and estrone (n=16), or treated with testosterone (n=15). The treatment groups were well-matched with respect to pretreatment total plasma cholesterol and plasma HDL-C concentrations.

Sex hormones were administered via silicone elastomer implants. To approximate the plasma sex hormone pattern seen in women with PCO,36 monkeys receiving androstenedione and estrone were implanted subcutaneously with silicone elastomer tubing (0.335 cm inner diameter, 0.465 cm outer diameter, 2.5 cm long) filled with crystalline androstenedione (Steraloids, Inc) and identical silicone elastomer tubing (2.0 cm long) filled with crystalline estrone (Steraloids, Inc). To approximate a male sex hormone pattern, females in the fourth group were implanted subcutaneously with three similar silicone elastomer implants 3.5 cm long and filled with crystalline testosterone. Untreated controls were implanted subcutaneously with empty silicone elastomer tubing 3.0 cm long. To determine the continued effectiveness of hormone delivery, plasma androstenedione, estrone, and testosterone concentrations37 were determined at 4- to 6-month intervals.

Effects of treatment on menstrual cyclicity and ovarian function were assessed.38 39 Briefly, animals were trained to leave their group pens and enter transfer cages, from which they moved through an enclosed ramp into individual squeeze cages. There they learned to present a leg for venipuncture and the perineum for vaginal swabbing, after which they were quickly returned to their group cages. Menstrual cyclicity and ovarian function were assessed by a combination of the presence or absence of menstrual bleeding and plasma estrogen and progesterone concentrations.38 39

Risk Variables
Total plasma cholesterol,40 triglycerides,40 and HDL-C41 concentrations were determined at 2-month intervals. At months 6 and 18, plasma lipoprotein patterns were assessed. Lipoprotein fractions were separated by ultracentrifugation and agarose column chromatography,42 and the cholesterol content of each fraction was quantified.43 In macaques, four major fractions are obtained. In addition to VLDL, LDL, and HDL, a fourth peak, IDL, intermediate in molecular weight to VLDL and LDL, is seen. In addition, average LDL molecular weight was determined for each sample by including a trace amount of iodinated LDL of known molecular weight.44

Polyacrylamide gradient gel electrophoresis (4% to 30%; Pharmacia) was used at months 6 and 18 to assess HDL subfractional size heterogeneity.45 46

Plasma concentrations of apoB47 and apo A-I48 were determined at months 6 and 18 by using enzyme-linked immunosorbent assays. Blood pressure49 was determined at 6-month intervals.

Fasting blood glucose50 and insulin51 and the blood glucose and insulin responses to intravenous glucose challenge51 were determined at months 6 and 18.

Body weight was determined at 2-month intervals. Body mass index was calculated as the ratio of body weight to body length (the distance from the suprasternal notch to the pubic symphysis). Skinfold thicknesses were measured with calipers, and a ratio of subscapular skinfold thickness to triceps skinfold thickness was calculated to represent a ratio of central to peripheral fat distribution.52

Since we have shown that social status is a significant predictor of atherosclerosis in this species, social status was assessed53 54 by direct observation of the outcomes of aggressive interactions. Frequencies of agonistic and affiliative behaviors also were quantified.54

Coronary Vasomotor Responses
Eight testosterone-treated and seven untreated control animals were chosen randomly for vasomotion studies just before euthanasia and necropsy. Animals were anesthetized, and skin incisions were made in the right and left femoral triangles. A catheter was inserted into the right femoral artery and advanced to the midthoracic aorta for measurement of blood pressure and heart rate. A custom-designed 3F (tapered to 1.8F) catheter was inserted into the left femoral artery and advanced into the left main coronary artery by using fluoroscopic guidance. Blood pressure was monitored from the tip of this catheter to exclude damping and obstruction of coronary flow.

Endothelium-dependent and -independent coronary artery responses were determined.55 Using an infusion pump, serial 2.5-minute intracoronary infusions were made in the following sequence: 5% dextrose in water (control); acetylcholine 10-6 mol/L (assumes coronary blood flow of 10 mL/min56 ); and nitroglycerin (40 µg/min). After each infusion, cineangiographic images were obtained in the 30° right anterior oblique projection at 60 frames/s. Images were taken during hand injection of 2 mL nonionic contrast solution (Omnipaque, Squibb) into the left main coronary artery. Approximately 10 minutes elapsed between drug infusions.

