ILAR Journal Online, Volume 32(2) 1990: Animal Models in Biomedical Research

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ILAR Journal V32(2) 1990
Animal Models in Biomedical Research

The Contribution of Nonhuman Primates to Understanding Coronary Artery Atherosclerosis in Humans
Thomas B. Clarkson and Sherry A. Klumpp

Thomas B. Clarkson is professor and chairman of Comparative Medicine and director of the Arteriosclerosis Research Center, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina. Sherry A. Klumpp is associate scientist and veterinary pathologist, Division of Pathobiology and Immunobiology, Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia.

Introduction

Critics of the use of animal models often claim that the dramatic decline in morbidity and mortality from coronary heart disease in our country has resulted from changes in diet and life-style, not from animal research. In this brief review, evidence is presented to illustrate that the principal life-style changes that have decreased morbidity and mortality resulting from coronary heart disease derive much of their rationale from observations made from studies on animal models. For brevity, this review is limited to the use of nonhuman primates and how they have contributed to understanding the importance of diet and life-style changes.

Atherogenic Effects of Dietary Cholesterol

For more than eight decades the excessive ingestion of dietary cholesterol has been recognized as a major factor in the rapid progression of coronary artery atherosclerosis. Nonhuman primates have contributed in a major way to our understanding of the detailed characteristics of dietary cholesterol exacerbation of atherosclerosis. Examples of these contributions will be restricted to three species.

Squirrel Monkeys

Squirrel monkeys (Saimiri sciureus; Figure 1) were critical in understanding individual differences in response to dietary cholesterol. Elevations in serum cholesterol concentrations among squirrel monkeys consuming cholesterol-containing diets range from slight (hyporesponders) to large (hyperresponders) (Clarkson et al., 1971). This marked individual variability in response to cholesterol-containing diets was found to be determined by the response of each animal's parents (Clarkson et al., 1976).

Hyporesponder squirrel monkeys develop minimal atherosclerosis in response to dietary cholesterol, but hyperresponder monkeys develop large, frequently complicated, atherosclerotic plaques (Clarkson et al., 1976). Fibromuscular plaques containing lipid are found in the abdominal aorta and in the mesenteric, coronary, and cerebral arteries. Foam cell lesions are oftell found at the base of and within the aortic valve leaflets.

Complications in hyperresponders eating a cholesterol-containing diet include cholelithiasis and cutaneous and tendinous xanthomas (Clarkson et al., 1976). Among the end-organ complications are cerebral (Bullock and Moossy, 1972) and gut infarcts and renal disease secondary to intra-cellular lipid accumulations in glomeruli (Clarkson et al., 1971). Lipid-laden, sclerotic aortic valves can cause congestive heart failure (Clarkson et al., 1976).

Rhesus Monkeys

Atherosclerosis of rhesus monkeys (Macaca mulatta;Figure 2) induced by the consumption of cholesterol-con-taining diets has been well characterized. Morphologic descriptions of diet-induced lesions have been reported by several investigators (Clarkson and Alexander, 1980; Taylor et al., 1962, 1963). In addition, Scott et al. (1967a,b) have made gross light microscopic and electron microscopic descriptions of diet-induced atherosclerosis in rhesus monkeys. The distribution and pathogenesis of diet-induced atherosclerotic lesions in rhesus monkeys have been described by Manning and Clarkson (1972), and the cellular and chemical composition of the lesions has been analyzed by Scott et al. (1967b) and by Manning and Clarkson (1972).

The first atherosclerotic lesions are seen in the aorta and in the carotid and iliac arteries. Later, lesions develop in the proximal coronary arteries and then in the distal coronary arteries; the mesenteric, splenic, and femoral arteries; and the circle of Willis. Fatty streaks are the most common lesions. Other lesions range from uncomplicated fibrous plaques to lesions containing areas of necrosis and, sometimes, mineralization, hemorrhage, and medial destruction. Atherosclerosis is frequently more severe in the coronary arteries and at the carotid bifurcations. The composition and extent of atherosclerotic lesions at different arterial sites depend, at least in part, on the age of the animal, type of diet, length of time an atherogenic diet is consumed, and serum cholesterol concentrations. Fibrotic aortic lesions have also been induced experimentally in rhesus monkeys through the use of intermittent hypercholesterolemia (Bullock et al., 1976), hypertension (McGill et al., 1961), and pyridoxine deficiency (Kim, 1961; Rinehart and Green-berg, 1949).

Rhesus monkeys consuming an atherogenic diet share with humans a relatively high frequency of myocardial infarction. Taylor et al. (1963) described a case of fatal myocardial infarction in a rhesus monkey that had been consuming an atherogenic diet, and Bond et al. (1980) described several cases of myocardial infarction in a large colony of rhesus monkeys being fed an atherogenic diet. From these reports one can conclude that roughly 1 rhesus monkey per 300 monkeys at risk per year will experience a myocardial infarction, an incidence quite similar to that seen in humans in North America.

