Biologic Features

Hypercholesterolemia is an important risk factor for premature atherosclerosis. One of the most severe manifestations of coronary and peripheral atherosclerosis occurs in homozygous familial hypercholesterolemia (FH), a genetic disorder characterized by elevated plasma low-density lipoproteins (LDL) and total plasma cholesterol.1

The disease is associated with xanthoma and premature atherosclerosis, and myocardial infarction occurs at an early age. The genetic basis for the disorder has been found to reside in the receptor for LDL.2 Homozygotes for FH have few or no functional LDL receptors, and LDL is removed from the plasma at one-third the normal rate. 

The ideal animal model for atherosclerosis should include well-defined genetic characteristics and features that are found in human disease, ranging from fatty streaks in early disease to complicated plaques in ad vanced disease.3 Lesions that occur in cholesterol-fed rabbits, a widely used model for atherosclerosis, are dif ferent from those in humans, sometimes resembling more of a lipid-storage disorder rather than atherosclero sis.4 An animal model for FH has been discovered in a strain of rabbits, designated Watanabe heritable hyper- lipidemic (WHHL) rabbits.5 This model is similar clinically and biochemically to patients with FH and fulfills, for the most part, criteria that are considered desirable for an atherosclerotic animal model.

The WHHL rabbit was first described by Watanabe, a veterinarian at Kobe University in Japan, who made the observation that one male rabbit in his colony had a ten- fold elevation in plasma cholesterol.5 He bred this rabbit with a normal female and obtained offspring with mild elevations of plasma cholesterol; subsequent breeding resulted in offspring with both mild and severe hypercholesterolemia. The pattern was established as a simple Mendelian trait with complete expression in the homozy gous state and partial expression in the heterozygous state, similar to humans. Homozygous WHHL rabbits have plasma cholesterol levels up to 900 mg/dl, most of which is in the LDL fraction, whereas heterozygous WHHL rabbits have marginally elevated plasma choles terol (Table 1 ). Cultured fibroblasts from homozygous rabbits express less than 5% of the expected number of LDL receptors.6

Homozygous WHHL rabbits have a pattern of athero sclerosis that is similar to that found in patients with FH.7,8 The earliest and most easily detectable lesions occur in the aortic arch at 2 months1 with deposition of cholesterol esters in intimal and medial smooth muscle cells and macrophage foam cells. At approximately 6 months, nu merous raised lesions in all portions of the aorta are evident, and by 1 0 to 1 2 months, advanced atherosclerotic plaques can be seen (Figure 1 ). Advanced lesions contain a necrotic cholesterol-filled core, calcification, and a fibrous cap (Figure 2). Electron microscopy shows a cellular population in these intimal lesions, comprised of smooth muscle and macrophages with cytoplasmic lipid droplets containing neutral lipid and multilamellar bodies (Figure 3).7 Coronary artery disease can be detected by 5 months of age and is most severe at the ostia, particularly the left coronary artery (Figure 4). Coronary thrombosis is rarely seen. Manifestations of ischemic heart disease may appear as early as 5 months of age, and virtually all rabbits have evidence of myocardial ischemia by 10 months.8 WHHL homozygotes have a lifespan of 2—4 years compared with normal rabbits, with lifespans of 5 to 6 years.8 

Homozygous WHHL rabbits have small subcutaneous xanthoma, usually adjacent to tendons of the hind paws, which contain typical foamy histiocytes that stain positive for lipid. Arcus cornea also occurs, but lipid deposits in other tissues are limited, usually consisting of rare foam cells in lymph nodes, spleen, and Kupffer cells of liver.7 

Heterozygous WHHL rabbits develop minimal atherosclerosis,9 probably since their diets are low in fat and cholesterol. WHHL heterozygotes fed up to 1% cholesterol and 2% peanut oil develop atherosclerotic lesions similar to WHHL homozygotes, with advanced plaques in the aorta and coronary arteries that exhibit necrosis, cholesterol clefts, fibrous caps, and calcification.9 These lesions differ from those observed in normal rabbits that are fed the same amount of dietary cholesterol, in which foam cell lesions predominate.

Table 1. Plasma Lipids and Lipoprotein Cholesterol in Control (New Zealand White), Heterozygous WHHL, and Homozygous WHHL Rabbits

 Values are expressed as mg/dl; plasma was obtained from males aged 8-12 months. t From Goldstein et al 1 and values are expressed as mg/dl; plasma was obtained from male rabbits aged 3 to 5 months.

Comparison with Human Disease

Table 2. Summary of Morphologic Findings in Homozygous WHHL Rabbits Compared with Cholesterol-fed WHHL Heterozygous and Cholesterol-fed Control (New Zealand White, NZW) Rabbits and Humans with Advanced

* NZW regression = NZW rabbits fed 2% cholesterol for 8 weeks, followed by 5 months of normal diet (age at time of sacrifice, 12 months).

t Associated with pulmonary hypertension in elderly patients.

4 Not examined.

11 From Atschul R: Selected Studies on Atherosclerosis, Springfield, IL, Charles C. Thomas, 1950.

¶ Kjaernes et al. (Acta Pathol Microbiol Scand (A) 1981, 89:35) have noted a difference between human and cholesterol-fed animals for the localization of lipid at arterial branches.

