新生血管与斑块稳定性的关系及VEGF基因干预的研究
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摘要
目的:构建携带人VEGF_(165)基因的重组腺病毒。方法:应用EcoRⅠ和XbaⅠ分别对质粒PDC-VEGF和PDC315进行酶切,凝胶电泳鉴定、回收纯化目的DNA片段,连接目的片段并转化大肠杆菌DH5α构建重组质粒PDC315-VEGF,对重组质粒进行EcoRⅠ和XbaⅠ、EcoRⅠ和BamHⅠ酶切和测序鉴定。通过脂质体将质粒PDC315-VEGF与含有腺病毒右臂的质粒pBHGE3共转染293细胞,9～12天出现病毒空斑效应。提取重组腺病毒DNA,通过PCR扩增法鉴定构建的腺病毒。大量扩增、氯化铯梯度离心纯化、浓缩重组腺病毒,微量滴定法测定腺病毒滴度。结果:通过连接反应将3973bp VEGF_(165)片段正确插入538bp PDC315载体,酶切电泳分析和测序证明正确构建重组质粒PDC315-VEGF。经细胞内同源重组构建携带人VEGF_(165)基因的重组腺病毒,以重组腺病毒DNA为模板,扩增出421bp VEGF_(165)基因片段,证实携带人VEGF_(165)基因成功克隆到复制缺陷型腺病毒载体,腺病毒滴度为5.0×10~9pfu/ml。结论:通过双质粒细胞同源重组,生产携带人VEGF_(165)基因的重组腺病毒ad.VEGF,为动物试验奠定基础。
目的:利用RNA干扰(RNA interference,RNAi)技术,以小鼠VEGFa基因(NM_001025257)靶基因,构建携带短发夹状干扰RNA(siRNA)的慢病毒载体。
方法:
1.根据小鼠VEGFa mRNA序列(NM_001025257),选择针对VEGF cds区3个19nts靶序列,设计并合成三对包含正反义靶序列的互补DNA链(siRNA1、siRNA2、siRNA3),同时设计并合成一对针对无关序列的互补DNA链为阴性对照(Negative)。退火与线性化pRNAT-U6.2/Lenti质粒连接、转化感受态细胞DH5α,扩增、纯化质粒,通过PCR扩增以及基因测序鉴定片段大小及插入片段序列。
2.钓取小鼠NIH/3T3细胞总RNA,利用RT-PCR方法大量扩增、纯化小鼠NIH/3T3细胞VEGFa cDNA,将BamHⅠ和EcoRⅠ酶切VEGFa基因片段与线性化处理pCDH1质粒进行连接、转化感受态细胞DH5α,构建携带小鼠VEGFa基因表达质粒pCDNA-VEGF。扩增、纯化质粒,通过PCR扩增后进行琼脂糖电泳以及基因测序鉴定片段大小及插入片段序列。
3.实验分6组,用脂质体将pCDNA-VEGF质粒或/和siRNA慢病毒质粒共转染NIH/3T3细胞,通过荧光Real time PCR和Western blot检测NIH/3T3细胞VEGF基因表达水平,筛选最有效抑制VEGF基因表达siRNA慢病毒表达质粒。
4.通过脂质体分别将最有效抑制小鼠VEGFa基因表达的siRNA慢病毒表达质粒和pRNAT-Negative慢病毒表达质粒与慢病毒包装质粒混合物混合,共转染293T细胞,24h后观察绿色荧光表达情况,转染48h后收集慢病毒上清。用慢病毒液转染96孔板培养的293T细胞,荧光显微镜计数GFP阳性细胞法测定慢病毒滴度。
结果:
1.特异性合成DNA链退火后定向克隆到线性化pRNAT-U6.2/Lenti质粒,PCR扩增出316bp或317bp目的基因片断,重组质粒测序结果与设计siRNA序列完全一致,成功构建针对小鼠VEGFa基因的特异性siRNA慢病毒表达质粒。
2.自NIH/3T3细胞获取高质量总RNA,通过RT-PCR扩增VEGFa cDNA,克隆VEGFa cDNA长885bp。构建pCDNA-VEGF质粒为1077bp(含有864bpVEGFa基因片断和213bp pCDNA载体片断),插入小鼠VEGFa寡核苷酸序列与设计序列完全相符。
3.pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平,siRNA1、siRNA2、siRNA3慢病毒表达质粒抑制pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白的表达水平,特别是pRNAT-siRNA3抑制作用最明显,VEGFa mRNA和蛋白表达均下降70%以上,pRNAT-negative慢病毒表达质粒对pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平无抑制作用。因此,pRNAT-siRNA3慢病毒表达质粒基因沉默效果最好。
4.通过脂质体分别将pRNAT-siRNA3质粒和pRNAT-negative质粒与慢病毒包装质粒混合物共转染293T细胞,24h后观察293T细胞有绿色荧光表达,成功建立慢病毒载体的包装细胞。转染48h收集慢病毒上清,浓缩、纯化后的慢病毒滴度均为l×10~8 ifu/ml。
结论:通过RNA干扰技术,成功设计、筛选、生产针对小鼠VEGFa基因沉默的特异性siRNA慢病毒,为动物试验奠定基础。
背景
动脉粥样斑块内新生血管内皮细胞连接不紧密、内皮下缺乏基底膜、管壁无平滑肌细胞,具有通透性高和脆性大的特点,易于破裂出血。VEGF是高度特异性血管内皮细胞有丝分裂素,在血管新生发挥重要作用,是促进或抑制新生血管形成的靶点。目前,动脉粥样斑块破裂与粥样斑块内新生血管之间的关系有争议。因此,建立apoE~(-/-)。小鼠颈动脉粥样斑块模型,干预粥样斑块内新生血管形成,有助于阐明粥样斑块内血管新生、斑块内出血、粥样斑块破裂的相互关系。
目的
建立apoE~(-/-)小鼠颈动脉粥样斑块模型,将携带目的基因病毒经动脉外膜转染动脉粥样斑块,通过促进或抑制粥样斑块内新生血管形成,明确粥样斑块内新生血管形成、斑块内出血、粥样斑块破裂的相互关系及其机制。
方法
1.动物模型:
164只8w雄性apoE~(-/-)小鼠,均给予高脂高胆固醇喂养。通过缩窄性颈动脉套管建立apoE~(-/-)小鼠左颈总动脉粥样斑块模型。
按20%浓度(V/V)将pluronic-127 gel加入Ad.VEGF(5×10~9pfu/ml)、Ad.lacZ(5×10~9pfu/ml)、siRNA.VEGF(1×1O~8ifu/ml)、siRNA.Negative(1×10~8ifu/ml),制成转染用腺病毒或慢病毒溶液。
缩窄性颈动脉套管4w后,去除缩窄性硅胶套管。将100ul病毒液均匀涂抹左颈总动脉外膜,室温孵育20min。术毕,继续高脂饲养4w。
局部基因转染2w时,自Ad.lacZ组、siRNA.Negative组分别随机处死2只apoE~(-/-)小鼠,观察粥样斑块内β-半乳糖苷酶和GFP表达,判断局部基因转染的可行性和有效性。
2.血脂水平测定:检测各组apoE~(-/-)小鼠血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)的含量。
3.病理学检测:每组20只apoE~(-/-)小鼠,对左颈总动脉粥样斑块分别进行HE染色、油红O染色、Masson染色、天狼猩红染色、Perl's染色(含铁血黄素染色),通过免疫组织化学方法检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)、血管内皮生长因子(VEGF)、vWF、纤维蛋白原、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)的蛋白表达。统计粥样斑块破裂(斑块内出血和血栓形成)发生率,计算易损指数=(巨噬细胞+脂质)阳性面积百分比/(平滑肌细胞+胶原)阳性面积百分比。
4.实时荧光定量RT-PCR检测基因表达:每组12只apoE~(-/-)小鼠左颈总动脉,每3只混合提取新鲜粥样斑块组织RNA,实时荧光定量PCR检测VEGF、MMP2、MMP9 mRNA表达水平。
5.免疫印迹技术(Western blot)检测蛋白表达:每组8只左颈总动脉,混合后提取粥样斑块组织蛋白,Western blot检测VEGF、MMP9的蛋白表达水平。
结果
1.ApoE~(-/-)小鼠基本特征:缩窄性颈动脉套管、去除套管以及颈动脉外膜局部转染病毒的手术顺利,实验期间无apoE~(-/-)小鼠意外死亡。各组apoE~(-/-)小鼠血脂含量及体重比较差异无统计学意义(p＞0.05)。
2.颈动脉外膜局部有效转染腺病毒或慢病毒液:颈动脉局部转染ad.LacZ腺病毒2w,颈动脉外膜、中膜、新生内膜均分布β-半乳糖苷酶蓝色颗粒;携带GFP基因的慢病毒转染2w,颈总动脉粥样斑块有强GFP表达,极少分布于其他脏器。腺病毒和慢病毒可将携带目的基因,通过血管外膜成功转染粥样斑块。
局部转染基因影响粥样斑块内VEGF合成,免疫组化示Ad.VEGF组粥样斑块内VEGF呈弥漫性强阳性表达(69.97±2.74%),荧光实时定量RT-PCR和Western blot示粥样斑块VEGF mRNA和VEGF蛋白(1.76±0.32)表达最高;siRNA.VEGF组粥样斑块内VEGF表达最弱(14.33±1.37%),荧光实时定量PCR和Western blot均显示siRNA.VEGF组粥样斑块VEGF mMRNA和VEGF蛋白(0.22±0.02)表达最弱。
3.局部基因转染对粥样斑块大小及斑块成分的影响:颈动脉局部转染Ad.VEGF组粥样斑块面积最大,siRNA.VEGF组粥样斑块面积最小,Ad.LacZ组和siRNA.Negative组粥样斑块面积差异无统计学意义。各组均形成以脂质沉积为主粥样斑块,脂质含量差异有统计学意义。Ad.VEGF组斑块α-SM actin含量最少(8.35±0.3%),siRNA.VEGF组粥样斑块内α-SM actin含量最多(19.01±1.17%)。Ad.VEGF组粥样斑块纤维帽最薄(6.07±0.25um),siRNA.VEGF组粥样斑块纤维帽最厚(15.40±1.46um)。粥样斑块内胶原分布呈类似表现,Ad.VEGF组粥样斑块胶原含量最少(10.11±1.05%),siRNA.VEGF组粥样斑块胶原含量最多(25.61±1.69%)。Ad.VEGF组粥样斑块内巨噬细胞含量最多(22.18±1.87%),siRNA.VEGF组粥样斑块巨噬细胞含量最少(8.25±0.86%)。Ad.VEGF组粥样斑块内MMP2、MMP9呈弥漫性强阳性分布,荧光实时定量RT-PCR显示Ad.VEGF组粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最高,Western blot显示Ad.VEGF组粥样斑块内MMP9以具有酶活性蛋白表达为主:siRNA.VEGF组粥样斑块内MMP9呈局限性弱阳性分布,粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最弱,斑块内以不具有酶活性的MMP9蛋白前体为主。
Ad.VEGF组粥样斑块易损指数显著升高(3.59±0.42),高于Ad.LacZ组(2.08±0.17)、siNRA.Negative组(2.03±0.18)、siRNA.VEGF组(1.12±0.08)(P均＜0.001)。siNRA.Negative组和siRNA.VEGF组粥样斑块易损指数差异无统计学意义(p＞0.05)。
