Establishment Of Lp-PLA₂ Knockout Rabbit Model And Study Of The Impact On Atherosclerosis

Atherosclerosis is the major cause of cardiovascular diseases,such as myocardial infarction and stroke.Due to its wide prevalence and high mortality,atherosclerosis has emerged as an important global public health problem.Lipoprotein-associated phospholipase A₂(Lp-PLA₂)was once widely regarded as a biomarker of cardiovascular disease risk and a potential therapeutic target.Subsequently,an oral and selective Lp-PLA₂ inhibitor(Darapladib)has been applied to some animal experiments and clinical trials to assess whether it could protect against atherosclerosis.The results of preclinical and phase II clinical trials showed that Darapladib could effectively prevent the development of atherosclerosis and necrotic core expansion.However,two large-scale phase III clinical trials missed the primary endpoints in atherosclerosis patients.Therefore,it is very important to clarify the specific functions and mechanisms of Lp-PLA₂ under physiological and pathological conditions.First,a high cholesterol diet(HCD)was used to induce atherosclerosis in rabbits.We found that plasma Lp-PLA₂ activity increased significantly with the progress of atherosclerosis in rabbits.At the end of induction,Lp-PLA₂ m RNA levels were significantly higher in arterial tissues of HCD rabbits than in normal chow diet(NCD)controls.And in rabbits with atherosclerosis,Lp-PLA₂ transcripts were higher in aortas with plaques compared with those without plaques.In addition,fast protein liquid chromatography(FPLC)was used to separate plasma lipoproteins at different induction stages,and we detected the distribution of cholesterol and Lp-PLA₂ in various lipoprotein fractions.The results revealed that the cholesterol contents in very low-density lipoprotein(VLDL)and intermediate-density lipoprotein/low-density lipoprotein(IDL/LDL)increased rapidly with the development of atherosclerosis in rabbits.Distribution pattern of Lp-PLA₂ activity changed with the stimulation of HCD,the proportions associated with VLDL and IDL/LDL were increased significantly.In order to clarify the role and mechanism of Lp-PLA₂ in the process of atherosclerosis,we successfully generated Lp-PLA₂ gene knockout rabbits using CRISPR/Cas9 system.Plasma Lp-PLA₂ activity assays showed that Lp-PLA₂heterozygous knockout(Lp-PLA₂^+/-)reduced Lp-PLA₂ activity by half and Lp-PLA₂homozygous knockout(Lp-PLA₂^-/-)resulted in complete absence of Lp-PLA₂ activity.Lp-PLA₂ knockout rabbits(including heterozygotes and homozygotes)showed a significant reduction of plasma lipid levels,including total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),triglyceride(TG)and nonesterified fatty acids(NEFA).The results of FPLC further proved that the deficiency of Lp-PLA₂ reduced the cholesterol contents in plasma IDL/LDL fractions to a lower level.Preliminary studies on the mechanism of Lp-PLA₂ regulating blood lipid metabolism showed that Lp-PLA₂ deficiency did not affect the clearance of LDL by livers,it did affect LDL production by reducing VLDL production in Lp-PLA₂^-/-rabbits.In addition,Lp-PLA₂ deficiency could accelerate the catabolism of triglycerides.Analysis of aortic lesions at the induction endpoint showed that complete absence of Lp-PLA₂significantly reduced the aortic plaque areas and inhibited the occurrence of advanced lesions,such as calcification and necrotic core formation.However,partially reduced Lp-PLA₂ could not significantly inhibit the development of atherosclerosis,the lesion areas of Lp-PLA₂^+/-rabbits showed a large variation.Analysis of many inflammatory genes in arterial plaques showed that complete absence of Lp-PLA₂ exerted a general anti-inflammatory effect.In vitro cell experiments demonstrated that the reduction or complete absence of Lp-PLA₂ could affect the behaviors of key cells involved in atherosclerosis,such as the adhesion of monocytes to endothelial cells and uptake of low-density lipoprotein(LDL)and oxidized low-density lipoprotein(ox LDL)by macrophages or endothelial cells.In summary,Lp-PLA₂ knockout rabbits were used as carriers in this study.We determined that Lp-PLA₂ deficiency significantly reduced plasma lipid levels on a standard diet.The clearance of LDL by livers was not affected by Lp-PLA₂ deficiency,while the production of LDL was decreased in Lp-PLA₂^-/-rabbits.In addition,Lp-PLA₂ deficiency accelerated the catabolism of triglycerides.With a high-cholesterol diet,the complete absence of Lp-PLA₂ could resist atherosclerosis and significantly inhibit the expression of inflammatory genes in the arteries;while the partial reduction of Lp-PLA₂could not effectively inhibit the development of atherosclerosis.This study provides new insights into whether Lp-PLA₂ can be used as a therapeutic target for cardiovascular disease.