The Impact Of VEGF Together With The SiRNA And AP-2α On The Atherosclerotic Plaque

PartⅠAP-2αstimulate expression of vaseular endothelial growth factor in primary culture of mouse vascular endothelial cellsBackground:The occurrence,development and evolution of Atherosclerosis (atherosclerosis,As) are general pathological process induced by a variety of mediators.Vascular endothelial appears to play a key role,and endothelial dysfunction marks the beginning of an insidious disease process,which silently progresses to a point where it can only be slowed but not reversed.Reducing endothelial damage and restoring endothelial function in atherosclerosis of the occurrence and development of the role has gradually been given attention.An identification of susceptibility gene should provide novel insights into improved effective prevention and treatment of acute cardiovascular syndromes.Recent studies have showed that AP-2αcould affect the expression of VEGF in HaCaT keratinocytes through protein-DNA interaction,and AP-2αinvolved in the development of AS disease.Vascular endothelial cells layed over the inner surface of blood vessels at the simple squamous cells,and possessed the material transfer, autocrine,paracrine and many other functions.Therefore,It is extremely important to investigate the role of vascular biology through the study of vascular endothelial cells.It has not been reported that AP-2αcould regulate the expression of VEGF in aortic endothelial cells,but we known VEGF promoter contains AP-2 binding site.This study will explore how AP-2αregulated the expression of VEGF and the mechanisms of its involved in the formation of As.Methods:1.The aorta was removed from C57BIV6J mice(weight 20-25g) executed by cervical dislocation mice after thoroughly eliminate adventitial connective tissue in bacteria free condition.The vessel was opened longitudinally and cut into 1 mm×1 mm pieces,then placed on culture plates with the intima side down.Mouse aortic endothelial cells were primary cultrued(containing 20%FBS DMEM culture medium) by explant method and subcultred(containing 10%FBS DMEM culture medium).Morphological(growth characteristic) and immunological(VDI factor-related antigen) criteria were identified under inverted phase contrast microscope.2.Full length cDNA of AP-2αgene was amplified using PCR from cDNA library of mouse,the PCR production was ligated to pMD18-T vector,and obtained pMD18-T-AP-2αrecombinant expression vector, and then the pMD18-T-AP-2αwas digested with EcoRⅠand XhoⅠ.After electrophoresis,the segments of 1320 bp were recovered and ligated to the pCMV-Myc vector digested also with EcoRⅠand XhoⅠto obtain the pCMV-Myc-AP-2αvector.3.From 2 to 4 generation culture cells were transfected with plasmid pCMV-Myc,pCMV-Myc-AP-2α,AP-2α-SiRNA and Control-Si-AP-2αto the primary culture cells,respectively,and then investigated the expression level of mRNA and protein of VEGF using quantitative real-time RT-PCR and western blot.Results:1.After explanting culture for 4 days in DMEM culture medium containing 20%FBS,the vascular explants were removed and formed confluent monolayers,it showed a cobblestone shape under inverted phase contrast microscope approximately 12 days.The growth velocity of passaged cells were more fast than that of the primary cells,the former would form confluent monolayers after 4-5 days.Characteristic granular cytoplasmic staining pattern were revealed after immunohistochemical labeling for von Willebrand factor.Nuclei did not stain with special color for the negative control stained with PBS.2.Mouse vascular endothelial cells were transfected transiently with pCMV- Myc-AP-2αand pCMV-Myc,respectively,and the mRNA levels of AP-2αwere detected.The quantitative real-time RT-PCR analysis showed that AP-2αexpression levels increased significantly when the cells were transfected with pCMV-Myc-AP-2α,while decreased significantly after suppressing AP-2a by RNA interference. This indicated that the system worked correctly.When transfected with pCMV-Myc-AP-2α,pCMV-Myc,AP-2α-SiRNA and the Control-SiRNA,the mRNA expression levels of VEGF were significantly increase after over-expressing Ap-2αand decreased significantly after suppressing AP-2a by RNA interference.Western blot detection also showed similar results that the protein expression level of VEGF increased markedly after over-expressing AP-2α,while decreased significantly after suppressing AP-2a by RNA interference.Conclusion:1.We