4.基因转染对斑块内新生血管形成及斑块稳定性的影响:根据vWF染色结果,粥样斑块内新生血管形成,新生血管管壁薄,多由单个或数个内皮细胞构成,无SMC组成。Ad.VEGF组斑块内新生血管密度最高(24.0±1.9),siRNA.VEGF组斑块内新生血管密度最低(6.6±1.2)。Ad.VEGF组粥样斑块破裂发生率最高,分别有8例(40%)发生斑块内出血和6例(30%)血栓形成,其中3例直视下可见粥样斑块近心端附壁血栓形成。Ad.LacZ组分别有2例(10%)斑块内出血和1例(5%)血栓形成,siRNA.VEGF组和siRNA.Negative组分别有2例(10%)斑块内出血,Pearson Chi-Square检验差异有统计学意义。
结论
(1)通过颈动脉外膜局部转染方式,可将目的基因高效转染粥样斑块。
(2)粥样斑块局部转染Ad.VEGF_(165),促进斑块内新生血管形成,增加粥样斑块破裂发生率。
(3)VEGFa基因沉默显著抑制粥样斑块进展,降低粥样斑块易损指数,增加粥样斑块稳定性。
目的
人类动脉粥样硬化病变主要发生在大、中动脉弯曲、分又及狭窄部位,如主动脉弓、颈动脉分支、冠状动脉、腹主动脉分支和股动脉分支等血流缓慢或涡流部位,即低管壁剪切力区。ApoE~(-/-)小鼠是研究动脉粥样硬化较理想动物模型,但小鼠主动脉管壁剪切力约为人主动脉管壁剪切力的20倍。利用Visualsonics Vevo770显微超声仪实时无创性观察小鼠血流动力学变化,研究管壁剪切力在apoE~(-/-)小鼠动脉粥样斑块形成过程中的作用。
方法
高脂高胆固醇饲料饲养apoE~(-/-)小鼠,将内径0.3mm缩窄性或内径0.6mm非缩窄性硅胶管套扎apoE~(-/-)小鼠左颈总动脉。静脉注射HRP(50mg/kg体重)观察缩窄性套管和非缩窄性套管对apoE~(-/-)小鼠动脉管壁通透性的影响。应用显微超声仪于相应观察时间点进行颈动脉超声检查,根据τ(dyne/cm~2)=4·V·η/ID公式计算动脉管壁剪切力。将颈动脉标本进行拍照和免疫组织化学分析。
结果
apoE~(-/-)小鼠颈总动脉内经0.51±0.02mm,管壁剪切力33.4±2.5dynes/cm~2,内经0.3mm缩窄性套管导致颈总动脉狭窄≈64%,缩窄性套管近端颈总动脉管壁剪切力降至12.2±0.8dynes/cm~2,而缩窄性套管段颈总动脉管壁剪切力显著升高至98.5±7.7dynes/cm~2。静脉注射HRP显示缩窄性套管段和缩窄性套管近端颈总动脉管壁通透性均增加。缩窄性套管近端颈总动脉形成以脂质沉积和单核/巨噬细胞黏附浸润为主粥样斑块,缩窄性套管段颈总动脉形成以收缩型平滑肌细胞为主的中膜增生,但无脂质沉积和单核/巨噬细胞黏附浸润。
结论
①在高脂喂养基础上,通过缩窄性套管加速apoE~(-/-)小鼠颈总动脉粥样斑块形成;②apoE~(-/-)小鼠颈总动脉管壁剪切力较人类高;③低管壁剪切力有促动脉粥样斑块形成作用;④高管壁剪切力抑制动脉粥样斑块形成;⑤显微超声仪可以实时无创性检测apoE~(-/-)小鼠血流动力学在动脉粥样斑块形成中的作用。
Objective:To construction of recombinant adenoviruses encoding human vascular endothelial growth factor165(VEGF_(165))gene.Methods:The PDC315 plasmid and PDC-VEGF plasmid were digested with EcoR I and Xba I,then 538bp of CMV and 3973bp VEGF_(165)cDNA 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 VEGF_(165)are 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 VEGF_(165)cDNA fragment.The replication deficient recombinant adenoviruses vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR which showed that VEGF_(165) mRNA was transcripted from the VEGF gene.The virus concentration reach to 5.0×10~9 pfu/ml.Conclusion:The recombinant adenoviruses vector encoding human VEGF_(165)gene 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~(TM)2000,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 1×10~8 ifu/ml.
Conclusion:The recombinant lentiviruses,which can express siRNA hairpin aimed at VEGFa gene,have been constructed successfully.
Background
Intraplaque 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.
Objectives
In 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.
Methods
1.Animal model
Male 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(5.0×10~9 pfu/ml),or Ad.LacZ(5.0x 10~9 pfu/ml), siRNA.VEGF(1.0×10~8 ifu/ml),siRNA.Negative(1.0×10~8 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 pattern
To 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 measurement
Before perfusion-fixation,blood samples were collected and serum total cholesterol, LDL cholesterol,HDL cholesterol,and triglycerides concentrations measured.
4.Histological and morphometric analysis
For 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 Analysis
For 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 Blotting
For 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.
Results
1.General characteristic
Throughout 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.
Conclusions
1.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.
Objective
It 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.
Methods
Male 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,TypeⅡ,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:τ(dyne/cm~2)=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.
Conclusions
This 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.
目的:利用RNA干扰(RNA interference,RNAi)技术,以小鼠VEGFa基因(NM_001025257)靶基因,构建携带短发夹状干扰RNA(siRNA)的慢病毒载体。
方法:
1.根据小鼠VEGFa mRNA序列(NM_001025257),选择针对VEGF cds区3个19nts靶序列,设计并合成三对包含正反义靶序列的互补DNA链(siRNA1、siRNA2、siRNA3),同时设计并合成一对针对无关序列的互补DNA链为阴性对照(Negative)。退火与线性化pRNAT-U6.2/Lenti质粒连接、转化感受态细胞DH5α,扩增、纯化质粒,通过PCR扩增以及基因测序鉴定片段大小及插入片段序列。
2.钓取小鼠NIH/3T3细胞总RNA,利用RT-PCR方法大量扩增、纯化小鼠NIH/3T3细胞VEGFa cDNA,将BamHⅠ和EcoRⅠ酶切VEGFa基因片段与线性化处理pCDH1质粒进行连接、转化感受态细胞DH5α,构建携带小鼠VEGFa基因表达质粒pCDNA-VEGF。扩增、纯化质粒,通过PCR扩增后进行琼脂糖电泳以及基因测序鉴定片段大小及插入片段序列。
3.实验分6组,用脂质体将pCDNA-VEGF质粒或/和siRNA慢病毒质粒共转染NIH/3T3细胞,通过荧光Real time PCR和Western blot检测NIH/3T3细胞VEGF基因表达水平,筛选最有效抑制VEGF基因表达siRNA慢病毒表达质粒。
4.通过脂质体分别将最有效抑制小鼠VEGFa基因表达的siRNA慢病毒表达质粒和pRNAT-Negative慢病毒表达质粒与慢病毒包装质粒混合物混合,共转染293T细胞,24h后观察绿色荧光表达情况,转染48h后收集慢病毒上清。用慢病毒液转染96孔板培养的293T细胞,荧光显微镜计数GFP阳性细胞法测定慢病毒滴度。
结果:
1.特异性合成DNA链退火后定向克隆到线性化pRNAT-U6.2/Lenti质粒,PCR扩增出316bp或317bp目的基因片断,重组质粒测序结果与设计siRNA序列完全一致,成功构建针对小鼠VEGFa基因的特异性siRNA慢病毒表达质粒。
2.自NIH/3T3细胞获取高质量总RNA,通过RT-PCR扩增VEGFa cDNA,克隆VEGFa cDNA长885bp。构建pCDNA-VEGF质粒为1077bp(含有864bpVEGFa基因片断和213bp pCDNA载体片断),插入小鼠VEGFa寡核苷酸序列与设计序列完全相符。
3.pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平,siRNA1、siRNA2、siRNA3慢病毒表达质粒抑制pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白的表达水平,特别是pRNAT-siRNA3抑制作用最明显,VEGFa mRNA和蛋白表达均下降70%以上,pRNAT-negative慢病毒表达质粒对pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平无抑制作用。因此,pRNAT-siRNA3慢病毒表达质粒基因沉默效果最好。
