Thomas B. Clarkson

Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone.

Although controversy continues, the preponderance of evidence indicates that estrogen replacement therapy favorably influences the risk of coronary heart disease in postmenopausal women. It remains uncertain how this effect is mediated and whether the cyclic addition of a progestin may influence adversely an estrogen-related cardioprotective effect. We investigated the influence of sex hormone replacement therapy on diet-induced coronary artery atherosclerosis in estrogen-deficient (ovariectomized) adult female cynomolgus monkeys. Monkeys were assigned randomly to one of three treatment groups: 1) no hormone replacement (n = 17), 2) continuously administered 17-beta estradiol plus cyclically administered progesterone (n = 20), and 3) continuously administered 17-beta estradiol (n = 18). The physiologic patterns of plasma estradiol and progesterone concentrations were maintained by administering the hormones in sustained-release subcutaneous Silastic implants. The experiment lasted 30 months. At necropsy, coronary artery atherosclerosis was inhibited similarly (reduced by approximately one-half) in animals in both hormone replacement groups (p less than or equal to 0.05). Antiatherogenic effects of hormone replacement were independent of variation in total plasma cholesterol, lipoprotein cholesterol, apoprotein A-1 and B concentrations, high density lipoprotein subfraction heterogeneity, and low density lipoprotein molecular weight. We conclude that physiologic estrogen replacement therapy with or without added progesterone inhibits atherosclerosis progression in ovariectomized monkeys. This may explain why estrogen replacement therapy results in reduced risk of coronary heart disease in postmenopausal women.

Estrogen replacement therapy, atherosclerosis, and vascular function

There is strong evidence from both human and nonhuman primate studies supporting the conclusion that estrogen deficiency increases the progression of atherosclerosis. More controversial is the conclusion that postmenopausal estrogen replacement inhibits the progression of atherosclerosis. Estrogen treatment of older women (>65 years) with pre-existing coronary artery atherosclerosis had no beneficial effects. In contrast, estrogen treatment of younger postmenopausal women or monkeys in the early stages of atherosclerosis progression has marked beneficial effects. Whether progestogens attenuate the cardiovascular benefits of estrogen replacement therapy has been controversial for more than a decade. Current evidence from studies of both monkeys and women suggest little or no attenuation of estrogen benefits for coronary artery atherosclerosis. Lack of compliance with estrogen replacement therapy, usually because of fear of breast cancer, remains a major problem. Future regimens may overcome that fear by the co-administration of a breast cancer preventive agent (i.e., selective estrogen receptor modulators, phytoestrogens) with low dose estrogen.

Short-term administration of estrogen and vascular responce of atherosclerotic coronary arteries

OBJECTIVES This experiment sought to determine the effect of short-term administration of estrogen on endothelium-dependent dilation in the coronary arteries of 13 surgically postmenopausal female cynomolgus monkeys. BACKGROUND Long-term estrogen replacement therapy prevents impaired endothelium-dependent dilation of atherosclerotic coronary arteries in postmenopausal female monkeys. However, it remains unclear whether this action of estrogen is due to long-term effects on plasma lipids and atherogenesis or to direct short-term effects on the endothelium. METHODS The monkeys consumed an atherogenic diet for 18 months after bilateral ovariectomy. Vascular responses were measured just before euthanasia and necropsy. Dextrose in water (control), acetylcholine, 10(-6)M, and nitroglycerin were infused for 2.5 min each both before and 20 min after intravenous injection of 54 ng ethinyl estradiol. RESULTS Quantitative coronary angiography revealed that the arteries constricted (-17 +/- 3%) in response to intracoronary infusion of acetylcholine before estrogen treatment but dilated (+5 +/- 3%) 20 min after intravenous injection of ethinyl estradiol (p less than 0.05). Coronary arteries dilated in response to nitroglycerin both before and after administration of estrogen (p greater than 0.05). Vascular responses of coronary arteries, both before and after administration of estrogen, were not associated with variation in plasma lipid concentrations, blood pressure, heart rate or plaque size. CONCLUSIONS Estrogen affects endothelium-dependent coronary dilation within 20 min of administration and may have rapid direct effects on the vascular endothelium.

Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques.

OBJECTIVE To examine the effects of soy phytoestrogens on coronary vascular reactivity in atherosclerotic male and female rhesus monkeys. DESIGN A prospective, randomized, blinded, controlled study. SETTING Comparative Medicine Clinical Research Center of an academic medical center. PATIENT(S) Twenty-two young adult rhesus monkeys with pre-existing diet-induced atherosclerosis. INTERVENTION(S) Monkeys were fed soy-based diets for 6 months identical in composition, except that the isoflavones were extracted from one flow-isoflavone) and intact in the other (high-isoflavone). Quantitative coronary angiography was performed at the end of the study period. Females in the low-isoflavone group under went a second angiography after an acute IV dose of genistein. MAIN OUTCOME MEASURE(S) Percent change in diameter of the proximal left circumflex coronary artery in response to intracoronary acetylcholine and nitroglycerin, compared with control diameter. RESULT(S) Arteries from males constricted in response to acetylcholine. Arteries from females in the low-isoflavone group constricted (-6.2% +/- 2.8%, mean +/- SEM), whereas arteries from females in the high-isoflavone group dilated (6.4% +/- 1.2%, mean +/- SEM). Intravenous administration of genistein caused dilation in the previously constricting low-isoflavone females (3.3% +/- 2.8%). CONCLUSION(S) Like mammalian estrogens, dietary soy isoflavones enhance the dilator response to acetylcholine of atherosclerotic arteries in female monkeys.

Effects of hormone replacement therapy on reactivity of atherosclerotic coronary arteries in cynomolgus monkeys

OBJECTIVES We attempted to determine whether continuous and cyclic medroxyprogesterone acetate modulates the effects of estrogen on dilation of atherosclerotic coronary arteries in surgically postmenopausal female monkeys. BACKGROUND Estrogen replacement in postmenopausal women preserves normal dilator responses of atherosclerotic coronary arteries. The effects of progestins on coronary artery reactivity have not been determined. METHODS Repeated quantitative coronary angiography was used to study the effects after 1 month of 1) no hormone replacement (control) or oral administration of 2) continuous conjugated equine estrogens, 3) cyclic high dose medroxyprogesterone acetate (MPA) given on days 16 to 26 of the month, 4) conjugated equine estrogens plus continuous low dose MPA, or 5) conjugated equine estrogens plus cyclic high dose MPA on endothelium-mediated dilation of atherosclerotic coronary arteries in 12 cynomolgus monkeys. Change in diameter of the left circumflex coronary artery was measured in response to intracoronary infusions of acetylcholine (10(-6) mol/liter per min) and nitroglycerin (15 micrograms/min). RESULTS Coronary arteries constricted during no hormone treatment (-8 +/- 3% [mean +/- SEM]), dilated during conjugated equine estrogen treatment (+3 +/- 1%, p < 0.05 vs. control) and constricted during cyclic MPA treatment (-3 +/- 2%). Addition of cyclic or continuous MPA to the conjugated equine estrogen regimen inhibited acetylcholine responses by 50% (p < 0.05 vs. conjugated equine estrogens). There was no effect of treatment on vascular response to nitroglycerin (p > 0.05). CONCLUSIONS Treatment with conjugated equine estrogens, but not MPA, augmented endothelium-mediated dilation of atherosclerotic coronary arteries. Addition of cyclic or continuous MPA to the conjugated equine estrogen regimen diminished endothelium-mediated dilation.

Social status, environment, and atherosclerosis in cynomolgus monkeys.