Cynomolgus Macaques

Diet-induced atherosclerotic lesions in cynomolgus monkeys (Macaca fascicularis; Figure 3) have been described by several investigators (Armstrong, 1976; Kramsch and Hollander, 1968; Malinow et al., 1976; Prathap, 1975; Wagner et al., 1978). In general, the diet-induced lesions in cynomolgus macaques are similar to the lesions in other macaques except they tend to have a higher connective tissue and mineral content.

There are several characteristics of cynomolgus macaques that make them useful models of atherosclerosis. First, the occurrence of myocardial infarction is greater in Malaysian cynomolgus macaques than other macaques, including the Philippine cynomolgus monkey (Bond et al., 1980). Second, there is a male-female difference in both plasma lipoproteins and coronary artery atherosclerosis (Hamm et al., 1983; Rudel and Pitts, 1978). Like human females, the female cynomolgus macaque has higher concentrations of high density lipoprotein (HDL) cholesterol than does the male. The extent of coronary artery atherosclerosis of female cynomolgus macaques, as in human females, is two to three times less than males. Third, intracranial cerebral artery atherosclerosis does occur in cynomolgus macaques eating a cholesterol-con-taining diet, but generally only when exacerbated by other risk factors such as hypertension (Angelo et al., 1980).

Regression of Coronary Artery Atherosclerosis with Lowering of Plasma Cholesterol Concentrations

Based primarily on studies of nonhuman primates and pigs, there is now ample evidence to suggest that regression of coronary artery atherosclerosis can occur with reductions in plasma cholesterol concentrations. Our own experimental evidence concerning regression comes from a large, long-term study of diet-induced atherosclerosis in rhesus monkeys (Clarkson et al., 1979, 1981, 1984; Wagner et al., 1980a,b). In that study, atherosclerosis was induced by diet to a stage comparable to male humans of either 35 or 50 years of age. This was done by extending the period of atherosclerosis induction from 19 to 38 months. Following the induction of atherosclerosis, a baseline sample of animals was examined to determine the extent and severity of the atherosclerosis at the time when the regression regimen was begun. Diets were fed to the animals that maintained plasma cholesterol concentrations either in the ranges in which regression was presumed to occur (180 to 220 mg/dl) or in ranges generally associated with atherosclerosis progression (above 220 mg/dl). The regression regimens were maintained for four years, after which the animals were necropsied, and the extent and severity of coronary artery atherosclerosis were compared with that of the baseline animals before the regression regimens were begun.

The following observations about regression of coronary artery atherosclerosis in these nonhuman primates have affected public health policies in the United States as well as diet and life-style of humans:

Reduction of total plasma cholesterol concentrations to about 220 mg/dl will result in regression of coronary artery atherosclerosis among monkeys with lesions comparable to young men. In contrast, reductions to below 200 mg/dl are required to induce regression of atherosclerosis in the coronary arteries of monkeys with lesions comparable to men in their midlife.
The best predictor of whether coronary artery atherosclerosis is likely to progress or regress is the ratio of the total plasma cholesterol concentration to the HDL cholesterol concentration. At ratios above 4.5, coronary artery atherosclerosis is progressive. In contrast, at ratios less than 4.5, coronary artery atherosclerosis regresses. There does not appear to be any added benefit to regression of ratios in the range of 3 to 4.
The area of the coronary artery lumen increases considerably during regression. The extent to which coronary artery lumens increase during regression relates 75 percent to increases in coronary artery size associated with the remodeling process. We estimate that 25 percent of the improvement in lumen areas is directly related to reduction in atherosclerosis plaque size.

Cigarette Smoking

Two smoking-related studies have been conducted with squirrel monkeys. Webster et al. (1970) reported that carbon monoxide (CO) increases intramyocardial coronary artery lumen stenosis, heart weight, and the prevalence of right bundle branch block in Peruvian squirrel monkeys consuming an atherogenic diet (Webster et al., 1970). The results of that study suggested that CO affects the rate of coronary artery plaque development rather than inducing atherosclerosis; no effect of CO on aortic atherosclerosis was found. In the second study, Topping and Turner (1975) described the effects of intravenous administration of nicotine on plasma triglycerides in the squirrel monkey.

Raymond and coworkers (1982) used stumptail macaques to investigate the effects of chronic exposure to cigarette smoke on plasma lipoprotein concentrations. The animals were fed a standard monkey chow diet and divided into high-dose, low-dose, and control groups. The investigators found a dose-related increase in peak blood carboxy-hemoglobin levels but no significant differences in total plasma cholesterol or low density lipoprotein (LDL) cholesterol concentrations; they concluded that chronic exposure to cigarette smoke had no effect on plasma lipoprotein levels in stumptail macaques fed a low-fat diet.

The most comprehensive study of cigarette smoking and experimental atherosclerosis has been done by Rogers and coworkers (1980). In that study, baboons were trained to smoke in a human-like manner. Cigarette smoking caused baboons to have higher LDL/HDL ratios.

Psychosocial Stress and Coronary Artery Atherosclerosis in Males

Reduction of stress is a life-style change widely believed to be beneficial in retarding the progression of coronary heart disease. Controlled, prospective studies have been difficult with men; however, nonhuman primates have proven to be excellent models for such studies.