4.通过脂质体分别将pRNAT-siRNA3质粒和pRNAT-negative质粒与慢病毒包装质粒混合物共转染293T细胞,24h后观察293T细胞有绿色荧光表达,成功建立慢病毒载体的包装细胞。转染48h收集慢病毒上清,浓缩、纯化后的慢病毒滴度均为l×10~8 ifu/ml。
结论:通过RNA干扰技术,成功设计、筛选、生产针对小鼠VEGFa基因沉默的特异性siRNA慢病毒,为动物试验奠定基础。
背景
动脉粥样斑块内新生血管内皮细胞连接不紧密、内皮下缺乏基底膜、管壁无平滑肌细胞,具有通透性高和脆性大的特点,易于破裂出血。VEGF是高度特异性血管内皮细胞有丝分裂素,在血管新生发挥重要作用,是促进或抑制新生血管形成的靶点。目前,动脉粥样斑块破裂与粥样斑块内新生血管之间的关系有争议。因此,建立apoE~(-/-)。小鼠颈动脉粥样斑块模型,干预粥样斑块内新生血管形成,有助于阐明粥样斑块内血管新生、斑块内出血、粥样斑块破裂的相互关系。
目的
建立apoE~(-/-)小鼠颈动脉粥样斑块模型,将携带目的基因病毒经动脉外膜转染动脉粥样斑块,通过促进或抑制粥样斑块内新生血管形成,明确粥样斑块内新生血管形成、斑块内出血、粥样斑块破裂的相互关系及其机制。
方法
1.动物模型:
164只8w雄性apoE~(-/-)小鼠,均给予高脂高胆固醇喂养。通过缩窄性颈动脉套管建立apoE~(-/-)小鼠左颈总动脉粥样斑块模型。
按20%浓度(V/V)将pluronic-127 gel加入Ad.VEGF(5×10~9pfu/ml)、Ad.lacZ(5×10~9pfu/ml)、siRNA.VEGF(1×1O~8ifu/ml)、siRNA.Negative(1×10~8ifu/ml),制成转染用腺病毒或慢病毒溶液。
缩窄性颈动脉套管4w后,去除缩窄性硅胶套管。将100ul病毒液均匀涂抹左颈总动脉外膜,室温孵育20min。术毕,继续高脂饲养4w。
局部基因转染2w时,自Ad.lacZ组、siRNA.Negative组分别随机处死2只apoE~(-/-)小鼠,观察粥样斑块内β-半乳糖苷酶和GFP表达,判断局部基因转染的可行性和有效性。
2.血脂水平测定:检测各组apoE~(-/-)小鼠血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)的含量。
3.病理学检测:每组20只apoE~(-/-)小鼠,对左颈总动脉粥样斑块分别进行HE染色、油红O染色、Masson染色、天狼猩红染色、Perl's染色(含铁血黄素染色),通过免疫组织化学方法检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)、血管内皮生长因子(VEGF)、vWF、纤维蛋白原、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)的蛋白表达。统计粥样斑块破裂(斑块内出血和血栓形成)发生率,计算易损指数=(巨噬细胞+脂质)阳性面积百分比/(平滑肌细胞+胶原)阳性面积百分比。
4.实时荧光定量RT-PCR检测基因表达:每组12只apoE~(-/-)小鼠左颈总动脉,每3只混合提取新鲜粥样斑块组织RNA,实时荧光定量PCR检测VEGF、MMP2、MMP9 mRNA表达水平。
5.免疫印迹技术(Western blot)检测蛋白表达:每组8只左颈总动脉,混合后提取粥样斑块组织蛋白,Western blot检测VEGF、MMP9的蛋白表达水平。
结果
1.ApoE~(-/-)小鼠基本特征:缩窄性颈动脉套管、去除套管以及颈动脉外膜局部转染病毒的手术顺利,实验期间无apoE~(-/-)小鼠意外死亡。各组apoE~(-/-)小鼠血脂含量及体重比较差异无统计学意义(p＞0.05)。
2.颈动脉外膜局部有效转染腺病毒或慢病毒液:颈动脉局部转染ad.LacZ腺病毒2w,颈动脉外膜、中膜、新生内膜均分布β-半乳糖苷酶蓝色颗粒;携带GFP基因的慢病毒转染2w,颈总动脉粥样斑块有强GFP表达,极少分布于其他脏器。腺病毒和慢病毒可将携带目的基因,通过血管外膜成功转染粥样斑块。
局部转染基因影响粥样斑块内VEGF合成,免疫组化示Ad.VEGF组粥样斑块内VEGF呈弥漫性强阳性表达(69.97±2.74%),荧光实时定量RT-PCR和Western blot示粥样斑块VEGF mRNA和VEGF蛋白(1.76±0.32)表达最高;siRNA.VEGF组粥样斑块内VEGF表达最弱(14.33±1.37%),荧光实时定量PCR和Western blot均显示siRNA.VEGF组粥样斑块VEGF mMRNA和VEGF蛋白(0.22±0.02)表达最弱。
3.局部基因转染对粥样斑块大小及斑块成分的影响:颈动脉局部转染Ad.VEGF组粥样斑块面积最大,siRNA.VEGF组粥样斑块面积最小,Ad.LacZ组和siRNA.Negative组粥样斑块面积差异无统计学意义。各组均形成以脂质沉积为主粥样斑块,脂质含量差异有统计学意义。Ad.VEGF组斑块α-SM actin含量最少(8.35±0.3%),siRNA.VEGF组粥样斑块内α-SM actin含量最多(19.01±1.17%)。Ad.VEGF组粥样斑块纤维帽最薄(6.07±0.25um),siRNA.VEGF组粥样斑块纤维帽最厚(15.40±1.46um)。粥样斑块内胶原分布呈类似表现,Ad.VEGF组粥样斑块胶原含量最少(10.11±1.05%),siRNA.VEGF组粥样斑块胶原含量最多(25.61±1.69%)。Ad.VEGF组粥样斑块内巨噬细胞含量最多(22.18±1.87%),siRNA.VEGF组粥样斑块巨噬细胞含量最少(8.25±0.86%)。Ad.VEGF组粥样斑块内MMP2、MMP9呈弥漫性强阳性分布,荧光实时定量RT-PCR显示Ad.VEGF组粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最高,Western blot显示Ad.VEGF组粥样斑块内MMP9以具有酶活性蛋白表达为主:siRNA.VEGF组粥样斑块内MMP9呈局限性弱阳性分布,粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最弱,斑块内以不具有酶活性的MMP9蛋白前体为主。
Ad.VEGF组粥样斑块易损指数显著升高(3.59±0.42),高于Ad.LacZ组(2.08±0.17)、siNRA.Negative组(2.03±0.18)、siRNA.VEGF组(1.12±0.08)(P均＜0.001)。siNRA.Negative组和siRNA.VEGF组粥样斑块易损指数差异无统计学意义(p＞0.05)。
4.基因转染对斑块内新生血管形成及斑块稳定性的影响:根据vWF染色结果,粥样斑块内新生血管形成,新生血管管壁薄,多由单个或数个内皮细胞构成,无SMC组成。Ad.VEGF组斑块内新生血管密度最高(24.0±1.9),siRNA.VEGF组斑块内新生血管密度最低(6.6±1.2)。Ad.VEGF组粥样斑块破裂发生率最高,分别有8例(40%)发生斑块内出血和6例(30%)血栓形成,其中3例直视下可见粥样斑块近心端附壁血栓形成。Ad.LacZ组分别有2例(10%)斑块内出血和1例(5%)血栓形成,siRNA.VEGF组和siRNA.Negative组分别有2例(10%)斑块内出血,Pearson Chi-Square检验差异有统计学意义。
结论
(1)通过颈动脉外膜局部转染方式,可将目的基因高效转染粥样斑块。
(2)粥样斑块局部转染Ad.VEGF_(165),促进斑块内新生血管形成,增加粥样斑块破裂发生率。
(3)VEGFa基因沉默显著抑制粥样斑块进展,降低粥样斑块易损指数,增加粥样斑块稳定性。
目的
人类动脉粥样硬化病变主要发生在大、中动脉弯曲、分又及狭窄部位,如主动脉弓、颈动脉分支、冠状动脉、腹主动脉分支和股动脉分支等血流缓慢或涡流部位,即低管壁剪切力区。ApoE~(-/-)小鼠是研究动脉粥样硬化较理想动物模型,但小鼠主动脉管壁剪切力约为人主动脉管壁剪切力的20倍。利用Visualsonics Vevo770显微超声仪实时无创性观察小鼠血流动力学变化,研究管壁剪切力在apoE~(-/-)小鼠动脉粥样斑块形成过程中的作用。
方法
高脂高胆固醇饲料饲养apoE~(-/-)小鼠,将内径0.3mm缩窄性或内径0.6mm非缩窄性硅胶管套扎apoE~(-/-)小鼠左颈总动脉。静脉注射HRP(50mg/kg体重)观察缩窄性套管和非缩窄性套管对apoE~(-/-)小鼠动脉管壁通透性的影响。应用显微超声仪于相应观察时间点进行颈动脉超声检查,根据τ(dyne/cm~2)=4·V·η/ID公式计算动脉管壁剪切力。将颈动脉标本进行拍照和免疫组织化学分析。
结果
apoE~(-/-)小鼠颈总动脉内经0.51±0.02mm,管壁剪切力33.4±2.5dynes/cm~2,内经0.3mm缩窄性套管导致颈总动脉狭窄≈64%,缩窄性套管近端颈总动脉管壁剪切力降至12.2±0.8dynes/cm~2,而缩窄性套管段颈总动脉管壁剪切力显著升高至98.5±7.7dynes/cm~2。静脉注射HRP显示缩窄性套管段和缩窄性套管近端颈总动脉管壁通透性均增加。缩窄性套管近端颈总动脉形成以脂质沉积和单核/巨噬细胞黏附浸润为主粥样斑块,缩窄性套管段颈总动脉形成以收缩型平滑肌细胞为主的中膜增生,但无脂质沉积和单核/巨噬细胞黏附浸润。
结论
①在高脂喂养基础上,通过缩窄性套管加速apoE~(-/-)小鼠颈总动脉粥样斑块形成;②apoE~(-/-)小鼠颈总动脉管壁剪切力较人类高;③低管壁剪切力有促动脉粥样斑块形成作用;④高管壁剪切力抑制动脉粥样斑块形成;⑤显微超声仪可以实时无创性检测apoE~(-/-)小鼠血流动力学在动脉粥样斑块形成中的作用。
Objective:To construction of recombinant adenoviruses encoding human vascular endothelial growth factor165(VEGF_(165))gene.Methods:The PDC315 plasmid and PDC-VEGF plasmid were digested with EcoR I and Xba I,then 538bp of CMV and 3973bp VEGF_(165)cDNA 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 VEGF_(165)are 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 VEGF_(165)cDNA fragment.The replication deficient recombinant adenoviruses vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR which showed that VEGF_(165) mRNA was transcripted from the VEGF gene.The virus concentration reach to 5.0×10~9 pfu/ml.Conclusion:The recombinant adenoviruses vector encoding human VEGF_(165)gene 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~(TM)2000,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 1×10~8 ifu/ml.