A single frame from the cineangiogram was selected for analysis based on clarity of the image of the proximal 2 to 3 cm of the left circumflex coronary artery. Criteria for clarity included maximal opacification, no overlapping structures, and minimal motion artifact. The frame was subsequently digitized by using a Cipro cine film projector and a Digitron remote work station (Siemens). QCA software (Gammasonics) was used to detect edges of the artery segment of interest and measure its average pixel diameter. The same segment of artery was analyzed after each infusion. The entire process of frame selection and QCA analysis was repeated by an observer who was blinded to the results of the first analysis. The results from the first and second analyses were highly correlated (r=.92). The average result of the two analyses was used to calculate the percentage of change in pixel diameter from the baseline resulting from each infusion.

Variations in total plasma cholesterol and HDL-C concentrations,57 58 arterial blood pressure,59 and degree of arterial atherosclerotic involvement60 are associated with altered vasomotor responsivity. Accordingly, these variables, adjusted for variation in these predictors, were used in an ANCOVA to determine the effects of testosterone on vasomotion.

Necropsy and Measurement of Atherosclerosis
After 24 months of treatment, the animals were anesthetized deeply with pentobarbital (30 mg/kg IV), and the cardiovascular system was flushed with normal saline. The heart was excised after ligation of the vena cava and pulmonary arteries and was perfusion-fixed via the aorta with 4% paraformaldehyde at a pressure of 100 mm Hg. The heart was then immersed in 4% paraformaldehyde. After fixation, five serial tissue blocks were cut from each of the left circumflex, left anterior descending, and right coronary arteries. One section from the first block of each artery was used for immunohistochemical assessment of intimal cellular composition. One section from each block was stained with Verhoeff–van Gieson stain, sections were projected, and the cross-sectional area occupied by intimal lesion (plaque size), the area encompassed by the internal elastic lamina (artery size), and the lumen area were measured by using a digitizer. Plaque size, artery size, and lumen area were expressed as the mean of the 15 sections of coronary arteries.

The aorta and the carotid and iliofemoral arteries were opened longitudinally and immersion-fixed in 4% paraformaldehyde. Four equally spaced cross sections were taken from the thoracic and abdominal aorta. Three serial sections were taken from each common carotid and iliofemoral artery. Mean intimal area was determined for each artery by using the digitizer.

Immunohistochemistry
To determine if the cellular composition of the coronary atherosclerotic lesions was affected by treatment, one 5-µm section from each coronary artery was stained to identify macrophages and smooth muscle cells (SMCs).61 Antibodies used were HHF35 (Enzo Diagnostics) for smooth muscle and HAM56 (Enzo) for macrophages. The double-labeling procedure employed sequential streptavidin-horseradish-peroxidase conjugate (GIBCO-BRL) for the HHF35 and streptavidin-beta-galactosidase (GIBCO-BRL) for macrophages. Purified murine IgG primary antibodies (Zymed Laboratories) were used as species-matched, isotype-matched, concentration-matched negative controls for the appropriate corresponding antibody. All sections were counterstained with nuclear fast red.

Relative numbers of intimal cells staining positive for HAM56 or HHF35 were estimated by an observer who was unaware of animals' experimental group assignment. Scores were assigned by using a scoring system in which 0=no cells positive, 1=1% to 25% cells positive, 2=26% to 50% cells positive, 3=51% to 75% cells positive, and 4=76% to 100% cells positive.

Statistical Analysis
To reduce skewness and equalize group variances, all atherosclerosis data underwent square-root transformation prior to analysis. ANOVA, repeated-measures ANOVA, ANCOVA, multiple regression, and Pearson's product-moment correlation were used for the statistical analyses. Duncan's new multiple range test was used for post hoc comparisons. Statistical analyses used BMDP statistical software (University of California).


*    Results
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 *Results
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Atherosclerosis Extent
ANOVA revealed a significant main effect of experimental condition on coronary artery atherosclerosis extent (F3,57=3.95, P<.02) (Fig 1Down). Post hoc comparisons revealed that atherosclerosis was more extensive in the testosterone-treated monkeys than either the untreated controls or estrone- and androstenedione-treated animals (P<.05). Atherosclerosis in ovariectomized monkeys was intermediate in extent and did not differ statistically from the other three groups.



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  		Figure 1. Bar graph showing extent of diet-induced atherosclerotic plaque in coronary arteries of female cynomolgus monkeys either untreated (control), ovariectomized, treated with subcutaneous crystalline androstenedione and estrone, or treated with crystalline testosterone. Open bars indicate values unadjusted before analysis; solid bars, values adjusted for significant risk predictors. *P<.05 vs control and androstenedione groups; **P<.05 vs control, ovariectomy, and androstenedione and estrone groups.

