Association Between Intraplaque Angiogenesis And Plaque Instability: The Role Of Local Interference For Vascular Endothelial Growth Factor-A

Objective:To construction of recombinant adenoviruses encoding human vascular endothelial growth factor165(VEGF₁₆₅)gene.Methods:The PDC315 plasmid and PDC-VEGF plasmid were digested with EcoR I and Xba I,then 538bp of CMV and 3973bp VEGF₁₆₅cDNA fragment were recovered and ligated by T4 DNA ligase.The ligation products were transformed into DH5a competent cells,resulting in the recombinant plasmid PDC315-VEGF which was confirmed by restriction enzyme digestion and DNA sequencing.The plasmid PDC315-VEGF was cotransfected together with pBHGE3 into 293 cells,where a near-complete cytopathic effect appeared 9～12 days later.Then 293 cells were exposed to 3 freeze-thaw cycles, and centrifuged.Three cycles of plaque purification and virus expansion were performed to ensure a single viral clone.The DNA of the adenovirus was extracted and then verified by PCR.The PCR primer sequences for VEGF₁₆₅are as follows: 5'-CCTTGC TGCTCTACCTCC-3'(sense),5'-AAATGCTTTCTCCGCTCTG-3' (antisense).The adenoviruses were purified using a double cesium chloride banding procedure according to standard techniques,dialyzed extensively against TBS buffer at 4℃and sterilized using a 0.22μm filter.The functional PFU titers were determined by plaque assays in 293 cells.Results:The recombinant plasmid PDC315-VEGF was correctly constructed and confirmed by restriction enzyme digestion analysis and DNA sequencing for full length of human VEGF₁₆₅cDNA fragment.The replication deficient recombinant adenoviruses vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR which showed that VEGF₁₆₅ mRNA was transcripted from the VEGF gene.The virus concentration reach to 5.0×10⁹ pfu/ml.Conclusion:The recombinant adenoviruses vector encoding human VEGF₁₆₅gene was successfully constructed. Objective:To design the small interference RNA(siRNA)specific to mouse endothelial vascular growth factor A(VEGFa)gene by RNA interfering technique, and construct its recombinant lentiviral expression vector.Methods:1.According to Tuschl's principle,three target sequences of mouse VEGFa gene were selected,and an irrelevant siRNA with a random combination of the mouse VEGFa gene was used as negative control sequence.Then four couples of complementary oligonucleotides of each sequence with hairpin loop of siRNA were synthesized.After annealing of the complementary strands,the DNA fragments were ligated into linearized plasmid pRNAT-U6.2/Lenti which linearized by restriction endonucleases BamH I and Xho I.The recombinant plasmid was transformed into competent E.coli.DH5a cells to amply and then purified.The purified plasmids were identified by PCR amplification and DNA sequencing.2.According to mouse vascular endothelial growth factor A(VEGFa)cDNA sequence,a pair of specific primers which contained respectively digestion site of EcoR I and BamH I on the 5' end were designed and constructed.Then revert transcript polymerase chain reaction(RT-PCR)was employed to clone VEGFa cDNA from mouse cells NIH/3T3 strain.After being purified,the VEGFa fragment was subcloned into linearized plasmid pKCDNA-EF1-Puro which linearized by restriction endonucleases BamH I and EcoR I.The recombinant plasmid pCDNA-VEGF was transformed into competent E.coli.DH5a cells to amply and then purified.The purified plasmids were identified by PCR amplification and DNA sequencing.3.Six groups were assigned.After plasmid pCDNA-VEGF and siRNA lentiviral plasmids were cotransfected into NIH/3T3 cells with the liposome mediation,then the cells were collected 48h late.The effect of RNAi on the protein and mRNA expression of VEGFa was examined with western blot and real time fluorescence quantitative reverse transcriptase PCR,respectively.4.With the help of lipofectamine^TM2000,recombinant lentiviruses were produced by 293T cells following the cotransfection of plasmid pRNAT-siRNA3 or pRNAT-negative with three package plasmid compound which consists of