E. A. Thorndike and A. S. Turner

Department of Clinical Sciences, Colorado State University, Ft.Collins ,CO 80523

Received 9/25/97 Accepted 4/10/98

4. CORONARY ARTERY DISEASE (CAD)

4.1 Animal Models Used to Study CAD

Many key questions regarding CAD and estrogen replacement therapy (ERT) have been answered with the assistance of animal models. In the 1950’s, for instance, the Michael Reese Research Institute used the hen as a model (8). They were the first to provide evidence that estrogen inhibited the progression of the disease.

Rodents and rabbits are practical and economical animals for the study of CAD. Most studies utilizing these species involve supraphysiologic lipid-concentrated diets with or without some form of vascular injury. In these models, the vascular injury (produced by intimal abrasion using an inflated balloon) induces a highly reproducible intimal migration and proliferation of vascular smooth muscle cells mimicking the early injury phase of atherosclerosis. However, there are still unanswered questions as to how these models relate to spontaneous human atherosclerosis. Mice (9), rats (10) and rabbits (11) have also been used to examine the effects of estrogen deficiency and arterial lipoprotein metabolism.

In the 1970’s, Magill and colleagues used the OVX baboon as a model to determine the effects of exogenous estrogen on atherosclerosis (12). This work led to the discovery that baboons, unlike many other primate species and humans, are resistant to dietary-induced atherosclerosis. There were no significant effects of estrogen replacement therapy, and no difference between male and female in the extent of dietary-induced coronary artery disease (11). This research contradicted data that had already been established with human studies (11) and suggested that the baboon was a poor model for some of the conditions seen after menopause.

The cynomolgus macaque (Macaca fascicularis) has traditionally been the non-human primate used most extensively in CAD research because of its susceptibility to dietary-induced coronary atherosclerosis (11). Its reproductive physiology is also comparable to that of humans, with a 28-day menstrual cycle, similar circulating hormonal patterns, and naturally occurring menopause (11). However, the acquisition of aged female monkeys is becoming more difficult and more costly (1). In addition, sophisticated housing facilities and technicians specially trained are required to handle the sometimes aggressive primate. One of the greatest disadvantages of using primates as an animal model is the potential for transmission of zoonotic diseases such as the Marburg virus disease, Ebola virus disease, viral hepatitis, Herpes virus simae, and tuberculosis (1). Thus, despite the large amount of productive research that has resulted from using the monkey, this model is extremely expensive and potentially dangerous. Additionally, use of this model is also complicated by the general distaste of the public for primate research.

4.2. The Use of Sheep to Study CAD

To date, few studies have used sheep as an animal model for CAD because of researchers’ unfamiliarity with ovine physiology, husbandry and veterinary requirements. In our laboratory, we examined the terminal aorta of OVX, sham-operated, and ovariectomized estrogen-supplemented (OVXE) ewes to determine the effects of estrogen deficiency and estrogen therapy. Histological analysis by light microscopy demonstrated that there was diffuse subintimal thickening in the distal aorta of animals that were estrogen deficient when compared to the control groups (figure 1). This marked subintimal thickening was significantly less in the sham-operated group and the estrogen supplemented group. With the use of an actin stain it was confirmed that smooth muscle proliferation was in fact partially responsible for the subintimal hyperplasia in the OVX animals .

Figures. 1. Representative light micrographs of the distal aorta in a sham-operated ewe (a) and an ovariectomized (estrogen-deficient) ewe after 6 months (b). There is marked subintimal thickening in the ovariectomized ewe.. E, endothelium; IEL, internal elastic lamina; E(N), endothelial nuclei; TM, tunica media; IH, intimal hyperplasia. Trichrome stain. Original magnification, X 40.

In the same study, we also compared serum cholesterol and triglyceride levels at four time points ( 0, 4 , 8, and 12 months) following OVX or sham surgery and found no effect of treatment on lipid profiles. The results strongly suggested that the myointimal thickening observed in OVX sheep was caused by estrogen deficiency rather than unfavorable changes in lipid profiles. In other words, intimal hyperplasia occurred despite normolipemia, and the hormonal influence on the vasculature was independent of lipid metabolism.

Numerous studies in humans have shown that increases in plasma total cholesterol or low density lipoprotein (LDL) cholesterol can increase the risk of CAD (13). Furthermore, it is believed that the effect of estrogen deficiency on CAD is partially mediated by an increase in high density lipoprotein (HDL) cholesterol. Although vascular pathology occurred without measurable changes in plasma lipids in sheep, these changes strongly suggest a link to alterations in reproductive hormone levels and it is likely that smooth muscle proliferation causing subintimal thickening was a result of OVX.

Future research using the sheep model should focus on whether increasing dietary fat can induce atherogenic changes. The diet of the grazing ruminant animal consists of pasture grasses and legumes which are low in lipid content (5-10 g lipid per 100g dry plant tissue) (14). Much larger intake of dietary lipid may be achieved in ruminant animals such as sheep by feeding diets containing "protected" lipid supplements. Previously, these have been used to increase energy intake or to produce polyunsaturated meat or milk. By spraying a casein/vegetable-oil emulsion with formaldehyde, the product provides an encapsulated form of lipid that is protected against lipolysis and hydrogenation by the rumen microflora but is well digested and absorbed from the lower gastrointestinal tract.

Providing "protected" lipid supplements would add little to the cost of the sheep model. Therefore, one of the most exciting questions regarding the use of the sheep model for CAD is: What would be the effect of a high fat diet (using "protected" lipid supplements) superimposed upon estrogen deficiency?

Phytoestrogens are nonsteroidal plant compounds, most of which are weakly estrogenic unless ingested in large quantities. They were responsible for infertility in sheep in southwestern Australia in the 1940s when red clover (with large quantities of estrogenic isoflavones) was ingested. Estrogenic isoflavones have been found in commercial feline diets containing soymeal, and there has also been a growing interest in the use of the estrogenic response of dietary soy in postmenopausal women (15). Unless the source of the sheep is from pastures with clover and there is evidence suggesting infertility, researchers using sheep can usually be assured that phytoestrogens are unlikely to be a confounding factor.