# Aorta.

** Transmural infarction was found in 8 of 67 hearts from WHHL homozygotes examined by Hatanaka et al.8 tt Homozygous familial hypercholesterolemia.

NZW rabbits were fed 0.5% cholesterol and 2% peanut oil for 24 weeks and were 7 months of age at the time of sacrifice.9 WHHL homozygous rabbits were 12 months of age.

Plasma cholesterol levels are comparable between patients with FH and homozygous WHHL rabbits, both of whom have marked increases in LDL. However, plasma triglycerides are elevated in the rabbit but not in patients with FH. This is due to the fact that WHHL rabbits have elevations in the levels of all lipoproteins that contain apoprotein B-100 (very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL, all of which contain substantial triglycerides).10 High density lipoproteins are reduced in both WHHL rabbits and patients. The expression of functional LDL receptors in WHHL rabbits and patients is similarly low, and intravenously injected 125I-labeled LDL disappears slowly from the circulation of homozygous WHHL rabbits, a finding that is similar to patients with homozygous FH.6 There are qualitative parallels between lipoprotein metabolism in the WHHL rabbit and patients with FH, although certain quantitative differences exist.1 

Usefulness of the Model

The WHHL rabbit as an animal model for endogenous hypercholesterolemia has contributed to our understanding of the LDL receptor in the production and clearance of lipoproteins that carry endogenous cholesterol. Newer uses for the WHHL rabbit as an experimental model include upregulation of LDL receptors in heterozygous WHHL rabbits by pharmacologic agents,1 and lowering LDL levels in animals that do not have the LDL receptor.1 The latter area is being approached by using agents that might prevent oxidative modification of LDL (i.e., probucol),11 and by using inhibitors of HMG CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis.12 Others have used the WHHL rabbit to study the role of agents not directly involved in lipoprotein metabolism, such as calcium-channel blockers, in preventing or retarding atherogenesis.13 The WHHL rabbit can serve as a model to investigate any number of factors that have been implicated in atherogenesis, including platelets, monocytes, and the endothelium.1’14 The recent development of the cholesterol-fed heterozygous WHHL rabbit as a model for atherosclerosis may even be more relevant for the majority of humans, in whom both diet and genetic predisposition appear to play interactive roles in atherogenesis.9

Availability of the Model

A breeding colony of WHHL rabbits has been established at the National Institutes of Health and animals canbe purchased by contacting Dr. Carl T. Hanson, Miss Damara Bolte, Small Animal Section, Bldg. 14, Room 101, National Institutes of Health, Bethesda, MD 20892.

References

1. Goldstein JL, Kita T, Brown MS: Defective lipoprotein receptors and atherosclerosis, N Engl J Med 1983, 309:288-296

2. Brown MS, Goldstein JL: A receptor-mediated pathway for cholesterol homeostasis. Science 1986, 232:34-47

3. Jokinen MP, Clarkson TB, Prichard RW: Recent advances in molecular pathology. Animal models in atherosclerosis research. Exp Mol Pathol 1985, 42:1—28

4. Prior JT, Kurtz DM, Ziegler DD: The hypercholesterolemic rabbit. An aid to understanding arteriosclerosis in man? Arch Pathol 1961, 71:672—684

5. Kondo T, Watanabe Y: A heritable hyperlipidemic rabbit. Exp Anim 1975, 24:89—94

6. Tanzawa K, Shimada Y, Kuroda M, Tsujita Y, Arai M, Watanabe H: WHHL-rabbit: a low density lipoprotein receptor- deficient animal model for familial hypercholesterolemia. FEBS Lett 1980, 118:81—84

7. Buja LM, Kita 1, Goldstein JL Watanabe Y, Brown MS: Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia. Arteriosclerosis 1983, 3:87-101

8. Hatanaka K, Ito T, Shionii M, Yamamoto A, Watanabe Y. lschemic heart disease in the WHHL rabbit: a model for myocardial injury in genetically hyperlipidemic animals. Am Heart J 1987, 113:280—288

9. Atkinson JB, Hoover RL Berry KK, Swift LL: Cholesterol-fed heterozygous Watanabe heritable hyperlipidemic rabbits: a new model for atherosclerosis. Atherosclerosis 1989,78:123-136

10. Havel RJ, Kita T, Kotite L, Kane JP, Hamilton RL Goldstein JL, Brown MS: Concentration and composition of lipoproteins in blood plasma of the WHHL rabbit. Arteriosclerosis 1982, 2:467—474

11. Carew TE, Schwenke DC, Steinberg D: Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Nail Acad Sci USA 1987, 84:7725-7729

12. Watanabe Y, Ito T, Shiomi M, Tsujita Y, Kuroda M, Arai M,Fukami M, Tamura A: Preventive effect of parvastatin sodium, a potent inhibitor of 3-hydroxy-3-methy1glutarl coenzyme A reductase, on coronary atherosclerosis and xanthoma in WHHL rabbits. Biochim Biophys Acta 1988, 960:294-302

13. Van Niekerk JLM, Hendricks 1, De Boer HHM, Van’t Laar A:Does nifedipine suppress atherogenesis in WHHL rabbits? Atherosclerosis 1984, 53:91—98

©1999, Janet Becker Rodgers, DVM, MS

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