Conclusion:The recombinant lentiviruses,which can express siRNA hairpin aimed at VEGFa gene,have been constructed successfully.
Background
Intraplaque 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.
Objectives
In 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.
Methods
1.Animal model
Male 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(5.0×10~9 pfu/ml),or Ad.LacZ(5.0x 10~9 pfu/ml), siRNA.VEGF(1.0×10~8 ifu/ml),siRNA.Negative(1.0×10~8 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 pattern
To 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 measurement
Before perfusion-fixation,blood samples were collected and serum total cholesterol, LDL cholesterol,HDL cholesterol,and triglycerides concentrations measured.
4.Histological and morphometric analysis
For 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 Analysis
For 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 Blotting
For 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.
Results
1.General characteristic
Throughout 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.
Conclusions
1.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.
Objective
It 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.
Methods
Male 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,TypeⅡ,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:τ(dyne/cm~2)=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.
Conclusions
This 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.
引文
1. Virmani R, Kolodgie FD, Burke AP, et al. Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol. 2005; 25(10): 2054-2061.
2. Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003; 349(24): 2316-2325.
3. Jeziorska M, Woolley DE. Local neovascularization and cellular composition within vulnerable regions of atherosclerotic plaques of human carotid arteries. J Pathol. 1999; 188(2): 189-196.
4. Leung DW, Cachiance G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989,246(4935):1306-1309.
5. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003; 9(6): 669-676.
6. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor: Multiple protein forms are encoded through alternative exon splicing. J Bio Chem. 1991; 266(18): 11947-11954.
7. Benjamin LE, Golijanin D, Itin A, et al. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest. 1999; 103(2): 159-165.
8. Eppler SM, Combs DL, Henry TD, et al. A target-mediated model to describe the Pharmacokinetics and hemodynamic effects of recombinant human vascular endothelial growth factor in humans. Clin Pharmacol Ther. 2002; 72(1): 20-32.
9. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999; 10(5): 440-447.
10. Baker AH, Kritz A, Work LM, et al. Cell-selective viral gene delivery vectors for the vasculature. Exp Physiol. 2005; 90(1): 27-31.
11. Robinson CJ, Srtinger SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001; 114 (Pt 5): 853-865.
12. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 2003; 4(5): 346-358.
13. He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998; 95(5): 2509-2514.
14. Mittal SK, McDermott MR, Johnson DC, et al. Monitoring foreign gene expression by a human adenovirus-based vector using the firely luciferase gene as a reporter. Virus Res. 1993; 28(1): 67-90.
1. Brand S. Antisense-RNA regulation and RNA interference. Biochem Biophys Acta, 2002; 1575(1-3): 15-25.
2. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001; 411(6836): 494-498.
3. Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000; 404(6775): 293-296.
4. Brummelkamp TR, BemardsR, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002; 296(5567): 550-553.
5. Berezhna SY, Supekova L, Supek F, et al. siNRA in human cells selectively localizes to target RNA sites. Proc Natl Acad Sci USA. 2006; 103(20): 7682-7687.
6. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001; 15(2): 188-200.
7. Miyagishi M, Taira K. RNAi expression vectors in mammalian cells. Methods Mol Biol. 2004; 252(1): 483-491.
8. Sui G, Soohoo C, Affarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA. 2002; 99(8): 5515-5520.
9. Naldini L, Blomer U, Gallay P. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259):263-267.
10. Bartosch B, Cosset FL. Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr Gene Ther. 2004; 4(4): 427-443.
11. Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther. 2001; 1(1): 17-40.
12. Misteli T, Spector DL. Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol. 1997; 15(10): 961-964.
13. Tiscornia G, Singer O, Verma IM. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc. 2006; 1(1): 234-240.
14. Morris KV, Rossi JJ. Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection. Hum Gene Ther. 2006; 17(5): 479-486.
15. Westerhout EM, Vink M, Haasnoot PC, et al. A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication. Mol Ther. 2006; 14(2):268-275.
16. Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Ther. 2003; 10:912-927.
1. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I . Circulation. 2003; 108: 1664-1672.
2. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation, 2003; 108: 1772-1778.
3. Libby P. Molecular bases of the acute coronary syndromes. Circulation, 1995;91: 2844-2850.
4. Burke AP, Farb A, Malcom GT, et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N Engl J Med.1997; 36: 1276-1282.
5. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275.
6. Kolodgie FD, Virmani R, Burke AP, et al. Pathologic assessment of the vulnerable human coronary plaque. Heart. 2004; 90:1385-1391.
7. Burke AP, Virmani R, Galis Z, et al. 34th Bethesda Conference: Task force #2-What is the pathologic basis for new atherosclerosis imaging techniques? J Am Coll Cardiol. 2003; 41: 1874-1886.
8. Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003; 349: 2316-2325.
9. Virmani R, Kolodgie FD, Burke AP, et al. Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol. 2005; 25:2054-2061.
10. Jeziorska M, Woolley DE. Local neovascularization and cellular composition within vulnerable regions of atherosclerotic plaques of human carotid arteries. J Pathol. 1999; 188: 189-196.
11. Kockx MM, Cromheeke KM, Knaapen MW, et al. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 440-446.
12. Virmani R, Narula J, Farb A. When neoangiogenesis ricochets. Am Heart J. 1998; 136: 937-939.
13. Khurana R, Simons M, Martin JF, et al. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation. 2005; 112: 1813-1824.
14. Ware JA. Too many vessels? Not enough? The wrong kind? The VEGF debate continues. Nat Med. 2001; 7:403-404.
15. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836): 494-498.
16. Wolinsky H, Glagov S. Nature of species differences in the medial distribution of aortic vasa vasorum in mammals. Circ. Res. 1967; 20: 409-421.
17. Shiomi M Ito T, Hirouchi Y, Enomoto M. Fibromuscular cap composition is important for the stability of established atherosclerotic plaques in mature WHHL rabbits treated with statins. Atherosclerosis. 2001; 157: 75-84.
18. StaryH C, ChandlerAB, DinsmoreR E, et al. A definition of initial, fatty streak, and intermediate lesions of the atherosclerosis: A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994; 89:2462-2478.
19. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of the advanved types of atherosclerotic lesions and a histological classification of atherosclerosis: A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation, 1995;92:1355-1374.
20. Gough PJ, Gomez IG, Wille PT, et al. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest, 2006, 116: 59-69.
21. Williams H, Johnson JL, Carson KG, Jackson CL. Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2002; 22:788-792.
22. Johnson J, Carson K, Williams H, et al. Plaque rupture after short periods of fat feeding in the apolipoprotein E-knockout mouse: model characterization and effects of Pravastatin treatment. Circulation. 2005; 111:1422-1430.
23. Rosenfeld ME, Polinsky P, Virmani R, et al. Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse. Arterioscler Thromb Vasc Biol. 2000; 20: 2587-2592.
24. Douglas JT. Adenovirus-mediated gene delivery: an overview. Methods Mol Biol, 2004; 246:3-14.
25. Yla-Herttuala S, Alitalo K. Gene transfer as a tool to induce therapeutic vascular growth. Nat Med. 2003; 9: 694-701.
26. Schneider DB, Sassani AB, Vassalli G, et al. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999; 29(3): 543-550.
27. Laitinen M, Pakkanen T, Donetti E, et al. Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther. 1997; 8(14): 1645-1650.
28. Lim K, Chae CB. A simple assay for DNA transfection by incubation of the cells in culture dishew with substrates for galatosidase. Bio Techniques, 1989; 7: 576-579.
29. Kain SR, Adams M, Kondepudi A, et al.Green fluorescent protein as a reporter of gene expression and protein licalization.Biotechniques,1995;19(4):650-655.
30. De Nooijer R, Verkleij CJ, von der Thusen JH, et al. Lesional overexpression of matrix metalloproteinase-9 promotes intraplaque hemorrhage in advanced lesions but not at earlier stages of atherogenesis. Arterioscler Thromb Vasc Biol. 2006; 26(2): 340-346.
31. Bloom W, Fawcett DW. A Textbook of Histology. Philadelphia, Pa: Saunders Company; 1962: 281-282.
32. Heistad DD, Marcus ML. Role of vasa vasorum in nourishment of the aorta, Blood Vessels. 1979, 16: 225-238.
33. Vancov V. Structural basis of the microcirculation in the wall of arterial vessels. BiblAnat. 1973; 11: 383-388.
34. Barger AC, Beeuwkes R 3rd, Lainey LL, et al. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. N Engl J Med. 1984; 310: 175-177.
35. Sueishi K, Yonemitsu Y, Nakagawa K, et al. Atherosclerosis and angiogenesis: its pathophysiological significance in humans as well as in an animal model induced by the gene transfer of vascular endothelial growth factor. Ann N Y Acad Sci. 1997; 811:311-324.
36. Kwon HM, Sangiorgi G, Ritman EL, et al. Enhanced coronary vasa vasorum neovascularization in experimental hypercholesterolemia. J Clin Invest; 1998; 101: 1551-1556.
37. Zhang Y, Cliff WJ, Schoefl GI, et al. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993; 143: 164-172.
38. Moulton KS, Heller E, Kondrding MA, et al. Angioenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipopotein E-deficient mice. Circulation. 1999; 99(13):1726-1732.
39. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003; 9: 677-684.
40. Zemplenyi T, Crawford DW, Cole MA. Adaptation to arterial wall hypoxia demonstrated in vivo with oxygen microcathodes. Atherosclerosis. 1989; 76: 173 -179.
41. Chen YX, Nakashima Y, Tanaks K, et al. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimals of human coronary arterie. Arterioscler Thromb Vasc Biol. 1999; 19: 131 -139.
42. Khurana R, Zhuang Z, Bhardwaj S, et al. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation. 2004; 110: 2436-2443.
43. Ramos MA, Kuzuya M, Esaki T, et al. Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions. Arterioscler Thromb Vase Biol. 1998; 18:1188-1196.
44. Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerosis lesions: possible pathophysiological significance of VEGF in progression of atheroselrosis. Circulation. 1998; 98(20): 2108-2116.