The purpose of this experiment was to examine the effects of social environment and social status on coronary artery and aortic atherosclerosis In adult male cynomolgus monkeys (Macaca fascicularls). Thirty experimental animals were assigned to six groups of five members each, and all animals were fed a moderately atherogenic diet (43% of calories as fat, 0.34 mg cholesterol/Cal) for 22 months. Group memberships were changed periodically among 15 monkeys (unstable social condition) and remained fixed throughout the experiment In the remaining animals (stable social condition). Within each condition, individual monkeys were classified as either dominant or subordinate animals, based on dyadic patterns of aggression and submission. At necropsy, the coronary arteries were subjected to pressure fixation and five sections each were taken from the left anterior descending, left circumflex, and right coronary arteries. The mean Intimal area measurement, based on all arterial sections, served as a coronary Index for each animal. Results Indicated that dominant animals in the unstable condition had significantly greater coronary artery atherosclerosis than dominant monkeys housed In stable social groups. Coronary artery atherosclerosis in the unstable dominants was also greater than among similarly housed (I.e., unstable) subordinates. A similar pattern was observed In the abdominal aorta, but was not statistically significant. No significant differences or similar patterns were seen In the thoracic aorta. Additional analyses revealed that the coronary artery effects were not due to concomitant differences In total serum cholesterol or high density llpoproteln cholesterol concentrations, blood pressures, ponderosity, or fasting glucose concentrations among the experimental animals. Behaviorally, manipulation of group memberships intensified agonistic encounters and disrupted patterns of affiliative Interaction between dominant and subordinate monkeys. Overall, these results suggest that social dominance (an Individual behavioral characteristic) Is associated with increased coronary artery atherosclerosis, but only under social conditions that provide recurrent threats to the status of dominant animals (I.e., under behavioral challenge).

Soy Protein Versus Soy Phytoestrogens in the Prevention of Diet-Induced Coronary Artery Atherosclerosis of Male Cynomolgus Monkeys

Soy protein, long recognized as having cardiovascular benefits, is a rich source of phytoestrogens (isoflavones). To distinguish the relative contributions of the protein moiety versus the alcohol-extractable phytoestrogens for cardiovascular protection, we studied young male cynomolgus macaques fed a moderately atherogenic diet and randomly assigned to three groups. The groups differed only in the source of dietary protein, which was either casein/lactalbumin (casein, n = 27), soy protein with the phytoestrogens intact (soy+, n = 27), or soy protein with the phytoestrogens mostly extracted (soy-, n = 28). The diets were fed for 14 months. Animals fed soy+ had significantly lower total and LDL plus VLDL cholesterol concentrations compared with the other two groups. They soy+ animals had the highest HDL cholesterol concentrations, the casein group had the lowest, and the soy- group was intermediate. A subset was necropsied for atherosclerosis evaluations (n = 11 per group). Morphometric and angiochemical measures were done to quantify atherosclerosis. Coronary artery atherosclerotic lesions were smallest in the soy+ group (90% less coronary atherosclerosis than the casein group and 50% less than the soy- group), largest in the casein group, and intermediate in the soy- group. The effects of the diets on lesion size and arterial lipid measures of the peripheral arteries were similar to those in the coronary arteries, with greatest prevention of atherogenesis with soy+ and intermediate benefit with soy- relative to casein. We could not determine whether the beneficial effects seen in the soy- group relate to the protein itself or to the remaining traces of phytoestrogens. The beneficial effects of soy protein on atherosclerosis appear to be mediated primarily by the phytoestrogen component. Testicular weights were unaffected by the phytoestrogens.

Estrogen and progesterone replacement therapy reduces low density lipoprotein accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys.

