Abstract
ABSTRACTAtherosclerosis is a complex disease affecting arterial blood vessels and blood flow that could result in a variety of life-threatening consequences. Disease models with diverged genomes are necessary for understanding the genetic architecture of this complex disease. Non-obese diabetic (NOD) mice are highly polymorphic and widely used for studies of type 1 diabetes and autoimmunity. Understanding atherosclerosis development in the NOD strain is of particular interest as human atherosclerosis on the diabetic and autoimmune background has not been successfully modeled. In this study, we used CRISPR/Cas9 genome editing to genetically disrupt apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) expression on the pure NOD background, and compared phenotype between single-gene-deleted mice and double-knockout mutants with reference to ApoE-deficient C57BL/6 mice. We found that genetic ablation of Ldlr or Apoe in NOD mice was not sufficient to establish an atherosclerosis model, in contrast to ApoE-deficient C57BL/6 mice fed a high-fat diet (HFD) for over 12 weeks. We further obtained NOD mice deficient in both LDLR and ApoE, and assessed the severity of atherosclerosis and immune response to hyperlipidemia in comparison to ApoE-deficient C57BL/6 mice. Strikingly, the double-knockout NOD mice treated with a HFD developed severe atherosclerosis with aorta narrowed by over 60% by plaques, accompanied by destruction of pancreatic islets and an inflammatory response to hyperlipidemia. Therefore, we succeeded in obtaining a genetic model with severe atherosclerosis on the NOD background, which is highly resistant to the disease. This model is useful for the study of atherosclerosis in the setting of autoimmunity.
Highlights
Atherosclerosis results from chronic inflammation and remains the top-ranking cause of human mortality (Lusis, 2000)
Genetic ablation of apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) in Non-obese diabetic (NOD) mice by the CRISPR/Cas9 system Previous studies suggested that NOD mice could be resistant to high-fat diet (HFD)-induced atherosclerosis; genetic validation of atherosclerosis on the pure NOD background deficient in ApoE or LDLR was not performed (Keren et al, 2001)
The NOD ApoE- or LDLR-deficient mice were generated by targeting the Apoe and Ldlr genes in NOD fertilized eggs by using the CRISPR/Cas9 system (Fig. 1A,B), as described in the literature and in the Materials and Methods section (Yang et al, 2014)
Summary
Atherosclerosis results from chronic inflammation and remains the top-ranking cause of human mortality (Lusis, 2000). To genetically validate development of atherosclerosis on the pure NOD background we used the CRISPR/Cas genome-editing tool to target Apoe and Ldlr loci. The double-knockout mice deficient in both ApoE and LDLR developed severe HFD-induced atherosclerosis In these novel models on a pure NOD background, we found that single-gene deficiency of Apoe or Ldlr had significantly lower serum lipids than Apoe-deficient C57BL/6 mice; the double-knockout animals on the NOD background had higher lipid levels. The double-knockout mice had a pro-inflammatory immune response to hyperlipidemia, and severe destruction of pancreatic islets Such a model developed on the resistant NOD background could be valuable to study atherosclerosis with complication of autoimmunity such as T1D (Eckel and Eisenbarth, 2012; Mitchell, 2012)
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