Study On The Mechanism Of Macrophage-Derived MMP9 In Atherosclerotic Model

Objective:In this study,the structural gene of rabbit matrix metalloproteinase 9(MMP9)was linked by preparing a scavenger receptor promoter,and a prokaryotic microinjection technique was used to make a macrophages-specific transgenic rabbit model expressing MMP9 to study atherosclerosis.The role of macrophage-derived MMP9 in the pathological process of atherosclerotic plaque formation and progression was analyzed in vivo experiments.In vitro experiments to observe the over-expression of MMP9 gene in HepG2 cells,proprotein convertase subtilisin/kexin type9(PCSK9),and the level of low density lipoprotein receptor(LDLR)gene expression,reveal the interaction between MMP9,PCSK9 and LDLR and their effects on lipid metabolism.Methods:In this study,Japan rabbit MMP9 cDNA was introduced into the pAL-1 vector containing a scavenger receptor promoter,and insulators were placed at both ends of the structure to prevent stable macrophages in animals not to affect the position of inserted chromosomes in order to achieve high expression of cell-specific MMP9.The constructed MMP9 gene was directly injected into the male pronucleus of the fertilized egg,and then the fertilized egg carrying the MMP9 gene was transplanted into the oviducts of the female pigs of the pseudo-pregnant recipient strain of the same strain to obtain a newly-transfected pup-bearing rabbit,and the transgeneration gene was screened for one generation.Male rabbits are sperm donors for reproduction.New born transgenic rabbits were fed with 0.3%cholesterol diet for 4 months and continued for 16 weeks.After the whole aorta and heart were removed,they were fixed with 10%neutral formalin,and the aorta and coronary artery were observed by HE staining.Arterial pathological changes in molecular and protein levels.In the cell experiment,HepG2 cells were transfected with the lentiviral packaging plasmid with MMP9 gene,and a MMP9 overexpression stable cell line was established.Experimental groups were set for the control group and the experimental group.The optimal concentration was selected by verifying the fluorescence expression of the MMP9 gene in the cells.RT-PCR and Western Blot validated the MMP9 mRNA expression level and protein expression level.Puromycin concentration 5μg/μL was screened for 2 weeks,and then preserved the successfully passaged cells.After successful modeling,the expression levels of PCSK9 and LDLR at the protein level and mRNA level in HepG2 cells overexpressing MMP9 were detected.Results:Compared with non-transgenic rabbits fed a high-cholesterol diet,there was no statistical difference in serum lipids including low-density lipoprotein,triglyceride and high-density lipoprotein between MMP9 transgenic rabbits and non-transgenic rabbits,and aortic arteriosclerosis plaques were found in the aorta.There was no significant difference in the area and location of the blocks.Further pathological analysis revealed that there was a large amount of calcium deposits at the endometrial lesions in the transgenic rabbits,accompanied by unstable plaque formation.Immunohistochemical staining pathological analysis of the arterial lesions was observed inside the transgenic rabbits,compared with non-transgenic rabbits,more lipid-rich foam cells aggregated in the intima.The results of coronary artery analysis showed that compared with non-transgenic rabbits,coronary plaques in transgenic rabbits showed similar results to the aorta,enriched in more macrophages and the deposition of calcium salts.At the same time,the expression of MMP-2,MMP-12,TIMP-1 and TIMP-2 protein in the aortic arch of the transgenic rabbits was increased.In cell experiments,the lentiviral packaging plasmid with MMP9 gene was successfully transfected into HepG2 cells,and a stable transfection cell line with MMP9 overexpression was established.After overexpression of MMP9 gene,PCSK9 showed a downward trend in protein level and mRNA level.While LDLR showed an upward trend at the mRNA level,there was no significant statistical difference in LDLR protein levels.Conclusion:The occurrence of atherosclerosis is closely related to the overexpression of MMP9.On the one hand,the increase of macrophage-derived MMP9 promotes the formation of advanced atherosclerotic lesions and promotes the occurrence of vascular calcification.On the other hand,overexpression of MMP9 gene in cells leads to a down-regulation of the PCSK9 gene level and thus to affect LDLR expression which may affect the lipid metabolism.