EXPERIMENTAL MODELS OF THE ATHEROSCLEROSIS ON RABBITS

Atherosclerosis is the main cause of cardiovascular diseases, which, despite a number of new advances in their diagnosis and treatment, still occupy a leading position. Experimental modeling of atherosclerosis in laboratory animals plays an important role in the study of the fundamental pathophysiological processes and pathology of atherosclerosis. Rabbits are among the most suitable animals for simulating atherosclerosis, as they are widely available, inexpensive to maintain, and easy to manipulate. The key advantage of rabbits over other animals is that their lipid metabolism is practically similar to that of humans. The aim of the study was to analyze literature data on experimental models of atherosclerosis in rabbits. The review shows that the history of the study of atherosclerosis by means of experimental models is very rich and originates from the works of the well-known Russian pathologists A.I. Ignatovsky, N.N. Anichkov, S.S. Khalatov (1908-1915), who developed a cholesterol model of the formation of atherosclerosis in rabbits. The principle of this model is to feed laboratory animals with food containing elevated levels of lipids and cholesterol. The composition of the cholesterol (atherogenic) diet may vary, determining the existence of modifications of this model. Most often, a diet with a cholesterol content of 0.3-0.5% is used, in cases where it is necessary to accelerate the development of atherosclerosis, a short-term use of a diet with a 1% cholesterol content is allowed. In addition to cholesterol, it is recommended to use vegetable oils (soybean, coconut or corn) in the atherogenic diet as they improve the absorption of cholesterol in the intestine. In 1980, Japanese researcher Y. Watanabe deduced a new model of atherosclerosis formation - on hereditarily determined hyperlipidemic rabbits Watanabe (WHHL-rabbits). WHHL rabbits contain a genetic mutation in the gene encoding low-density lipoprotein receptors, which results in these animals having high plasma cholesterol levels with a normal diet. Thanks to modern genetic technologies, various genetic models of atherosclerosis in rabbits have also been created: transgenic and “knocked out” rabbits. The main method for obtaining transgenic rabbits is pronuclear microinjection, which allows the introduction of a transgene (additional DNA fragment) into their genome. To date, using this technology, it has been possible to introduce more than a dozen genes responsible for lipid metabolism. The principle of creating knocked out rabbits consists in specific inactivation using genome editing technologies (ZFN, TALEN, CRISPR / Cas9) of a certain working gene. Experimental models of atherosclerosis in rabbits have not lost their significance and continue to be used to study the fundamental morphological (pathological) and pathological mechanisms underlying atherosclerosis, to search for new diagnostic biomarkers and potential targets for therapeutic effects, as well as to conduct preclinical trials of newly developed drugs.

Genetically Modified Rabbits for Cardiovascular Research

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

Gene Editing in Rabbits: Unique Opportunities for Translational Biomedical Research

The rabbit is a classic animal model for biomedical research, but the production of gene targeted transgenic rabbits had been extremely challenging until the recent advent of gene editing tools. More than fifty gene knockout or knock-in rabbit models have been reported in the past decade. Gene edited (GE) rabbit models, compared to their counterpart mouse models, may offer unique opportunities in translational biomedical research attributed primarily to their relatively large size and long lifespan. More importantly, GE rabbit models have been found to mimic several disease pathologies better than their mouse counterparts particularly in fields focused on genetically inherited diseases, cardiovascular diseases, ocular diseases, and others. In this review we present selected examples of research areas where GE rabbit models are expected to make immediate contributions to the understanding of the pathophysiology of human disease, and support the development of novel therapeutics.

The Creation of a Multiallele Knockout Genotype in Rabbit Using CRISPR/Cas9 and Its Application in Translational Medicine

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected.

Transgenic Rabbit Models: Now and the Future

Transgenic rabbits have contributed to the progress of biomedical science as human disease models because of their unique features, such as the lipid metabolism system similar to humans and medium body size that facilitates handling and experimental manipulation. In fact, many useful transgenic rabbits have been generated and used in research fields such as lipid metabolism and atherosclerosis, cardiac failure, immunology, and oncogenesis. However, there have been long-term problems, namely that the transgenic efficiency when using pronuclear microinjection is low compared with transgenic mice and production of knockout rabbits is impossible owing to the lack of embryonic stem cells for gene targeting in rabbits. Despite these limitations, the emergence of novel genome editing technology has changed the production of genetically modified animals including the rabbit. We are finally able to produce both transgenic and knockout rabbit models to analyze gain- and loss-of-functions of specific genes. It is expected that the use of genetically modified rabbits will extend to various research fields. In this review, we describe the unique features of rabbits as laboratory animals, the current status of their development and use, and future perspectives of transgenic rabbit models for human diseases.

The rabbit papillomavirus model: a valuable tool to study viral–host interactions

Cottontail rabbit papillomavirus (CRPV) was the first DNA virus shown to be tumorigenic. The virus has since been renamed and is officially known as Sylvilagus floridanus papillomavirus 1 (SfPV1). Since its inception as a surrogate preclinical model for high-risk human papillomavirus (HPV) infections, the SfPV1/rabbit model has been widely used to study viral–host interactions and has played a pivotal role in the successful development of three prophylactic virus-like particle vaccines. In this review, we will focus on the use of the model to gain a better understanding of viral pathogenesis, gene function and host immune responses to viral infections. We will discuss the application of the model in HPV-associated vaccine testing, in therapeutic vaccine development (using our novel HLA-A2.1 transgenic rabbits) and in the development and validation of novel anti-viral and anti-tumour compounds. Our goal is to demonstrate the role the SfPV1/rabbit model has played, and continues to play, in helping to unravel the intricacies of papillomavirus infections and to develop tools to thwart the disease. This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.

Hypertrophic cardiomyopathy R403Q mutation in rabbit β-myosin reduces contractile function at the molecular and myofibrillar levels

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human β-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999–1006.]. Since then, more than 300 mutations in the β-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or β-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a β-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force–velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in β-cardiac myosin.