45. Ferrara N, Gerber HP, Le Couter J. The biology of VEGF and its receptors. Nat. Med. 2003; 9(6): 669-676.
46. Ferrara N, Winer J, Burton J. Aortic smooth muscle cells express and secrete vascular endothelial growth factor. Growth Factors, 1991, 5(2): 141-148.
47. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell. 1993; 4: 1317 -1326.
48. Pinhal-Enfield G, Ramanathan M, Hasko G, et al. An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am J Pathol. 2003; 163: 711-721.
49. Leibovich SJ, Chen JF, Pinhal-Enfield G, et al. Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin. Am J Pathol. 2002; 160: 2231- 2244.
50. Leung DW, Cachiance G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989; 246(4935): 1306-1309.
51. Gerrity RG, Richardson M, Somer JB, et al. Endothelial cell morphology in areas of in vivo Evans blue uptake in the aorta of young pigs, II: ultrastructure of the intima in areas of differing permeability to proteins. Am J Pathol. 1977; 89: 313-334.
52. Celletti FL, Waugh JM, Amabile PG, et al. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med. 2001; 7: 425-429.
53. Eppler SM, Combs DL, Henry TD, et al. A target-mediated model to describe the Pharmacokinetics and hemodynamic effects of recombinant human vascular endothelial growth factor in humans. Clin Pharmacol Ther. 2002; 72: 20-32.
54. Laitinen M, Zachary I, Breier G, et al. VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther. 1997; 8: 1737-1744.
55. Khurana R, Shafi S, Martin J, et al. Vascular endothelial growth factor gene transfer inhibits neointimal macrophage accumulation in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol. 2004; 24: 1074-1080:
56. Zachary I, Mathur A, Yla-Herttuala S, et al. Vascular protection: a novel nonangiogenic cardiovascular role for vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2000; 20: 1512-1520.
57. Bhardwaj S, Roy H, Gruchala M, et al. Angiogenic responses of vascular endothelial growth factors in periadventitial tissue. Hum Gene Ther. 2003; 14: 1451-1462.
58. Ozawa CR, Banfi A, Glazer NL, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest. 2004; 113: 516-527.
59. Dor Y, Djonov V, Abramovitch R, et al. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J. 2002; 21:1939-1947
60. Rissanen TT, Markkanen JE, Gruchala M, et al. VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res, 2003; 92(10):1098-1106.
61. Sirois MG,Edelman ER. VEGF effect on vascular permeability is mediated by synthesis of platelet activating factor. Am J Physiol. 1997, 272(6 pt 2): H2746-H2756.
62. Mannucci PM. von Willebrand factor: a marker of endothelial damage? Arterioscler Thromb Vasc Biol. 1998; 18: 1359-1362.
63. De Meyer GRY, Hoylaerts MF, Kockx MM, et al. Intimal deposition of functional von Willebrand factor in atherogenesis. Arterioscler Thromb Vasc Biol. 1999; 19: 2524-2534.
64. Kumamoto M, Nakashima Y, Sueishi K. Intimal neovascularization in human coronary atherosclerosis: its origin and pathophysiological significance. Hum Pathol. 1995; 26: 450-456.
65. O'Brien ER, Garvin MR, Dev R, et al. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol. 1994; 145: 883-894.
66. Muhlhauser J, Merrill MJ, Pili R, et al. VEGF 165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res. 1995; 77(6): 1077-1086.
67. Moulton KS, Vakili K, Zurakowski D, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003; 100: 4736-4741.
68. Davies MJ, Richardson PD, Woolf N, et al. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage and smooth muscle cell component. Br Heart. 1993; 69:377-381.
69. Halayko AJ, Solway J. Molecular mechanisms of phenotypic plasticity in smooth muscle cells. J Appl Physiol. 2001; 90: 358-368.
70. Regan CP, Adam PJ, Madsen CS, et al: Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury. J Clin Invest. 2000; 106: 1139-1147.
71. Feil S, Hofmann F, Feil R. SM22alpha modulates vascular smooth muscle cell phenotype during atherogenesis. Circ Res. 2004; 94(7): 863-865.
72. Faries PL, Rohan DI, Wyers MC, et al. Vascular smooth muscle cells derived from atherosclerotic human arteries exhibit greater adhesion, migration, and proliferation than venous cells. J Surg Res, 2002; 104: 22-28.
73. Lavigne MC, Ramwell PW, Clarke R. Growth and phenol-typic characterization of porcine coronary artery smooth muscle cells. In Vitro Cell Dev Biol Anim. 1999; 35: 136-143.
74. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801-809.
75. Glukhova MA, Kabakov AE, Frid MG, et al. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988; 85(24): 9542-9546..
76. von der Thusen JH, van Berkel TJ, Biessen EA. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
77. Lucerna M, Zernecke A, de Nooijer R, et al. Vascular endothelial growth factor-A induces plaque expansion in ApoE knock-out mice by promoting de novo leukocyte recruitment. Blood. 2007; 109(1):122-129.
78. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917): 868-874.
79. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95(9): 858-866.
80. Groszek E, Grundy SM. The possible role of the arterial microcirculation in the pathogenesis of aterosclerosis. J Chronic Dis,1998:33(11-12): 679-684.
81. de Boer OJ, van der Wal AC, Teeling P, et al. Leukocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role of neovascularization? Cardiovasa Res. 1999; 41: 443-449.
82. O'Brien KD, McDonald TO, Chait A, et al. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93: 672-682.
83. Kaartinen M, Penttila A, Kovanen PT. Mast cells accompany microvessels in human coronary atheromas: implications for intimal neovascularization and hemorrhage. Atherosclerosis. 1996; 123: 123-131
84. Couffinhal T, Kearney M, Witzenbichler B, et al. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997; 150(5): 1673-1685.
85. Newby A. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 2005; 85(1): 1-31.
86. Loftus IM, Naylor AR, Goodall S, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques. A potential role in acute plaque disruption. Stroke. 2000; 31: 40-47.
87. Glagov S, Weisenberg E, Zarins CK, et al. Compensatory enlargement of human atherosclerotic coronary arteries . N Engl J Med, 1987; 316: 371-375.
88. Pasterkamp G, Fitzgerald PF, de Kleijn DP. Atherosclerotic expansive remodeled plaques: a wolf in sheep's clothing. J Vasc Res, 2002; 39: 514-523.
89. Ivan E, Khatri JJ, Johnson C, et al. Expansive arterial remodelling is associated with increased neointimal macrophage foam cell content: the murine mode macrophage-rich carotid artery lesions. Circulation. 2002; 105: 2686-2691.
90. Godin D, Ivan E, Johnson C, et al. Remodeling of carotid artery is associated with increased expression of matrix metalloproteinases in mouse blood flow cessation model. Circulation. 2000; 102: 2861-2866,
91. Fryer J A, Myers PC, Appleberg M. Carotid intraplaque hemorrhage: the significance of neovascularity. J Vasc Surg. 1987; 6: 341-349.
92. Shen H, Clauss M, Ryan J, et al. Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes. Blood. 1993; 81:2767-2773.
93. MuroharaT, Horowitz JR, Silver M, et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacylin. Circulation, 1998; 97: 99-107.
94. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 1995; 146: 1029-1039.
95. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992; 13: 18-32.
96. Milei J, Parodi JC, Alonso GF, et al. Carotid rupture and intraplaque hemorrhage: immunophenotype and role of cells involved. Am Heart J. 1998; 136: 1096-1105.
97. McCarthy MJ, Loftus IM, Thompson MM, Jones L, London NJ, Bell PR, Naylor AR, Brindle NP. Angiogenesis and the atherosclerotic carotid plaque: an association between symptomatology and plaque morphology. J Vasc Surg. 1999; 30: 261-268.
98. Mofidi R, Crotty TB, McCarthy P, Sheehan SJ, Mehigan D, Keaveny TV. Association between plaque instability, angiogenesis and symptomatic carotid occlusive disease. Br J Surg. 2001; 88: 945-950.
99. Fleiner M, Kummer M, Mirlacher M, Sauter G, Cathomas G, Krapf R, Biedermann BC. Arterial neovascularization and inflammation in vulnerable patients: early and late signs of symptomatic atherosclerosis. Circulation. 2004; 110: 2843-2850.
100. Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation. 2004; 110: 2032-2038.
101. Felton CV, Crook D, Davies MJ, Oliver MR. Relation of plaque lipid composition and morphology to the stability of human aortic plaques. Arterioscler Thromb Vasc Biol 1997;17:1337-1345.
102. Takaya N, Yuan C, Chu B, et al. Presence of intraplaque hemorrhage stimulates progression of carotid atherosclerotic plaques: a high-resolution magnetic resonance imaging study. Circulation. 2005; 111: 2768-2775.
103. Ishikawa K, Sugawara D, Wang X, et al. Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice. Circ Res 2001; 88: 506-512.
104. Belotti D, Paganoni P, Manenti L, et al. Matrix metalloproteinases (MMP9 and MMP2) induce the release of vascular endothelial growth factor (VEGF) by ovarian carcinoma cells: implications for ascites formation. Cancer Res. 2003; 63: 5224-5229.
105. Cullen P, Baetta R, Bellosta S,et al. Rupture of the atherosclerotic plaque: does a good animal model exist? Arterioscler Thromb Vasc Biol. 2003, 23: 535-542.
106. der Thusen JH, van Vlijmen BJ, Hoeben RC, et al. Induction of atherosclerotic plaque rupture in apolipoprotein E-/- mice after adenovirus-mediated transfer of p53. Circulation. 2002; 105: 2064-2070.
107. Caligiuri G, Groyer E, Khallou-Laschet J, et al. Reduced immunoregulatory CD31+ T cells in the blood of atherosclerotic mice with plaque thrombosis. Arterioscler Thromb Vasc Biol. 2005; 25: 1659-1664.
108. Sasaki T, Kuzuya M, Nakamura K, et al. A simple method of plaque rupture induction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2006; 26: 1304-1309.
109. Reddick RL, Zhang SH, Maeda N. Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice. Atherosclerosis. 1998,140(2): 297-305
110. Leclercq A, Houard X, Philippe M, et al. Involvement of intraplaque hemorrhage in atherothrombosis evolution via neutrophil protease enrichment. J Leukoc Biol. 2007 Sep 7; [Epub ahead of print]
111. Kuzuya M, Satake S, Esaki T, et al. Induction of angiogenesis by smooth muscle cell-derived factor: possible role in neovascularization in atherosclerotic plaque. J Cell Physiol. 1995, 164(3):658-667.