The pattern of experimental condition–associated differences in atherosclerosis extent in the thoracic and abdominal aorta and iliofemoral arteries was similar to that of the coronary arteries (Table 1Down); however, none of the differences were significant. Experimental condition was associated with differences in atherosclerosis extent in both left (F3,57=2.73; P=.05) and right (F3,57=3.79; P<.02) common carotid arteries. Ovariectomized monkeys had more extensive atherosclerosis in the carotid arteries (P<.05; Table 1Down).


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  		Table 1. Extent of Atherosclerosis in Aorta, Carotid, and Iliofemoral Arteries in Female Cynomolgus Monkeys

Characteristics of Atherosclerotic Coronary Arteries
Atherosclerotic lesions ranged in extent from fatty streaks to large advanced plaques with necrosis, hemorrhage, or mineralization. In addition to its effect on plaque size, testosterone treatment was associated with a 13% increase in artery size (F3,57=2.62; P=.06) and a 10% increase in lumen size (F3,57=2.54; P=.06). Among intact and ovariectomized controls, there were positive correlations between plaque size and sizes of both the artery and lumen. These correlations were not found among monkeys in either androgen treatment group.

There were no apparent treatment-associated differences in histopathological appearance of atherosclerotic lesions. In all sections of coronary arteries examined, 90% or more of intimal cells stained positively as either macrophages or SMCs. All intimal lesions contained both cell types. Macrophages predominated in approximately half of lesions, and SMCs predominated in the other half. However, intimal cellular composition was not affected by treatment.

Total Plasma Cholesterol, HDL-C, and Triglycerides
Total plasma cholesterol, HDL-C, and triglyceride concentrations (Table 2Down) represent means of all values for each animal during the 24-month treatment period. There was a main effect of experimental condition on total plasma cholesterol concentrations (F3,57=3.19; P=.03). Total plasma cholesterol concentrations were highest in ovariectomized monkeys, although this group differed statistically only from untreated controls and estrone- and androstenedione-treated animals. Testosterone-treated animals had intermediate total plasma cholesterol concentrations that did not differ statistically from any of the other three groups. Experimental condition was not associated with differences in plasma HDL-C concentrations (F3,57=1.00; P>.39) or total plasma triglyceride concentrations (F3,57=0.11; P>.95).


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  		Table 2. Cholesterol and Triglyceride Concentrations in Female Cynomolgus Monkeys

Plasma Lipoprotein Patterns
Plasma LDL cholesterol concentrations were increased in ovariectomized monkeys and decreased in testosterone-treated monkeys (F3,57=6.31; P=.0009) relative to untreated controls at month 6. However, these differences were not apparent at month 18. Plasma LDL molecular weight was relatively increased in ovariectomized monkeys (F3,57=4.44; P<.008) at month 6 and in both ovariectomized and testosterone-treated animals at month 18 (F3,57=6.46; P<.0008). Plasma VLDL+IDL cholesterol concentrations did not differ among groups at month 6. At month 18, VLDL+IDL was two to three times higher (F3,57=7.13; P<.0004) in both ovariectomized and testosterone-treated monkeys relative to controls. Plasma HDL-C concentrations were unaffected by treatment.

Plasma apoA-I concentrations were decreased in ovariectomized monkeys, relative to controls and testosterone-treated monkeys, at month 6 (F3,57=3.31; P<.03) and month 18 (F3,57=2.00, P<.12). Plasma apoB concentrations were unaffected by treatment.

At month 6, differences in HDL subfractional heterogeneity were apparent. Testosterone-treated animals had relative increases in HDL2a (F3,57=7.15; P=.0004) and HDL2b (F3,57=3.94; P<.02) fractions, whereas the HDL3b fraction was decreased (F3,57=7.13; P=.0004). Ovariectomized animals had a relative increase in HDL2a (F3,57=7.15; P=.0004) and a decrease in HDL3b (F3,57=7.13, P=.0004) fractions. These effects were transient; by month 18, the only difference was a relative decrease in HDL3b in testosterone-treated animals (F3,57=2.84; P<.05).

Other Risk Variables
Blood pressure was not affected by treatment and was not correlated with atherosclerosis extent.

