Hyperlipidemia induces typical atherosclerosis development in Ldlr and Apoe deficient rats
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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