pKCPACK-GAG,pKCPACK-REV,pKCPACK-VSV-G.The expression of GFP was examined under fluorescent microscope 24h after transfection.After 48h transfection, the lentivirus supernatant on 293T cells was collected.The titers of the recombinant lentiviruses were determined by scoring GFP expression following serial dilutions of the viral supernatant.Results:1.Three recombinant lentiviral plasmids of siRNA specific to mouse VEGFa gene and one negative were constructed successfully.The results of the gel electrophoresis and their DNA sequence analysis completely coincided with their designed sequences.2.The product of RT-PCR contained the mouse VEGFa cDNA.The recombinant plasmid pCDNA-VEGF contained correct nucleotide sequence for full length of mouse VEGFa cDNA fragment by DNA sequence analysis.3.After plasmid pCDNA-VEGF was transferred into NIH/3T3 ceils,the VEGFa expression effectively increased at the level of mRNA and protein.VEGFa siRNA knocked down VEGFa expreesion in NIH/3T3 cells obviously.However,the RNA interference effects showed a significant disparity.Compared with pRNAT-negative, recombinated siRNA lentiviral plasmids(pRNAT-siRNA1,pRNAT-siRNA2 and pRNAT-siRNA3)inhibited the VEGFa expression at the different levels of mRNA and protein.Among them,pRNAT-siRNA3 could causes more efficient down regulation of VEGFa expression,resulting in down regulation of VEGFa mRNA and protein by approximately 80.0%and 66.3%respectively.4.The transfected 293T cells were found containing strong expression of GFP 24h after transfection,confirming that the four plasmid system of the lentiviral vector and its packaging cell line were successfully constructed.After 48h of transfection, the lentivirus supernatant was collected and the titer of the recombinant lentiviruses reached 1×10⁸ ifu/ml.Conclusion:The recombinant lentiviruses,which can express siRNA hairpin aimed at VEGFa gene,have been constructed successfully. BackgroundIntraplaque hemorrhage(IPH)is believed to arise from the disruption of thin-walled microvessels that are lined by a discontinuous endothelium without supporting smooth-muscle cells.Therefore,the immature neovascular may be more fragile and probably contribute to intraplaque hemorrhage and cause plaque rupture.However, the role of angiogenesis in plaque destabilization and rupture has emerged as a major unresolved issue.Vascular endothelial growth factor-A(VEGF-A),which is the most important and dominant proangiogenic cytokines,plays a major role in neovascularization.This study aims to assess the effect of neoangiogenesis on plaque stability in shear-induced advanced atherosclerotic plaques in apoE^-/-mice.ObjectivesIn this study we analyzed the effects of VEGF overexpression in advanced atherosclerotic plaques in the carotid artery of apoE^-/-mice.On the mean while,we also sought to assess the ability of RNA interference targeting VEGF to inhibit plaque neovascularization.Then we analyzed the effects of neoangiogenesis on plaque stability.Methods1.Animal modelMale apoE-deficient mice(n=164),8 to 10weeks of age,were placed on a Western-type diet.Atherosclerotic lesions were induced by perivascular constrictive collar placement on the left common carotid artery.The collar was removed after four weeks and 100μl of Ad.VEGF(5.0×10⁹ pfu/ml),or Ad.LacZ(5.0x 10⁹ pfu/ml), siRNA.VEGF(1.0×10⁸ ifu/ml),siRNA.Negative(1.0×10⁸ ifu/ml)suspension in 20% pluronic-127 gel was added locally on the adventitia surface of carotid artery and incubated at room temperature for 20 minutes.Four weeks late,atherosclerotic lesions from carotids were analyzed.2.Recombinant adenoviral and lentiviruses expression patternTo evaluate the efficiency and distribution of adenoviral vascular transduction,the gene transfer efficiency was examined using 5-bromo-4-chloro-3-indolyl-β-D-galactopyransidase(X-gal)staining assay. To evaluate the efficiency and distribution of recombinant lentiviruses vascular transduction,the expression of green fluorescence protein was observed which auto-fluorescence was suppressed with 0.5%Chicago Sky Blue.3.Serum