Menstrual cyclicity was affected greatly by treatment. Ovariectomy resulted in the complete absence of menstrual cycles. Treatment with androstenedione and estrone resulted in transient suppression of menstrual cyclicity in most monkeys. Over the course of treatment, these animals had 45% the number of ovulatory cycles as untreated controls. Testosterone treatment completely suppressed ovulation in 14 of 15 monkeys. As a group, these animals had 2% the number of cycles as untreated controls. Frequency of ovulation was not correlated with atherosclerosis extent.

Plasma steroid concentrations were in the ranges anticipated. In animals treated with androstenedione and estrone, plasma androstenedione concentrations averaged 73±12 pmol/L and plasma estrone concentrations, 4.30±1.55 pmol/L. In testosterone-treated animals, plasma testosterone concentrations averaged 0.62±0.09 nmol/L. Plasma steroid concentrations were not associated with atherosclerosis extent.

Body weight and body mass index were increased 40% (F3,57=11.1; P<.0001) and 30% (F3,57=16.5; P<.0001), respectively, in testosterone-treated animals relative to untreated controls. Testosterone treatment also resulted in a 19% increase in triceps skinfold thickness (F3,57=4.28; P<.009) and a 29% increase in subscapular skinfold thickness (F3,57=3.51; P<.03) relative to controls. This indicates that the increase in body mass of testosterone-treated animals was due, in part, to increased adiposity. Adiposity was generalized in nature, as opposed to primarily central, as there was no difference in the ratio of subscapular to triceps skinfold thickness. Increased muscle mass also was apparent in these animals, which further explains the observed increase in body mass. Consistent with this apparent anabolic effect of testosterone was a 40% increase in heart mass (F3,57=7.56; P=.0002) determined at necropsy. None of these variables was associated with atherosclerosis extent.

Social status also was not associated with atherosclerosis extent. Similarly, there were no associations between atherosclerosis extent and frequencies of either agonistic or affiliative behavior. Testosterone treatment was not associated with an increase in aggressive behavior.

Although fasting plasma glucose concentrations and glucose responsiveness to intravenous glucose challenge were unaffected by treatment, plasma insulin response to intravenous glucose challenge increased by an average of 37% in testosterone-treated animals relative to ovariectomized and estrone- and androstenedione-treated monkeys (F3,57=4.93; P<.005). Untreated controls did not differ from any of the three treatment groups with regard to insulin responsiveness. Plasma insulin and glucose concentrations were unassociated with atherosclerosis extent.

Risk Variables as Predictors of Coronary Artery Atherosclerosis Extent
Using multiple regression analysis, we determined that total plasma cholesterol and HDL-C concentrations were significant predictors of coronary artery atherosclerosis; together they accounted for 28% of the variability in lesion extent. These variables were used as covariates in an ANCOVA to determine whether they accounted for effects of treatment on atherosclerosis extent. Adjusted values are given in Fig 1Up. A significant main effect of treatment persisted (F3,57=3.81; P<.02); however, the between-group relationships changed slightly. Prior to adjustment, atherosclerosis extent in ovariectomized monkeys was intermediate in extent compared with the other groups. After adjustment, testosterone-treated monkeys had more extensive atherosclerosis than monkeys in the other three groups, which, in turn, did not differ from one another. Thus, the effect of testosterone on atherosclerosis progression remains unexplained by risk factors measured in this experiment.

Coronary Vasomotor Responsiveness
Intracoronary infusion of the endothelium-dependent vasodilator acetylcholine resulted in little change in coronary artery diameter of untreated controls, while arteries of testosterone-treated monkeys dilated (F1,10=6.50; P<.03) (Fig 2Down). Infusion of the endothelium-independent dilator nitroglycerin resulted in dilation in both groups of monkeys (Fig 2Down).



