Characterization of Lipid Metabolism and Atherosclerotic Phenotypes in Novel Humanized ApoE Knockin Rats

Abstract

Apolipoprotein E (ApoE) is one of the essential plasma apolipoproteins involved majorly in lipids’ metabolism. Human ApoE has three isoforms (E2, E3, and E4) differing by one or two amino acids. E3 is the most common one with a population allelic frequency of 78% and considered normal. E2 (7%) is associated with genetic disorder type III hyperlipoproteinemia. E4 (15%) is the major susceptibility gene related to the onset of Alzheimer's disease (AD) and cardiovascular diseases (CVDs). Gene modified mice carrying a mutant ApoE gene or different ApoE isoforms, such as ApoE knockout (ApoE KO) mouse and humanized ApoE knockin (hApoE KI) mice, provide a series of beneficial mouse models in the studies of atherosclerosis, hyperlipidemia, and AD. However, alternative animal models are necessary to investigate the roles and underlying mechanisms of ApoE in human diseases. Recently, the novel hApoE KI rat models created by Envigo RMS, Saint Louis, MO (preciously named Horizon Discovery and SAGE Labs respectively) by replacing endogenous rat ApoE with human ApoE2, E3, or E4 allele are available. Hypothesis The hApoE KI rats could recapitulate the diversities of metabolic phenotypes in human carrying polymorphic ApoE proteins, and will be ideal animal models to study the roles of ApoE proteins. Methods We examed the body weight, serum lipid levels, the mass of fat tissue, and vascular atherosclerotic lesions of these hApoE rat models in comparison to wide-type Sprague Dawley (WT) and ApoE KO rats, as well as the metabolic changes, death rate, and development of vascular atherosclerosis of these rats with 4-months atherogenic Paigen diet (PD) (40 kcal% Fat, 1.25% Cholesterol, 0.5% Cholic Acid). Results We investigate the body weight starting from week seven till month six, and found that all the hApoE KI and ApoE KO rats had a lower body weight than WT at the corresponding age. The hApoE2 KI rats showed lowest body weight. At six months (P6m), all hApoE KI and KO rats had lower anti-atherogenic lipoprotein HDL. The total cholesterol (TC), total triacylglycerol (TG), and LDL/VLDL of hApoE2 KI rats were higher than those of WT, hApoE3 KI and hApoE4 KI rats. ApoE KO rats also exhibited elevated total cholesterol and LDL/VLDL, but not total triacylglycerol. Surprisingly, the mass of white fat was less in hApoE2 KI rats. No obvious aortic plaques were identified in the en face aortas stained by Oil Red O of all normal diet-fed hApoE KI and ApoE KO rats at P6m. A mild atherosclerotic lesion was detected in frozen section of hApoE2 KI and ApoE KO aortic roots. After challenged with the PD, hApoE2 KI and ApoE KO rats died within two and half weeks. Causes of death were different: hApoE2 KI rats might be due to fat embolism in big vessels and heart chambers; ApoE KO rats might be caused by massive pulmonary alveolar hydrops (accompanying considerable lipid-laden macrophages accumulated in the lung). Thicker walls of small vessels in lung and heart, as well as necrosis of cardiomyocytes, were demonstrated in hApoE4 KI rats after four months PD. Conclusion hApoE2 rats develop hyperlipoproteinemia but are not a suitable animal model for the study of atherosclerosis. With the assistance of a PD challenge, hApoE4 KI rats could be a good model for the analysis of CVDs.