The effect of estrogen and progesterone replacement therapy on the initiating events in atherogenesis was studied in surgically postmenopausal cynomolgus monkeys. Monkeys were ovariectomized and divided randomly into two groups, one receiving 17 beta-estradiol and cyclic progesterone treatment (n = 9) and ovariectomized controls receiving no hormone replacement therapy (n = 8). The monkeys were fed a moderately atherogenic diet for 18 wk to accelerate the early pathogenic processes but not to be of sufficient duration to produce grossly visible atherosclerotic lesions. Sex hormone replacement therapy decreased the accumulation of LDL and products of LDL degradation in the coronary arteries by greater than 70% while having no significant effect on plasma lipid, lipoprotein, or apoprotein concentrations. Arterial intimal lesions were small with no difference between groups. The reduction in arterial LDL metabolism occurred very early in the pathogenesis of atherosclerosis and was independent of indices of endothelial cell injury, such as enhanced endothelial cell turnover or leukocyte adhesion to the endothelium. Results of this study suggest that one mechanism by which sex hormone treatment inhibits the initiation of atherosclerosis is a direct effect at the level of the arterial wall by suppressing the uptake and/or degradation of LDL.

behaviorally Induced Heart Rate Reactivity and Atherosclerosis in Cynomolgus Monkeys

&NA; It has been suggested that individual differences in behaviorally induced cardiovascular reactivity may mediate associations between behavioral factors and atherosclerotic disease. The present study provides data relevant to this hypothesis within an animal model. Experimental animals were 26 adult, male cynomolgus monkeys that had been fed a moderately atherogenic diet for 22 months. In the weeks preceding termination of these animals, monkeys were fitted with electrocardiogram (EKG) telemetry devices and their heart rates (HRs) recorded under baseline and stressed conditions. Stress‐period HR measures were obtained during a standard challenge involving threatened capture and physical handling of the animals. At necropsy, the coronary arteries were subjected to pressure fixation and sections taken from the left main, left anterior descending, left circumflex, and right coronary arteries. Mean intimal area measurements, calculated for each artery, were then compared between animals identified as High (n = 8) and Low (n = 8) HR reactors during stress. Results indicated that High HR reactors had significantly greater coronary artery atherosclerosis than did Low HR reactive animals, both in individual arteries and on an overall coronary index. Atherosclerosis in the thoracic aorta was found to differ similarly between High and Low HR reactors. Additional analyses revealed that High HR reactors were significantly more aggressive, more ponderous, and had greater heart weights than did Low HR reactors. Although groups did not differ in resting HRs, body weights, or lipid values, high‐density lipoprotein (HDL) cholesterol comprised a slightly smaller fraction of the total serum cholesterol of High, relative to Low, HR reactive monkeys. It is concluded that these findings provide initial support for the hypothesis that cardiovascular hyperresponsiveness under stress is related to the development of atherosclerosis.

Effects of soy isoflavones on atherosclerosis: potential mechanisms

It has long been recognized that coronary heart disease rates are lower in Japan, where soy consumption is common, than in Western countries. In experimental studies, atherosclerosis was reduced in animals fed diets containing soy protein compared with those fed diets with animal protein. Recently, several lines of evidence have suggested that the components of soy protein that lower lipid concentrations are extractable by alcohol (eg, the isoflavones genistein and daidzein). We recently evaluated the relative effect of the soy protein versus the alcohol-extractable components of soy on cardiovascular disease and its risk factors. Young male and female cynomolgus monkeys were fed diets that contained either 1) casein-lactalbumin as the source of protein (casein), 2) soy protein isolate from which the isoflavones were alcohol extracted (SPI-), or 3) isoflavone-intact soy protein (SPI+). The SPI+ group had significant improvements in LDL cholesterol and HDL cholesterol. Only HDL cholesterol was significantly improved in the SPI- group males compared with the casein group. The casein group had the most atherosclerosis, the SPI+ group had the least, and the SPI- group was intermediate but did not differ significantly from the casein group. Potential mechanisms by which soy isoflavones might prevent atherosclerosis include a beneficial effect on plasma lipid concentrations, antioxidant effects, antiproliferative and antimigratory effects on smooth muscle cells, effects on thrombus formation, and maintenance of normal vascular reactivity.