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  		Figure 2. Bar graph showing percent change in diameter of atherosclerotic coronary arteries of female monkeys either untreated or treated with subcutaneous crystalline testosterone in response to the endothelium-independent agent nitroglycerin and the endothelium-dependent agent acetylcholine. Data are adjusted for differences in total plasma and HDL cholesterol concentrations, blood pressure, and plaque size.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Effects of endogenous or exogenous androgens on atherosclerosis progression, vasomotor function, or risk of CHD have received little attention due, at least in part, to the great difficulty involved in addressing such questions directly in human subjects. For this reason, we have employed a nonhuman primate model. We have shown that, similar to human populations in most Western societies, coronary atherosclerosis develops more rapidly in male monkeys than similarly aged females.62 Also, there is an association between endogenous sex hormone patterns and the development of coronary atherosclerosis. In pregnancy, a hyperestrogenic state, atherosclerosis is inhibited.63 On the other hand, oophorectomy, which produces pronounced estrogen deficiency, results in exacerbated atherosclerosis.38 Furthermore, physiological concentrations of exogenously administered natural estrogen, with or without progesterone, inhibit atherosclerosis development.64 The current experiment extends these findings by implicating physiological (male) concentrations of exogenously administered testosterone in the exacerbation of atherosclerosis progression. Furthermore, testosterone treatment had potentially adverse effects on arterial remodeling, or compensatory enlargement, in response to atherosclerosis progression. This is suggested by the finding that failure of compensatory enlargement, or remodeling, was associated with increased CHD prevalence in a series of autopsy cases.35 Taken together, these findings suggest that gender differences in atherosclerosis extent may be accounted for not only by antiatherogenic effects of endogenous estrogen in females but by atherogenic effects of endogenous androgen in males.

Although atherosclerosis was more advanced in testosterone-treated animals, their coronary artery responses to acetylcholine were greatly improved relative to untreated controls. Responses to this endothelium-dependent dilator in the control animals were those expected of atherosclerotic vessels (either no response or "paradoxical vasoconstriction"), while those of testosterone-treated animals were, in all cases, dilation. This suggests that any adverse effects of male-like endogenous androgen patterns on risk of clinical CHD are not explained by adverse effects on coronary vasospasm or vasospasm-associated events such as plaque rupture, thrombosis, myocardial ischemia, or myocardial infarction.

While a male-like plasma androgen pattern resulted in worsened coronary artery atherosclerosis, experimentally induced hyperandrogenism similar to that seen in women with PCO did not have adverse effects on atherosclerosis. There are at least two possible explanations for this finding. The androgens elevated in PCO, such as androstenedione, are less potent than testosterone. Also, the hyperandrogenism is usually associated with acyclic elevations in plasma estrogen (primarily estrone) concentrations.36 Thus, while ovulation occurred less often in our primate model, as it does in women with PCO,36 antiatherogenic effects of the accompanying chronic elevation in circulating estrogen may predominate over any atherogenic effects of the relatively weak androgenic stimulus of androstenedione. This speculation is supported by findings that indicate that antiatherogenic effects of contraceptive estrogens are not antagonized by coadministration of androgenic progestins.65 66

The mechanisms by which atherogenic effects of testosterone are mediated are uncertain. In the current study, atherosclerosis extent was not associated with effects on plasma lipoprotein or nonlipoprotein risk variables. Total plasma cholesterol and HDL-C concentrations were not altered, and LDL cholesterol concentrations were decreased only transiently by testosterone treatment. Though plasma VLDL+IDL cholesterol and average LDL molecular weight were increased and HDL subfractional heterogeneity was altered in these animals, these changes were not correlated with atherosclerosis extent. Furthermore, statistical adjustment for significant risk variable predictors of atherosclerosis extent did not alter the conclusion that testosterone treatment resulted in exacerbated atherosclerosis. Effects of testosterone on aspects of lipoprotein metabolism or plasma lipoprotein patterns unassessed in this experiment (eg, plasma LDL heterogeneity or composition) may have played a role.

Similarly, effects of testosterone on other risk variables did not explain the increase in atherosclerosis extent. Blood pressure was not affected by treatment. Atherosclerosis was most extensive in the two groups in which menstrual cyclicity and ovulation were virtually absent, the ovariectomized and testosterone-treated groups. However, this increase was statistically significant only in the testosterone group. Also, plasma lipoprotein patterns were quite different in ovariectomized monkeys relative to testosterone-treated monkeys. Adjustment for significant lipoprotein risk factors resulted in a statistically significant difference in atherosclerosis extent between these two groups, with atherosclerosis extent doubled in the testosterone-treated monkeys. This suggests that any effect of anovulation we observed can be explained by the associated variation in total plasma cholesterol and HDL-C concentrations and that effects of testosterone occurred independently of effects on ovulation and plasma lipoproteins.

Testosterone treatment resulted in increased body weight, body mass index, and indices of generalized adiposity and muscle mass. Probably related to these changes was a relative insulin resistance. While it remains possible that these known CHD risk variables contributed to the atherogenic effects of testosterone, they were not correlated with extent of atherosclerosis.