successfully achieved the primary culture cells of mouse aorta vascular endothelial cells using explantation method,it was satisfied to the research of basic or clinical.2.The recombinant plasmid vector pCMV-Myc-AP-2αwas successfully constructed for the next step to carry out genetic experiments of the relationship between AP-2αand VEGF.3.AP-2αcan up-regulate the expression of VEGFA,which implied that VEGF was a downstream gene of AP-2α. PartⅡ.The effect of VEGF-SiRNA and AP-2αon the atherosclerotic plaqueobjectives:To investigate the effect of Vascular endothelial growth factor(VEGF) and small interfering fragment RNA(siRNA) on the formation of atherosclerotic plaque and the interactions of VEGF and AP-2αby interference effect on aortic atherosclerotic plaque in vivo in different periods of apoE^-/- mice.Methods:1.Mouse vascular endothelial cells were transiently transfected with small interfering fragment(VEGFA-siRNA1,VEGFA-siRNA2, VEGFA-siRNA3,VEGFA-siRNA4) and the control sequence (Control-SiVEGF A).The mice was divided into 5 groups,mouse aortic endothelial cells were transfected with lipofectamine^TM2000.The mRNA expression level of VEGF gene was detected using RT-PCR analysis.2.Males apoE^-/- mice of 6-week-old,16-week-old,28-week-old (twenty mice of each age) separately were raised with a western-type diet for 4 weeks.And then the same age mice were divided to 4 groups randomly,each age group was randomly divided into VEGFA-siRNA4 group,contro1-siRNA,negative control group and blank control group, the first three groups were injected with 1mg/kg/d VEGFA-siRNA4, 1mg/kg/d control-siRNA,0.2ml of normal saline on mouse tail vein on alternate days,respectively,and the fourth group was injected nothing. The mice continued being raised on a western-type diet for two weeks.3.Serum lipid and lipoprotein detectionBefore perfusion-fixation,blood samples were collected,and serum total cholesterol,LDL cholesterol,HDL cholesterol,and triglycerides concentrations were detected,respectively.4.immunohistochemical staining in mice aortic atherosclerotic plaqueAccording to the instructions of Envision^TM two-step immunohistochemical staining kit,atherosclerotic plaques were stained with Monoclonal antibody of VEGFA and AP-2αto detect the expression levels of them.5.Real-time Quantitative RT-PCR AnalysisFor each group of animals(n=20),department of aortic sinus and thoracic aorta from 2 mice were pooled and total RNA was extracted.The mRNA level of VEGFA and AP-2αwas performed by real-time RT-PCR using SYBR green PCR Master Mix,and the data were analyzed with the LightCyeler software version3.5.6.Western BlottingFor each group of animals,department of aortic sinus and thoracic aorta from 3 mice were pooled and total protein was extracted,protein expression levels of VEGFA and AP-2αwere detected using western blot,and the data were analyzed with the LightCyeler software version3.5.Results:1.Expression levels of VEGFA protein and mRNA can be inhibited by siRNA1,siRNA2,siRNA3,siRNA4 in mouse aortic endothelial cells. The interference effect of VEGFA-siRNA4 worked best.2.There were no significant differences(P＞0.05) in serum lipid levels and body weight among experimental groups of mice and we can get the opposite results in comparison with each group.3.Immunohistochemical staining indicated immunoreactive cells were stained brown in atherosclerotic plaque.3.Real-time quantitative RT-PCR and Western blottingThe expression level of VEGF mRNA and VEGF protein in plaque content were the weakest while injected with VEGF-siRNA among the parallel group.In comparison with each group,the expression level of VEGF mRNA,VEGF protein,AP-2αmRNA and AP-2αprotein increase gradually with the increase of the age and achieve the highest level in 28-week-old group.However,the expression level of AP-2αmRNA content and AP-2αprotein had no significant effect of interference suppression with VEGF-siRNA.Conclusions:1.The specific siRNA sequences of VEGFA were successfully screened for gene silencing in mice through RNA interference technology to lay a foundation for animal experiments.2.Both VEGF and AP-2αwere participate in the formation of aortic atherosclerotic plaque.3.Both VEGF and AP-2αcould progress neovascularization in atherosclerotic plaque.RNAi targeting VEGF significantly suppresses expression of VEGF in progress of thoracic aortic atherosclerotic plaque in the ApoE-/- mice but does not affect the expression of AP-2αmRNA and AP-2αprotein.