Investigation Of NARC-1 Expression In Atherosclerotic Lesion Of New Zealand Rabbit

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a new proprotein convertase (PC) gene discovered through the application of bioinformatics methods and DNA microarray, which encodes neural apoptosis-regulated convertase 1 (NARC-1), a member of PC family. NARC-1 degrades low density lipoprotein receptor (LDLR) and plays an important role in LDL internalization. PCSK9 gene mutation with gain of function results in a consequence that plasma LDL can not be removed efficiently, resulting in familial hypercholesterolemia. Thus, the mutation of this gene is regarded as the third type of familial hypercholesterolemia. Recently, a study has found that apart from the key role of PCSK9 in the regulation of cholesterol homeostasis and apoptosis, PCSK9 is also an important risk factor of atherosclerosis. Examining NARC-1/PCSK9 protein expression in atherosclerotic lesion will be helpful to clarify the relationship between the PCSK9 gene and formation of atherosclerosis, reveal a new mechanism for atherosclerosis development and provide new targets and ideas for the prevention and treatment of atherosclerotic diseases.OBJECTIVE To examine the expression of NARC-1 protein in atherosclerotic lesion of New Zealand rabbits.MEHTODS New Zealand male purebred rabbits, feeding adaptation for 1 week, were randomly divided into 2 groups, control group (n=8) and high-cholesterol group (n=8). The control group was given a normal control diet and the high-fat group was fed a high-cholesterol diet (2% cholesterol wt/wt). All rabbits were euthanized at the end of the 8-week experiment. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were determined by commercially enzymatic methods. Atherosclerosis index (AI) and the HDL-C/LDL-C ratio were calculated. Sudan IV staining was used to examine the aortic atherosclerotic lesions. The pathological changes of rabbit aortic preparations was quantified by computer-assisted image analysis of Oil red O and HE staining using Image-Pro Plus image analysis software. NARC-1 protein distribution and expression was analyzed by immunohistochemistry and Western blotting.RESULTS Plasma TC, HDL-C and LDL-C concentrations,and AI were markedly increased, but the HDL-C/LDL-C ratio was markedly decreased in high-cholesterol group. High-cholesterol diet induced significant aortic atherosclerotic lesions. Histopathology analysis showed that aortic intima (I) was obviously thickened and the intima/media thickness ratio was significantly increased in high-cholesterol group rabbits. Oil red O staining indicated that the number of foam cells in artery endothelium were markedly increased in the high-cholesterol group compared with the control group. HE staining revealed the aortic intima in control rabbits was integrity and thin, compered to thickened aortic intima and obvious atherosclerotic plaques in high-fat group rabbits. Immunohistochemical detection showed NARC-1 protein was found in the intima plaque and located in cytoplasma and plasma membrane of foam cells in high-fat group. Western blotting analysis also indicated NARC-1 protein expression was significantly elevated in the aortic atherosclerotic plaque of high-fat group rabbits.CONCLUSION The high-cholesterol diet induced aortic atherosclerotic lesion in New Zealand rabbits. NARC-1 protein was remarkably expressed in aortic atherosclerotic plaque and located in the cytoplasma and plasma membrane of the foam cells in high-fat group rabbits. The results indicate that NARC-1/PCSK9 is one of the important factors involved in atherosclerosis development.