Elsevier

Atherosclerosis

Volume 271, April 2018, Pages 26-35
Atherosclerosis

Hyperlipidemia induces typical atherosclerosis development in Ldlr and Apoe deficient rats

https://doi.org/10.1016/j.atherosclerosis.2018.02.015Get rights and content

Highlights

  • Rat models of atherosclerosis established using CRISPR/Cas9.

  • Systematic comparison of Apoe/Ldlr single and double knockout rats.

  • First demonstration for Apoe deficiency in rat led to typical atherosclerosis.

  • Sex difference profile similar to human disease in which males had heavier plaques.

Abstract

Background and aims

Low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) knockout (KO) mice have been widely used as animal models of atherosclerosis. However, data suggested that it is difficult to develop typical atherosclerosis in rats. To this end, Ldlr and Apoe KO rats were generated and the potential to develop novel atherosclerosis models was evaluated.

Methods

We established Apoe/Ldlr single and double KO (DKO) rats via the CRISPR/Cas9 system in the same background. Phenotypes of dyslipidemia and atherosclerosis in these KO rats were systematically characterized.

Results

Knockout of either gene led to severe dyslipidemia and liver steatosis. Significant atherosclerotic plaques were observed in the abdominal aorta of all mutant rats fed a normal diet for 48 weeks. Western diet greatly aggravated atherosclerosis and fatty liver. In addition, we found mononuclear cell infiltration in early lesions. Increased expression of inflammatory cytokines, as well as macrophage accumulation in lesions of mutants, was observed, indicating that mononuclear cell trafficking and endothelial inflammation affected atherogenesis. Moreover, mutant rats displayed a sex difference profile more similar to humans in which males had heavier plaque burdens than females.

Conclusions

Deficiency of either Ldlr or Apoe genes induced hyperlipidemia, which promoted endothelial inflammation and led to typical atherosclerosis in rats on normal or Western diets. These models display certain advantages, which will benefit future investigations of atherosclerotic pathology and antiatherosclerotic therapeutics.

Introduction

Atherosclerosis is the pathological and physiological basis, as well as a pre-symptom, of many cardiovascular diseases. Low-density lipoprotein receptor (LDLR) and apolipoprotein E (ApoE) participate in the transport of cholesterol-rich lipoproteins. Ldlr or Apoe deficiency in humans is related to elevated plasma total cholesterol and consequently higher risk of hypercholesterolemia, atherosclerosis and coronary artery disease [1,2]. Both Apoe and Ldlr knockout (KO) mice have become the most used animal models of atherosclerosis, and helped a great deal understand the biology of atherosclerosis [[3], [4], [5]]. However, the translation of discovery from mice to humans has been slow and unconvincing, largely due to differences between mice and humans in the pathogenesis and phenotype of atherosclerosis. For example, typical atherosclerosis in mice is usually induced by severe hypercholesterolemia. The most important clinical consequences of atherosclerosis in humans arise from lesions in the coronary, carotid, and cerebral arteries, whereas the focus in mice is on the aorta and proximal great vessels [6,7]. Intimal-thickening occurs in early lesions in human disease but not in mice [8]. Furthermore, data exclusively obtained from mouse models may be compromised by species-dependent effects and have limited value in identifying features unique to human disease. Therefore, it is necessary to develop novel animal models, to obtain a more comprehensive understanding of the initiation, progression and consequences of atherosclerosis, as well as to find effective therapies.

As certain characteristics of lipid metabolism in rats are in-between those of humans and mice [9], we speculated that rat atherosclerosis models might have advantages compared to mouse models. In fact, it is accepted that rat as a model animal has advantages over other animal models, especially for studying cardiovascular diseases such as hypertension and stroke [10]. So far, only two papers related to atherosclerosis in Apoe gene knockout rats have been published. One study described that occlusal disharmony accelerated atherosclerosis in Apoe KO rats, but only showed very early signs of atherosclerosis and lipid deposition [11]. Another report about Apoe KO rats generated through TALEN technology stated that Apoe KO rats were totally different from Apoe KO mice as the rats were resistant to hyperlipidemia-induced endothelial inflammation and did not develop atherosclerosis [12]. As for Ldlr, a paper in 2016 reported the generation of an Ldlr KO rat via Zinc-finger nuclease (ZFN) technology and stated that it did not develop plaques but could be a new model of hypercholesterolemia [13]. During the preparation of our paper, another Ldlr KO rat generated by ZFN was reported, which developed typical plaque formation when fed a Western diet, whereas no aortic lesions were found in the normal diet-fed group [14].

Here, using the CRISPR/Cas9 technique, we generated Apoe/Ldlr single and double KO rat models in the same genetic background. Phenotypes of the KO rats were systematically characterized. The three mutant rats developed major phenotypic and biochemical characteristics present in human atherosclerosis, including marked hyperlipidemia, different stages of atherosclerosis and liver steatosis. More importantly, heavier plaques were found in males, a sex difference more similar to humans than to mouse models, thus creating several promising alternative animal models for the study of human hyperlipidemia and atherosclerosis.

Section snippets

Animals and diet

All techniques and procedures were performed according to the NIH guidelines, and were approved by the Animal Ethics Committee of East China Normal University (Permit number: M20150505). Heritable total Ldlr or Apoe gene knockout (KO) rats were generated by CRISPR-Cas9 system in our lab according to the method described previously in Nature Protocol [15]. More detailed methods are provided in Supplementary material.

Biochemical analysis

Rats were fasted overnight (12–14 h) and blood samples from the retro-orbital

Generation of heritable total Ldlr and Apoe KO rats using CRISPR/Cas9 system

Two sgRNAs targeting Ldlr exon 4 were transcribed. Zygotes of SD rats were microinjected with a mixture of Cas9 mRNA (50 ng/μl) and sgRNA (25 ng/μl each). A total of 24 pups were born from 2 pseudopregnant female SD rats transferred with 100 injected zygotes. PCR analysis showed that nine rats (founder 2, 3, 7, 10, 16, 21–24) had an Ldlr deletion (Supplementary Fig. 1A). Further sequence analysis revealed that 23 rats (founder 1–4, 6–24) had a frameshift mutation (Supplementary Fig. 1B). In the

Discussion

This is the first report of typical atherosclerotic rat models based on CRISPR/Cas9 generated Ldlr/Apoe single or double knockouts. This work not only provides new insights into ApoE and LDLR functions in lipid metabolism and atherosclerosis related diseases, but also presents novel options for animal models of hyperlipidemia and atherosclerosis. The generated rat models had certain advantages, including a sex difference more similar to humans and the ability to develop lesions under normal

Conflict of interest

The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

Financial support

This work was supported by a grant from the National Natural Science Foundation of China [31271468] to HC.

Author contributions

YZ, HC, DL, WP and ML conceived and designed the experiments. YZ, YY, RX, XC, YX, LX, PY, TW and LZ conducted the experiments. HC, DL, YZ, YY, XC, and WP analyzed the results. HC, YZ, DL and ML wrote the manuscript. All authors critically revised the manuscript, read and approved the manuscript.

Acknowledgements

We thank Dr. Stefan Siwko, Texas A&M University Health Science Center, for scientific editing of the manuscript.

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