Associate editor: M. EndohRabbit models for the study of human atherosclerosis: From pathophysiological mechanisms to translational medicine
Introduction
Atherosclerotic diseases such as myocardial infarction and stroke are still major causes of mortality globally. Atherosclerosis develops slowly throughout the stages of human life and many genetic and environmental factors are concertedly involved in the pathogenesis of this disease (Libby et al., 2011). For study of the pathophysiology and also for the development of therapeutic modalities and diagnostic tools, utilization of appropriate experimental animals is essential. In general, the ideal animal models of human atherosclerosis should possess several important characteristics (Fan and Watanabe, 2000, Vesselinovitch, 1988). They should be easy to acquire and maintain at a reasonable cost, easy to handle, and the proper size to allow for all anticipated experimental manipulations. Ideally, the animal should reproduce in a laboratory setting and have a well-defined genetic background. Finally, the animal model should share with humans the most important aspects of lipid metabolism and cardiovascular pathophysiology. Atherosclerotic lesions should develop gradually while the animal consumes a special diet, and features of the lesions (from the fatty streaks to complicated plaques with calcification, ulceration, hemorrhage and thrombosis) should mimic those of human patients with clinical sequelae (such as myocardial infarction, stroke and gangrene). Unfortunately, there is no single animal model that fulfills all of these requirements; therefore, many different animals have been used to illustrate the diverse aspects of atherosclerosis (Moghadasian, 2002). Among those used to date, rabbits are the first and one of the best models for the study of lipoprotein metabolism and atherosclerosis.
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History of rabbit atherosclerosis models
The first experiment using rabbits to investigate atherosclerosis should date back to more than a century ago. In 1908, a Russian physician, Ignatowski, fed rabbits with a diet enriched in animal proteins (milk, meat and eggs) and observed intimal lesions with large clear cell (now called foam cell) accumulation in the aorta (Ignatowski, 1908). Later, a Russian experimental pathologist, Anitschkow, used a cholesterol diet dissolved in vegetable oil to produce aortic atherosclerosis in rabbits
Lipid metabolism features of normal rabbits
All laboratory rabbits (including NZW and JW) are originated from European rabbits (Oryctolagus cuniculus) and belong to the family Leporidae of the order Lagomorpha. The rabbit is an herbivore, and its typical laboratory chow diet contains ~15% protein, 40–50% carbohydrate, 2% vegetable fat and 15–25% fiber. Normally, the cholesterol (phytosterol) content in a regular chow diet is less than 0.01%. On this type of diet, plasma cholesterol levels for both New Zealand White (NZW) and Japanese
Cholesterol-fed rabbits
As mentioned above, rabbits are sensitive to dietary cholesterol and rapidly develop severe hypercholesterolemia leading to prominent aortic atherosclerosis. Therefore, cholesterol-fed rabbits are widely used for atherosclerosis studies. When rabbits are fed a chow diet containing up to 2% cholesterol, they show rapid elevation of plasma cholesterol, which can exceed 2000 mg/dl. This response can be further enhanced by adding extra fat in the diet, with saturated fats increasing both plasma
Watanabe heritable hyperlipidemic (WHHL) rabbits
WHHL rabbits as an excellent animal model of human familial hypercholesterolemia were established by Dr. Yoshio Watanabe at Kobe University, Japan, in 1980 (Kobayashi et al., 2011, Shiomi and Ito, 2009, Watanabe, 1980). WHHL rabbits are genetically deficient in LDL receptor functions due to a spontaneously arising deletion in exon 4 of the LDL receptor gene that encodes a 4-amino-acid deletion in the cysteine-rich ligand-binding domain (Yamamoto et al., 1986). Homozygous WHHL rabbits on a chow
Transgenic rabbits
In addition to cholesterol-fed rabbits and WHHL rabbits, transgenic (Tg) rabbits have also been used for the study of human cardiovascular disease during the last two decades. The technology for producing Tg rabbits was simultaneously developed by German (Brem et al., 1985) and US (Hammer et al., 1985) groups in 1985, but the actual use of Tg rabbits as an experimental tool in the field of cardiovascular diseases was not realized until 1994 when the first Tg rabbit expressing human hepatic
Knock out (KO) rabbits
Owing to the lack of embryonic stem (ES) cells and genome information in rabbits, for a long time, it has been impossible to generate knock out (KO) rabbits using homologous recombination-based genomic manipulation in ES cells as in mice. Lack of KO rabbits also constitutes another barrier for researchers to use gene deficiency rabbits for human disease study. We attempted to adopt somatic cell nuclear transfer for gene targeting in rabbits since the first cloned rabbit was reported 12 years ago
Considerations and limitations
Although rabbits are a valuable experimental model in cardiovascular research, they should not be considered as a simple substitute of rodents, as discussed above. Rabbits should only be used for those experiments aimed at answering questions or elucidating mechanisms that cannot be properly addressed in other animal models, such as translational research for the development of lipid-lowering drugs or diagnostic methods. Without these considerations, there may be less advantage of using rabbits
Conclusions
The rabbit is an important model for the study of human atherosclerosis and lipoprotein metabolism. Transgenic and KO rabbits will provide novel means not only for the elucidation of molecular mechanisms but also for translational research. We can expect that these novel models will provide new insights into the understanding of atherosclerosis and expand the power of the rabbit model for translational research in cardiovascular disease. In order to enhance the use of rabbit models in the
Sources of funding
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (71790514, 19390099, 21659078, and 22390068 to JF), the National Institutes of Health (HL105114, HL088391, NS066652, and HL068878 to Y.E.C.), and the American Heart Association (National Scientist Development 0835237N to J.Z.).
Disclosures
None.
Acknowledgments
We would like thank Drs. Shiomi M., Niimi M., Li S., Koike T., Yu Y., Liang J., Waqar BA, Ning B., and Nishijima K. for their contributions to this project.
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