We have completed several experiments concerning behavioral influences on coronary artery atherogenesis in male cynomolgus monkeys. In the first study (Kaplan et al., 1982), we studied the effect of the stress of social manipulation (periodic group reorganization) on the development of atherosclerosis. Coronary artery atherosclerosis of socially unstable dominant monkeys greatly exceeded that of subordinate monkeys. The psychosocial stress exacerbation of atherogenesis in cynomolgus monkeys cannot be explained by increase in the usual coronary heart disease risk factors. Although the mechanisms responsible for behaviorally induced atherosclerosis are unknown, many investigators have suggested that autonomic nervous system and neuroendocrine reactions to stress may contribute to lesion development (Manuck et al., 1983; Schneiderman, 1983). Of particular interest have been the potentially atherogenic effects of acute cardiovascular adjustments accompanying arousal of the sympathetic nervous system. It has been proposed that abrupt rises in heart rate and blood pressure promote injury to arterial endothelium (Clarkson et al., 1986). Accordingly, we have conducted a study (Kaplan et al., 1987) in which we administered pro-pranolol to monkeys at a dosage sufficient to lower heart rate by 20 to 25 beats per minute and blood pressure by about 15 mm Hg. These monkeys, in addition to untreated controls, were housed in unstable social groups, and all animals were fed a moderately atherogenic diet. We found a significant interaction between experimental condition (i.e., propranolol-treated, untreated) and social status. In the untreated condition, dominant animals had roughly double the amount of coronary artery atherosclerosis when compared to their subordinate counterparts, which closely replicated differences observed between unstable dominant and subordinate monkeys in the first experiment. Variability in serum lipid concentrations and blood pressure could not account for this effect.

Psychosocial Stress and Coronary Artery Atherosclerosis in Females

The relative protection against coronary heart disease accorded women during their reproductive years is an important aspect of the public health implications of this disease (Kannel, 1986). We were encouraged that our animal model could be used to examine the possibility of adverse effects of stress on coronary atherosclerosis of females. We indicated earlier that coronary artery atherosclerosis of female cynomolgus monkeys is less extensive than in males. In our initial study, female monkeys were fed a moderately atherogenic diet and housed in stable social groups. Stressed, subordinate monkeys were found to have greater atherosclerosis than did their dominant counterparts (Hamm et al., 1983).

Because the lesions seen among stressed subordinate females were as severe as those seen in males, we sought to understand in a second study (Kaplan et al., 1984) the effect of the stressful social status. Stressed, subordinate monkeys were found to have a disruption in normal ovarian function, as shown by high frequencies of anovulatory menstrual cycles and by luteal-phase progesterone deficiencies. It is noteworthy that surgical ovariectomy is associated similarly with an increased severity of coronary artery atherosclerosis in this species (Adams et al., 1985). These observations suggested to us that impairment of ovarian function, whether produced experimentally or as a result of the stress of social subordination, may compromise the female macaque's ordinary protection against coronary artery atherosclerosis.

Effect of Contraceptive Steroids on Plasma Lipoproteins and Coronary Artery Atherosclerosis in Female Monkeys

An important public health issue has been whether the lowering of plasma HDLs by certain oral contraceptives increases risk for coronary heart disease. For this reason, we have sought to determine whether contraceptive steroid-induced changes in the plasma lipids and lipoproteins of monkeys affected the progression of coronary artery atherosclerosis. The cynomolgus monkey model seemed critical to these studies, because, as previously discussed, they share with premenopausal white women a relative protection against coronary artery atherosclerosis, and endogenous estrogen is important in their "female protection.'' The cynomolgus female model seemed even more appropriate because we were able to show that the changes induced by higher-dose contraceptive steroids are similar to those reported for women (Adams et al., 1983, 1987a).

In our study on contraceptive steroid effects, nontreated, premenopausal cynomolgus monkeys were compared with premenopausal cynomolgus monkeys treated with an oral contraceptive regimen comparable with Ovral (Adams et al., 1987). Monkeys receiving oral contraceptive treatment had a slight increase in their total plasma cholesterol concentrations, a considerable increase in their LDL cholesterol, and a marked decrease in HDL cholesterol concentrations relative to control monkeys. Despite these changes, there was a clear reduction in the number of animals with coronary artery plaques and somewhat of a reduction in the extent of the plaques. We further examined a subset of treated and control animals at highest risk based on their lipid profiles. Although the mean total plasma cholesterol/HDL cholesterol ratios in the two groups were comparable, there was a striking difference in the extent of coronary artery atherosclerosis, with markedly smaller lesions among those animals treated with the contraceptive steroids.

References

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Figure 1 Squirrel monkey (Saimiri sciureus). Courtesy Oregon Regional Primate Research Center, Harry Wohlsein, photographer.

Figure 2 Rhesus monkey (Macaca mulatta). Courtesy Bernadette M. Marriott.

Figure 3 Cynomolgus monkeys (Macaca mulatta). Courtesy Office of Animal Care and Use, Division of Research Resources, National Institutes of Health.