Low social status has been associated with increased extent of atherosclerosis in females with intact ovaries but not ovariectomized females.38 In that experiment, evidence indicated that low social status was associated with relatively poor ovarian function and that effects of low social status on atherosclerosis were mediated by this relative estrogen deficiency. Thus, it was unexpected that, among untreated controls in the current experiment, there was no association between social status and atherosclerosis extent. However, it is perhaps not surprising that there was no association within the other three groups, since ovarian function was similarly suppressed in all animals within a group, regardless of status. As with social status, other behavioral variables (ie, frequencies of agonistic and affiliative behaviors) were unaffected by treatment and unassociated with atherosclerosis.

Other possible explanations for the atherogenic effects of testosterone involve direct effects on atherosclerosis initiation and progression at the level of the arterial intima. Although testosterone is an anabolic steroid and, in the current study, had an obvious anabolic effect on skeletal muscle and myocardium, there was no immunohistochemical evidence of treatment-associated differences in arterial cellular composition, suggesting that testosterone did not have a hyperplastic or hypertrophic effect on arterial SMCs.

Testosterone could influence metabolic events occurring in the arterial intima. Using the macaque model, we have found that estrogen-progestin combinations inhibit arterial uptake and catabolism of plasma LDL.67 68 Effects of androgens have not been studied, but they may have the opposite effect on arterial LDL metabolism, causing accelerated arterial cholesterol accumulation and atherosclerosis progression. In this regard, there is evidence that some estrogens (eg, estradiol) have in vitro antioxidant effects on isolated LDL, while testosterone does not.69 70 Since oxidative modification of LDL is associated with a marked increase in its uptake and metabolism by arterial macrophages, this represents a possible mechanism by which estrogens, such as estradiol, exert their antiatherosclerotic effect.

Sex steroid receptors exist in the cardiovascular system,71 72 73 74 which suggests a role for sex hormones in the regulation of endothelial cell, vascular SMC, and/or macrophage function. There is plentiful evidence that sex hormones are modulators of immune and inflammatory responses.75 76 77 78 Since locally mediated immune and inflammatory reactions are clearly involved in atherosclerosis initiation and progression, sex hormones may, through effects on autocrine, paracrine, or endocrine regulation of immune or inflammatory processes, influence atherosclerosis progression.

Androgens also may influence atherosclerosis progression or risk of clinical CHD events by affecting endothelium-dependent vasomotor responses. Atherosclerosis results in impaired endothelial function which, in turn, causes impaired vasodilator and augmented vasoconstrictor responses to neurohumoral stimuli. These alterations lead to increased incidence of vasospasm, which eventually may result in plaque instability or rupture, thrombosis, and myocardial ischemia or infarction. We55 and others79 have described evidence that exogenous estrogen administered either acutely or chronically protects coronary arteries against atherosclerosis-related impairment of endothelium-dependent vasomotor function. It was, therefore, of particular interest to find that chronic testosterone treatment had a similar protective effect in the current study, despite the fact that atherosclerosis was substantially more advanced in the testosterone-treated group than in the untreated control group. To our knowledge, there have been no previous studies of the effects of androgens on vasomotor responses of atherosclerotic arteries. While our results seem to contradict the results of some studies in nonatherosclerotic rats,80 81 82 the studies are not directly comparable due to marked differences in design and the fact that overwhelming evidence indicates that atherosclerotic vessels respond much differently than nonatherosclerotic vessels.

Although mechanisms by which testosterone may favorably modulate vasomotor responses were not addressed in the current study, there are a number of possibilities. There is evidence that porcine endothelial cells can convert testosterone to estradiol and estrone via aromatization.83 Thus, it seems possible that local conversion of androgen to estrogen may be responsible for the observed effects. Both estradiol and testosterone are reported to stimulate the production of vasodilatory prostanoids by endothelial cells obtained from female but not male piglets.83 Thus, endothelium-dependent vascular responses to estrogen may differ markedly between males and females. Finally, it is also possible that steroids other than estrogens may have favorable effects on coronary vasomotion.

Regardless of mechanism(s), we conclude that an experimentally induced physiological (male) plasma androgen pattern results in an exacerbation of diet-induced atherosclerosis in female monkeys, yet also results in an improvement in atherosclerosis-related impairment of coronary vasomotor function. The results indicate that testosterone may have a direct role in the increased rate of atherosclerosis progression and increased risk of CHD seen in men relative to women in most Western societies.


*    Acknowledgments
 
This study was supported in part by grants HL14164 and HL45666 from the National Heart, Lung, and Blood Institute, Bethesda, Md. The authors thank Deborah Golden for expert technical assistance.

Received December 12, 1994; accepted February 7, 1995.


*    References
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