112. Leong-Poi H, Christiansen J, Heppner P, et al. Assessment of endogenous and therapeutic arterio genesis by contrast ultrasound molecular imaging of integrin expression. Circulation. 2005; 111: 3248-3254.
1. VanderLaan PA, Reardon CA, Getz GS. Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators. Arterioscler Thromb Vasc Biol. 2004; 24(1): 12-22.
2. Fry DL. Certain histological and chemical responses of the vascular interface to acutely induced mechanical stress in the aorta of the dog. Circ Res.1969;24:93-108.
3. Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma and arterial wall shear: observation, correlation and proposal of a shear dependent mass transfer mechanism for atherogenesis. Proc R Soc Lond B Biol Sci. 1971; 177: 109-159.
4. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA. 1999,282: 2035-2042.
5. Chiu JJ, Wang DL, Chien S. Effects of disturbed flow on endothelial cells. J . Biomech Eng. 1998, 120(1): 2-8.
6. Nerem RM, Alexander RW, Chappell DC et al. The study of the influence of flow . on vascular endothelial biology. Am J Med Sci. 1998, 316(3): 169-175
7. Taylor CA, Hughes TJ, Zarins CK. Finite element modeling of three-dimensional pulsatile flow in the abdominal aorta: relevance to atherosclerosis. Ann Biomed Eng. 1998; 26(6): 975-987.
8. Weinbaum S, Chien S. Lipid transport aspects of atherogenesis. J Biomech Eng. 1993,115(4B): 602-610.
9. Matharu NM, Rainger GE, Vohra R, et al. Effects of disturbed flow on endothelial cell function: Pathogenic implications of modified leukocyte recruitment. Biorheology. 2006; 43(1):31-44.
10. Plump AS, Smith JD, Hayek T, et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E deficient mice created by homologous recombination in ES Cells. Cell. 1992; 71(2): 343-353.
11. Zhang SH, Reddick RL, Piedrahita JA, et al. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258(5081): 468-471.
12. Nakashima Y, Plump AS, Raines EW, Breslow JL, et, al. ApoE Deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994; 14(1): 133-140.
13. Rekhter MD, Hicks GW, Brammer DW, et al. Animal model that mimics atherosclerotic plaque rupture. Circ Res. 1998; 83(7):705-713.
14. Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis. 2001; 154(2): 399-406.
15. Majesky MW. Mouse model for atherosclerotic plaque rupture. Circulation. 2002; 105(17): 2010-2011.
16. Cullen P, Baetta R, Bellosta S, et al. Rupture of the atherosclerotic plaque: does a good animal model exist? Arterioscler Thromb Vasc Biol. 2003; 23(4): 535-542.
17. Breslow JI. Mouse models of atherosclerosis. Science. 1996, 272(5262): 685-688.
18. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1995; 92:1355-1374.
19. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb. 1994; 14(1): 141-147.
20 von der Thusen JH, van Berkel TJ, Biessen EA. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
21. Gan LM, Gronros J, Hagg U, et al. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis. 2007; 190(2): 313-320.
22. Zhou YQ, Foster FS, Nieman BJ, et al. Comprehensive transthoracic cardiac imaging in mice using ultrasound biomicroscopy with anatomical confirmation by magnetic resonance imaging. Physiol Genomics. 2004; 18: 232-244.
23. Weinberg PD, Ross Ethier C. Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40(7): 1594-1598.
24. Nederkoorn PJ, Mali WP, Eikelboom BC, et al. Preoperative diagnosis of carotid artery stenosis: accuracy of noninvasive testing. Stroke. 2002; 33(8):2003-2008.
25. Hunink MG, Polak JF, Barlan MM, et al. Detection and quantification of carotid artery stenosis: efficacy of various Doppler velocity parameters. Am J Roentgenol. 1993; 160(3): 619-625.
26 Gnasso A, Carallo C, Irace C, et al. Association between intima-media thickness and wall shear stress in common carotid arteries in healthy male subjects. Circulation. 1996; 94: 3257-3262.
27. Geary RL, Kohler TR, Vergel S, et al. Time course of flow-induced smooth muscle cell proliferation and intimal thickening in endothelialized baboon vascular grafts. Circ Res. 1994; 74(1): 14-23.
28. Castier Y, Brandes RP, Leseche G, et al. p47phox-dependent NADPH oxidase regulates flow-Induced vascular remodeling. Circ Res. 2005; 97(6):533-540.
29. Nicolaides AN, Shifrin EG, Bradbury A, et al. Angiographic and duplex grading of internal carotid stenosis: can we overcome the confusion? J Endovasc Surg. 1996; 3: 158-165.
. 30. Wolinsky H, Glagov S. Nature of species differences in the medial distribution of aortic vasa vasorum in mammals. Circ Res. 1967; 20(4): 409-421.
31. Booth RF, Martin JF, Honey AC, et al. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis. 1989; 76(2-3): 257-268.
32. Akishita M, Ouchi Y, Miyoshi H, et al. Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells. Atherosclerosis. 1997; 130(1-2):l-10.
33. Scott TM, Honey AC, Martin JF, et al. Perivascular innervation is lost in experimental atherosclerosis. Cardioscience. 1992;3(3):145-153.
34. De Meyer GR, Van Put DJ, Kockx MM, et al. Possible mechanisms of collar-induced intimal thickening. Arterioscler Thromb Vasc Biol. 1997; 17(10): 1924-1930.
35. Van Put DJM, Van Osselaer N, De Meyer GRY, et al. Role of polymorphonuclear leukocytes in collar-induced intimal thickening in the rabbit carotid artery. Arterioscler Thromb Vasc Biol. 1999; 18 :915-921.
36. Lubbers J, de Vries MP, Veldman AE, et al. Influence of a downstream narrowing on the flow profile in a tube. J Biomech. 2006; 39(1):70-77.
37. Stroud JS, Berger SA, Saloner D. Influence of stenosis morphology on flow through severely stenotic vessels: implications for plaque rupture. J Biomech. 2000; 33(4): 443-455.
38. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990; 66: 1045-1066.
39. Ku DN, Giddens DP, Zarins CK, et al. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis. 1985; 5(3): 293-302.
40. Glagov S, Zarins C, Giddens DP, et al. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112(10):1018-1031.
41 Buchanan JR Jr, Kleinstreuer C, Truskey GA, et al. Relation between non-uniform hemodynamics and sites of altered permeability and lesion growth at the rabbit aorto-celiac junction. Atherosclerosis. 1999; 143(1): 27-40.
42. Feintuch A, Ruengsakuirach P, Lin A, et al. Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am J Physiol Heart Circ Physiol. 2007; 292(2): H884-H892.
43. Suo J, Ferrara DE, Sorescu D, et al. Hemodynamic shear stresses in mouse aortas: implications for atherogenesis.Arterioscler Thromb Vasc Biol.2007;27(2):346-351.
44. Gronros J, Wikstrom J, Hagg U, et al. Proximal to middle left coronary artery flow velocity ratio, as assessed using color Doppler echocardiography, predicts coronary artery atherosclerosis in mice. Arterioscler Thromb Vasc Biol, 2006; 26(5): 1126-1131.
45. Womersley JR. Oscillatory flow in arteries: the constrained elastic tube as a model of arterial flow and pulse transmission. Phys Med Biol. 1957; 2(2):178-187.
46. Cheng C, Tempel D, van Haperen R, et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress, Circulation. 2006; 113(23): 2744-2753.
47. Cheng C, van Haperen R, de Waard M, et al. Shear stress affects the intracellular distribution of eNOS: direct demonstration by a novel in vivo technique. Blood. 2005; 106: 3691-3698.
48. Penn MS, Koelle MR, Schwartz SM, et al. Visualization and quantification of transmural concentration profiles of macromolecules across the arterial wall. Circ Res. 1990; 67: 11-22.
49. Rangaswamy S, Penn MS, Saidel GM, et al. Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo. Circ Res. 1997; 80: 37-44.
50. Himburg HA, Grzybowski DM, Hazel AL, et al. Spatial comparison between wall shear stress measures and porcine arterial endothelial permeability. Am J Physiol Heart Circ Physiol. 2004; 286: H1916-H1922.
51. Nielsen LB. Transfer of low density lipoprotein into the arterial wall and the risk of atherosclerosis. Arteriosclerosis. 1996; 123:1-15.
52. Schwenke DC. Selective increase in cholesterol at atherosclerosis-susceptible aortic sites after short-term cholesterol feeding. Arterioscler Thromb Vasc Biol. 1995; 15(11): 1928-1937.
53. Aviram M. The contribution of the macrophage receptor for oxidized LDL to its cellular uptake. Biochem Biophys Res Commu. 1991; 179(1):359-365.
54. Han J, Nicholson AC. Lipoproteins modulate expression of the macrophage scavenger receptor. Am JPathol. 1998; 152(6):1647-1654.
55. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917): 868-874.
56. Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999; 340(2): 115-126.
57. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362(6423): 801-809.
58. Chiu JJ, Lee PL, Chen CN, et al. Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-alpha in endothelial cells. Arterioscler Thromb Vasc Biol. 2004; 24(1): 73-79.
59. Chappell DC, Varner SE, Nerem RM, et al. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res. 1998; 82(5): 532-539.
60. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95(9): 858-866.
61. Cheng C, Tempel D, van Haperen R, et al. Shear stress-induced changes in atherosclerotic plaque composition are modulated by Chemokines. J Clin Invest. 2007; 117(3): 616-626.
62. Iiyama K, Hajra L, Iiyama M, et al. Patterns of vascular cell adhesion molecule-1 and intercellular molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res. 1999; 85: 199-207.
63. Topper JN, Gimbrone MA Jr. Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype.Mol Med Today.999;5: 40-46.
64. Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 1995; 9(10): 874-882.
65. Korenaga R, Ando J, Kosaki K, et al. Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells. Am J Physiol. 1997; 273(5 Pt 1): C1506-C1515.
66. Ben Driss A, Benessiano J, Poitevin P, et al. Arterial expansive remodeling induced by high flow rates. Am J Physiol. 1997; 272(2 Pt 2): H851-H858.