lipid and lipoprotein measurementBefore perfusion-fixation,blood samples were collected and serum total cholesterol, LDL cholesterol,HDL cholesterol,and triglycerides concentrations measured.4.Histological and morphometric analysisFor each group of animals(n=20),cross cryosections were stained with hematoxylin and eosin(H & E),oil red O,picrosirius red,Masson' s trichrome,Perl's staining. Immunohistochemical staining were performed for detecting macrophage,smooth muscle cells,von Willebrand factor(vWF),Fibrinogen,VEGF,MMP2,MMP9.The plaque component was expressed as a percentage of the total intimal area.5.Real-time Quantitative RT-PCR AnalysisFor each group of animals(n=12),carotids from 3 mice were pooled and total RNA was extracted.The gene expression analysis was performed by real-time RT-PCR using SYBR green PCR Master Mix and data were analyzed with the LightCycler software version 3.5.7.Western BlottingFor each group of animals,carotids from 8 mice were pooled and total protein was extracted.Equal amounts of protein lysate was loaded onto SDS-PAGE,and Western blot analyses using antibodies and visualization by ECL were performed.Results1.General characteristicThroughout the experiments,the mice remained in good health and gene transfer was well-tolerated.There were no significant differences in serum lipid levels and body weight among experimental groups of mice.2.Recombinant adenoviral and lentiviruses expression pattern Abundant strongly positive staining forβ-galactosidase activity was revealed in the neointimal compartment 2 week after periadventitial transduction with Ad.LacZ. Two weeks after lentiviruses transfection,the expression of green fluresence protein was observed in RNAi plaque. The relative area of VEGF staining,VEGF mRNA and VEGF protein in Ad.VEGF transferred atherosclerotic lesions was significantly increased which determined by immunostaining,real-time PCR,and Western blot,whereas the plaques transferred with VEGF-targeted siRNA expression lentiviruses significantly inhibited VEGF expression.3.Histological and morphometric analysis Morphometric analysis showed that Ad.VEGF delivery to shear-induced plaques significantly increased plaque size and led to an almost complete occlusion of the lumen,but there were considerably smaller plaques in siRNA.VEGF transferred carotid.Lipids were abundantly present in plaques and no difference in lipid content among groups.In Ad.VEGF transferred lesions,the collagen staining andα-SM-actin staining were decreased,macrophage positive areas were significantly larger,the increased expression of endogenous MMP-2 and MMP-9 were consistent with the distribution of macrophages,MMP2 mRNA and MMP9 mRNA synthesis and protein secretion were increased.However,in siRNA.VEGF transduction lesions,the collagen staining andα-SM-actin staining were increased,macrophage positive areas were significantly lower,the decreased expression of endogenous MMP-2 and MMP-9 were consistent with the distribution of macrophages,MMP2 mRNA and MMP9 mRNA synthesis and protein secretion were decreased.Subsequent analysis revealed that MMP9 and MMP2 protein expression was predominantly in the proteolytically activated form in Ad.VEGF transferred lesions,whereas MMP9 and MMP2 protein expression was predominantly as a proform in siRNA.VEGF transferred lesions. Vulnerable index from Ad.VEGF group was 3.59±0.42,significantly greater than other three group(Ad.LacZ:2.08±0.17,P＜0.001;siNRA.Negative:2.03±0.18, P＜0.001;siRNA.VEGF:1.12±0.08,P＜0.001).4.Angiogenesis and plaque stability Thin-walled,capillary-like vessels were observed in atherosclerotic lesions.The Ad.VEGF transferred lesions contained extensive areas of neovascularization (24.0±1.9),whereas the siRNA.VEGF transferred lesions contained local areas of neovascularization(6.6±1.2).Subsequently,the Ad.VEGF transduction led to a considerable increase in the incidence of IPH and fibrous cap rupture accompanying the thrombus formation in 14 mice(14 of 20),whereas in Ad.LacZ group, siRNA.Negative group and siRNA.VEGF group,a mere 15%(3/20),10%(2/20)and 10%(2/20)of such adverse events were observed.The hemorrhage mostly located in neovascularized areas.Conclusions1.The mean for gene transduction through local periadventitial is effective.2.VEGF overexpression promote angiogenesis in advanced plaques and induce plaque vulnerability.3.RNAi targeting VEGF significantly suppresses expression of VEGE, neovascularization and stability in advanced plaques. ObjectiveIt is well known that atherosclerotic plaques are preferentially present at lesion-prone sites and shear stresses play a key role in the pathogenesis of atherosclerosis in humans.However,artery shear stress is much higher in mice than in humans.The effect of shear stress on atherosclerosis in apoE^-/-mice is little to known because of technical difficulties.The aim of this study was to determine in vivo shear stress values in the development of rapid,site-controlled atherogenesis in apoE^-/-mice.MethodsMale apoE^-/-mice,age 8 weeks,were raised on Western-type diet two weeks before operation and continued after operation.A constricting silastic tube(0.30mm inner diameter)or a nonconstrictive silastic tube(0.60mm inner diameter)was placed on left carotid artery.Horseradish peroxidase(HRP,Typeâ…¡,50 mg/kg body wt), which as a marker of vascular permeability to proteins,intravenously into apoE^-/-mice and allowed to circulate for 10 minutes.At several time points,atherosclerotic lesions and local hemodynamic environment in the collar treated artery was analyzed using Vevo 770 ultrasound biomicroscopy(UBM),which with a transducer frequency of 40 MHz with B-scan imaging and Doppler flow measurement capabilities.Peak wall shear stress was calculated using the following formula:Ï„(dyne/cm²)=4·V·η/ID.The collared carotid artery was photographed under a stereomicroscope connected to a standard CCD.Then each vessel was assessed throughout the entire length of the carotid artery for histological analysis.Results Before placement of collar.common carotid diameter was 0.51±0.02mm and peak blood velocity was 1217.5±92.54mm/s.Placement of constrictive collar resulted in≈64%axisymmetric stenosis in collar region of right carotid artery and peak blood velocity decreased to 443.00±28.94 mm/s in the proximal to the collar region. Whereas peak blood velocity within the collar region was accelerate,high-velocity jet formed(2109.41±165.05mm/s,2w;2665.13±289.66 mm/s,4w).Accordingly,shear stress in the proximal to the constrictive collar region was decreased,on the other hand,shear stress within the constrictive collar region was sharply increased.After 2w placement of constrictive collar,the endothelial integrity was confirmed by staining for VWF throughout the entire length of carotid artery.However,the permeability of endothelium to macromolecules increased diffusely both in the region proximal to constrictive collar and intra constrictive collar region.At 2 weeks,the early atherosclerotic lesions contained diffuse deposition of monocyte/macrophages and extensive lipid deposits in proximal to the constrictive collar.At 8 weeks,the atherosclerotic plaques had grown markedly,with near-total occlusion of the lumen in the low shear-stress regions.A gradual thickening of the intimal lesion developed, which consisted predominantly ofα-actin positive SMCs within the constrictive collar region beginning at week 8.However,almost no lipid deposition and macrophage accumulation were observed in the elevated shear-stress regions at all time points.The UBM-observed lesions were highly correlated to those found on en face staining and histology.ConclusionsThis study provides in vivo noninvasive evidence for a causal relationship between shear stress and atherosclerosis in hypercholesterolemic apoE^-/-mice by placement of constrictive collar around carotid artery,even though actual carotid shear stress is much higher in mice than in humans.Then,the atherosclerotic plaque formed in the relatively lowered shear stress region,whereas higher shear stress caused an atheroprotective phenotype.