67. Yilmaz A, Lipfert B, Cicha I, et al. Accumulation of immune cells and high expression of Chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques. Exp Mol Pathol. 2007; 82(3):245-55.
68. Bentzon JF, Weile C, Sondergaard CS, et al. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler Thromb Vasc Biol. 2006; 26(12): 2696-2702.
69. Lardenoye JH, Delsing DJ, de Vries MR, et al. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87(3): 248-253.
1. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med 1991; 325: 445-53.
2. European Carotid Trial Collaborative Group. MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70-99%) or mild (0-29%) carotid stenosis. Lancet 1991; 337:1235-43.
3. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study. Endarterectomy for asymptomatic carotid artery stenosis. JAMA 1995; 273: 1421- 28.
4. Chassin MR. Appropriate use of carotid endarterectomy. N Engl J Med 1998;339: 1468-71.
5. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774-81.
6. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation 2003; 108:1664-72.
7. Hollander M, Bots ML, del Sol AI, et al. Carotid plaques increase the risk of stroke and subtypes of cerebral infarction in asymptomatic elderly: the Rotterdam study. Circulation 2002; 105:2872-77.
8. Bamford J, Sandercock P, Dennis M, et al. Classification and natural history of clinically identifiable subtypes of cerebral infarction. Lancet 1991 ;337:1521-26.
9. Gray-Weale AC, Graham JC, Burnett JR, et al. Carotid artery atheroma: comparison of preoperative B-mode ultrasound appearance with carotid endarterectomy specimen pathology. J Cardiovasc Surg 1988; 29:676-81.
10. Gronholdt ML, Nordestgaard BG, Schroeder TV, et al. Ultrasonic echolucent carotid plaques predict future stroke. Circulation 2001; 104:68-73.
11. Tegos TJ, Sohail M, Sabetai MM, et al. Echomorphologic and histopathologic characteristics of unstable carotid plaques. Am J Neuroradiol 2000; 21:1937 -44.
12. Gorgan JK, Shaalan WE, Cheng H, et al. B-mode ultrasonographic characterization of carotid atherosclerotic plaques in symptomatic and asymptomatic patients. J Vasc Surg 2005; 42:435-41.
13. Mathiesen EB, Bonaa KH, Joakimsen O. Echo lucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: the Tromso study Circulation 2001; 103: 2171-75.
14 Yamashiro K, Watanabe T, Tanaka R, et al. Clustering of risk factors increases the incidence of echolucent carotid plaque in stroke patients. Cerebrovasc Dis 2006; 22:432-8.
15. Sitzer M, Muller W, Siebler M, et al. Plaque ulceration and lumen thrombus are the main sources of cerebral microemboli in high-grade internal carotid artery stenosis. Stroke 1995; 26:1231-33.
16. Fisher M, Paganini-Hill A, Martin A, et al. Carotid plaque pathology: thrombosis, ulceration, and stroke pathogenesis. Stroke 2005; 36:253-7.
17. Prabhakaran S, Rundek T, Ramas R, et al. Carotid plaque surface irregularity predicts ischemic stroke: The Northern Manhattan Study. Stroke 2006;37: 2696-701.
18. Johnson JM, Ansel AL, Morgan S, et al. Ultrasonographic screening for evaluation and follow-up of carotid artery ulceration: a new basis for assessing risk. Am J Surg 1982; 144:614-18.
19. Jander S, Sitzer M, Schumann R, et al. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke 1998; 29:1625-30.
20. Sluijter JP, Pulskens WP, Schoneveld AH, et al. Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms. Stroke 2006; 37:235-9.
21. Loftus IM, Naylor AR, Bell PR, et al. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg 2002; 89:680-94.
22. Loftus IM, Naylor R, Goodall S, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke 2000; 31:40-7.
23. Sapienza P, di Marzo L, Borrelli V, et al. Metalloproteinases and their inhibitors are markers of plaque instability. Surgery 2005;137(3): 355-63.
24. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation 2003; 108:1772-78.
25. Fujii K, Kobayashi Y, Mintz GS, et al. Intravascular ultrasound assessment of ulcerated ruptured plaques: a comparison of culprit and nonculprit lesions of patients with acute coronary syndromes and lesions in patients without acute coronary syndromes.Circulation 2003;108:2473- 78.
26. Cherian P, Hankey GJ, Eikelboom JW, et al. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke 2003; 34:2132 -37.
27. Inwald DP, McDowall A, Peters MJ, et al. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res 2003; 92:1041-48.
28. Garlichs CD, Kozina S, Fateh-Moghadam S, et al. Upregulation of CD40-CD40 Ligand (CD154) in patients with acute cerebral ischemia. Stroke 2003; 34:1412-18.
29. Napoli MD, Papa F, Bocola V. Prognostic influence of increased C-reactive protein and fibrinogen levels in ischemic stroke. Stroke 2001;32:133-8.
30. Sabetai MM, Tegos TJ, Nicolaides AN, et al. Reproducibility of computer-quantified carotid plaque echogenicity: can we overcome the subjectivity? Stroke. 2000; 31:2189-96.
1 VanderLaan PA, Reardon CA, Getz GS. Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators. Arterioscler Thromb Vasc Biol. 2004; 24: 12-22.
2 Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking ApoE: evaluation of lesional development and progression. Arterioscler Thromb Vasc Biol. 1994; 14: 141-147.
3 Nakashima Y, Plump AS, Raines EW, et al. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb Vasc Biol. 1994; 14: 133-140.
4 Weinberg PD, Ross Ethier C.Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40(7): 1594-1598.
5 Castier Y, Brandes RP, Leseche G, et al. p47phox-dependent NADPH oxidase regulates flow-Induced vascular remodeling. Circ Res. 2005; 97:533-540.
6 Feintuch A, Ruengsakulrach P, Lin A, Zhang J et al. Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am J Physiol Heart Circ Physiol. 2007; 292(2):H884-H892.
7 Suo J, Ferrara DE, Sorescu D, Guldberg RE et al. Hemodynamic shear stresses in mouse aortas: implications for atherogenesis. Arterioscler Thromb Vasc Biol. 2007; 27(2): 346-351.
8 Gan LM, Gronros J, Hagg U, Wikstrom J, et al. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis. 2007; 190(2): 313-320.
9 von der Thusen JH, van Berkel TJ, Biessen EA, et al. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
10 Cheng C. Tempel D, van Haperen R, et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation. 2006; 113(23): 2744-53.
11 Ben Driss A, Benessiano J, Poitevin P, et al. Arterial expansive remodeling induced by high flow rates. Am J Physiol. 1997;272(Heart Circ Physiol 41):H851-H858.
12 Spielmann RP, Zhen J, Triebel HJ, et al. Magnetic resonance imaging and pulsed Doppler sonography of poststenotic jets: correlation between signal void and flow velocity distribution. Magn Reson Imaging. 1992; 10(6):893-901.
13 Lubbers J, de Vries MP, Veldman AE, et al. Influence of a downstream narrowing on the flow profile in a tube._J Biomech. 2006; 39(1):70-77.
14 Stroud JS, Berger SA, Saloner D. Influence of stenosis morphology on flow through severely stenotic vessels: implications for plaque rupture. J Biomech. 2000; 33(4): 443-455.
15 Chakravarty S, Mandal PK. Two-dimensional blood flow through tapered arteries under stenotic conditions International Journal of Non-Linear Mechanics. 2000; 35(5): 779-793.
16 Zand T, Majno G, Nunnari JJ, et al. Lipid deposition and intimal stress and strain: a study in rats with aortic stenosis. Am J Pathol. 1991;139:101-113.
17 Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990 ;66:1045-1066.
18 Himburg HA, Grzybowski DM, Hazel AL, et al. Spatial comparison between wall shear stress measures and porcine arterial endothelial permeability. Am J Physiol Heart Circ Physiol. 2004;286:H1916- H1922.
19 Nielsen LB. Transfer of low density lipoprotein into the arterial wall and the risk of atherosclerosis. Arteriosclerosis. 1996; 123:1-15.
20 Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95:858-866.
21 Cheng C, Tempel D, van Haperen R, et al. Shear stress-induced changes in atherosclerotic plaque composition are modulated by Chemokines. J Clin Invest. 2007; 117(3): 616-626.
22 Zarins CK, Bomberger RA, Glagov S. Local effects of stenoses: increased flow velocity inhibits atherogenesis. Circulation. 1981;64(suppl II): 221-227.
23 Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma and arterial wall shear: observation, correlation and proposal of a shear dependent mass transfer mechanism for atherogenesis. Proc R Soc Lond B Biol Sci. 1971; 177:109-159.
24 Schwenke DC. Selective increase in cholesterol at atherosclerosis-susceptible aortic sites after short-term cholesterol feeding. Arterioscler Thromb Vasc Biol. 1995;15:1928-1937.
25 Yilmaz A, Lipfert B, Cicha I, et al. Accumulation of immune cells and high expression of Chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques. Exp Mol Pathol. 2007; 82(3):245-55.
26 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362:801-809.
27 Bentzon JF, Weile C, Sondergaard CS, et al. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in apoE knockout mice. Arterioscler Thromb Vasc Biol. 2006; 26(12): 2696-2702.
28 Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998; 18: 677-685.
29 Booth RFG, Martin JF, Honey AC, et al. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis. 1989;76:257-268.
30 Lardenoye JH, Delsing DJ, de Vries MR, et al. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87:248-253.
31 Wang YX, Halks-Miller M, Vergona R, et al. Increased aortic stiffness by pulse wave velocity in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol. 2000;278: H428-H434.
2. Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003; 349(24): 2316-2325.
3. Jeziorska M, Woolley DE. Local neovascularization and cellular composition within vulnerable regions of atherosclerotic plaques of human carotid arteries. J Pathol. 1999; 188(2): 189-196.
4. Leung DW, Cachiance G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 1989,246(4935):1306-1309.
5. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003; 9(6): 669-676.
6. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor: Multiple protein forms are encoded through alternative exon splicing. J Bio Chem. 1991; 266(18): 11947-11954.
7. Benjamin LE, Golijanin D, Itin A, et al. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest. 1999; 103(2): 159-165.
8. Eppler SM, Combs DL, Henry TD, et al. A target-mediated model to describe the Pharmacokinetics and hemodynamic effects of recombinant human vascular endothelial growth factor in humans. Clin Pharmacol Ther. 2002; 72(1): 20-32.
9. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999; 10(5): 440-447.
10. Baker AH, Kritz A, Work LM, et al. Cell-selective viral gene delivery vectors for the vasculature. Exp Physiol. 2005; 90(1): 27-31.
11. Robinson CJ, Srtinger SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001; 114 (Pt 5): 853-865.
12. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 2003; 4(5): 346-358.
13. He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998; 95(5): 2509-2514.
14. Mittal SK, McDermott MR, Johnson DC, et al. Monitoring foreign gene expression by a human adenovirus-based vector using the firely luciferase gene as a reporter. Virus Res. 1993; 28(1): 67-90.
1. Brand S. Antisense-RNA regulation and RNA interference. Biochem Biophys Acta, 2002; 1575(1-3): 15-25.
2. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001; 411(6836): 494-498.
3. Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000; 404(6775): 293-296.
4. Brummelkamp TR, BemardsR, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002; 296(5567): 550-553.
5. Berezhna SY, Supekova L, Supek F, et al. siNRA in human cells selectively localizes to target RNA sites. Proc Natl Acad Sci USA. 2006; 103(20): 7682-7687.
6. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001; 15(2): 188-200.
7. Miyagishi M, Taira K. RNAi expression vectors in mammalian cells. Methods Mol Biol. 2004; 252(1): 483-491.
8. Sui G, Soohoo C, Affarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA. 2002; 99(8): 5515-5520.
9. Naldini L, Blomer U, Gallay P. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259):263-267.
10. Bartosch B, Cosset FL. Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr Gene Ther. 2004; 4(4): 427-443.
11. Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther. 2001; 1(1): 17-40.
12. Misteli T, Spector DL. Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol. 1997; 15(10): 961-964.
13. Tiscornia G, Singer O, Verma IM. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc. 2006; 1(1): 234-240.
14. Morris KV, Rossi JJ. Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection. Hum Gene Ther. 2006; 17(5): 479-486.
15. Westerhout EM, Vink M, Haasnoot PC, et al. A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication. Mol Ther. 2006; 14(2):268-275.
16. Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Ther. 2003; 10:912-927.
1. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I . Circulation. 2003; 108: 1664-1672.
2. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation, 2003; 108: 1772-1778.
3. Libby P. Molecular bases of the acute coronary syndromes. Circulation, 1995;91: 2844-2850.
4. Burke AP, Farb A, Malcom GT, et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N Engl J Med.1997; 36: 1276-1282.
5. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000; 20: 1262-1275.
6. Kolodgie FD, Virmani R, Burke AP, et al. Pathologic assessment of the vulnerable human coronary plaque. Heart. 2004; 90:1385-1391.
7. Burke AP, Virmani R, Galis Z, et al. 34th Bethesda Conference: Task force #2-What is the pathologic basis for new atherosclerosis imaging techniques? J Am Coll Cardiol. 2003; 41: 1874-1886.
8. Kolodgie FD, Gold HK, Burke AP, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003; 349: 2316-2325.
9. Virmani R, Kolodgie FD, Burke AP, et al. Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage. Arterioscler Thromb Vasc Biol. 2005; 25:2054-2061.
10. Jeziorska M, Woolley DE. Local neovascularization and cellular composition within vulnerable regions of atherosclerotic plaques of human carotid arteries. J Pathol. 1999; 188: 189-196.
11. Kockx MM, Cromheeke KM, Knaapen MW, et al. Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis. Arterioscler Thromb Vasc Biol. 2003; 23: 440-446.
12. Virmani R, Narula J, Farb A. When neoangiogenesis ricochets. Am Heart J. 1998; 136: 937-939.
13. Khurana R, Simons M, Martin JF, et al. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation. 2005; 112: 1813-1824.
14. Ware JA. Too many vessels? Not enough? The wrong kind? The VEGF debate continues. Nat Med. 2001; 7:403-404.
15. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836): 494-498.
16. Wolinsky H, Glagov S. Nature of species differences in the medial distribution of aortic vasa vasorum in mammals. Circ. Res. 1967; 20: 409-421.
17. Shiomi M Ito T, Hirouchi Y, Enomoto M. Fibromuscular cap composition is important for the stability of established atherosclerotic plaques in mature WHHL rabbits treated with statins. Atherosclerosis. 2001; 157: 75-84.
18. StaryH C, ChandlerAB, DinsmoreR E, et al. A definition of initial, fatty streak, and intermediate lesions of the atherosclerosis: A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994; 89:2462-2478.
19. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of the advanved types of atherosclerotic lesions and a histological classification of atherosclerosis: A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation, 1995;92:1355-1374.
20. Gough PJ, Gomez IG, Wille PT, et al. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest, 2006, 116: 59-69.
21. Williams H, Johnson JL, Carson KG, Jackson CL. Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2002; 22:788-792.
22. Johnson J, Carson K, Williams H, et al. Plaque rupture after short periods of fat feeding in the apolipoprotein E-knockout mouse: model characterization and effects of Pravastatin treatment. Circulation. 2005; 111:1422-1430.
23. Rosenfeld ME, Polinsky P, Virmani R, et al. Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse. Arterioscler Thromb Vasc Biol. 2000; 20: 2587-2592.
24. Douglas JT. Adenovirus-mediated gene delivery: an overview. Methods Mol Biol, 2004; 246:3-14.
25. Yla-Herttuala S, Alitalo K. Gene transfer as a tool to induce therapeutic vascular growth. Nat Med. 2003; 9: 694-701.
26. Schneider DB, Sassani AB, Vassalli G, et al. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999; 29(3): 543-550.
27. Laitinen M, Pakkanen T, Donetti E, et al. Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther. 1997; 8(14): 1645-1650.
28. Lim K, Chae CB. A simple assay for DNA transfection by incubation of the cells in culture dishew with substrates for galatosidase. Bio Techniques, 1989; 7: 576-579.
29. Kain SR, Adams M, Kondepudi A, et al.Green fluorescent protein as a reporter of gene expression and protein licalization.Biotechniques,1995;19(4):650-655.
30. De Nooijer R, Verkleij CJ, von der Thusen JH, et al. Lesional overexpression of matrix metalloproteinase-9 promotes intraplaque hemorrhage in advanced lesions but not at earlier stages of atherogenesis. Arterioscler Thromb Vasc Biol. 2006; 26(2): 340-346.
31. Bloom W, Fawcett DW. A Textbook of Histology. Philadelphia, Pa: Saunders Company; 1962: 281-282.
32. Heistad DD, Marcus ML. Role of vasa vasorum in nourishment of the aorta, Blood Vessels. 1979, 16: 225-238.
33. Vancov V. Structural basis of the microcirculation in the wall of arterial vessels. BiblAnat. 1973; 11: 383-388.
34. Barger AC, Beeuwkes R 3rd, Lainey LL, et al. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. N Engl J Med. 1984; 310: 175-177.
35. Sueishi K, Yonemitsu Y, Nakagawa K, et al. Atherosclerosis and angiogenesis: its pathophysiological significance in humans as well as in an animal model induced by the gene transfer of vascular endothelial growth factor. Ann N Y Acad Sci. 1997; 811:311-324.
36. Kwon HM, Sangiorgi G, Ritman EL, et al. Enhanced coronary vasa vasorum neovascularization in experimental hypercholesterolemia. J Clin Invest; 1998; 101: 1551-1556.
37. Zhang Y, Cliff WJ, Schoefl GI, et al. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993; 143: 164-172.
38. Moulton KS, Heller E, Kondrding MA, et al. Angioenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipopotein E-deficient mice. Circulation. 1999; 99(13):1726-1732.
39. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003; 9: 677-684.
40. Zemplenyi T, Crawford DW, Cole MA. Adaptation to arterial wall hypoxia demonstrated in vivo with oxygen microcathodes. Atherosclerosis. 1989; 76: 173 -179.
41. Chen YX, Nakashima Y, Tanaks K, et al. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimals of human coronary arterie. Arterioscler Thromb Vasc Biol. 1999; 19: 131 -139.
42. Khurana R, Zhuang Z, Bhardwaj S, et al. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation. 2004; 110: 2436-2443.
43. Ramos MA, Kuzuya M, Esaki T, et al. Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions. Arterioscler Thromb Vase Biol. 1998; 18:1188-1196.
44. Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerosis lesions: possible pathophysiological significance of VEGF in progression of atheroselrosis. Circulation. 1998; 98(20): 2108-2116.
45. Ferrara N, Gerber HP, Le Couter J. The biology of VEGF and its receptors. Nat. Med. 2003; 9(6): 669-676.
46. Ferrara N, Winer J, Burton J. Aortic smooth muscle cells express and secrete vascular endothelial growth factor. Growth Factors, 1991, 5(2): 141-148.
47. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell. 1993; 4: 1317 -1326.
48. Pinhal-Enfield G, Ramanathan M, Hasko G, et al. An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am J Pathol. 2003; 163: 711-721.
49. Leibovich SJ, Chen JF, Pinhal-Enfield G, et al. Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin. Am J Pathol. 2002; 160: 2231- 2244.
50. Leung DW, Cachiance G, Kuang WJ, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989; 246(4935): 1306-1309.
51. Gerrity RG, Richardson M, Somer JB, et al. Endothelial cell morphology in areas of in vivo Evans blue uptake in the aorta of young pigs, II: ultrastructure of the intima in areas of differing permeability to proteins. Am J Pathol. 1977; 89: 313-334.
52. Celletti FL, Waugh JM, Amabile PG, et al. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med. 2001; 7: 425-429.
53. Eppler SM, Combs DL, Henry TD, et al. A target-mediated model to describe the Pharmacokinetics and hemodynamic effects of recombinant human vascular endothelial growth factor in humans. Clin Pharmacol Ther. 2002; 72: 20-32.
54. Laitinen M, Zachary I, Breier G, et al. VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther. 1997; 8: 1737-1744.
55. Khurana R, Shafi S, Martin J, et al. Vascular endothelial growth factor gene transfer inhibits neointimal macrophage accumulation in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol. 2004; 24: 1074-1080:
56. Zachary I, Mathur A, Yla-Herttuala S, et al. Vascular protection: a novel nonangiogenic cardiovascular role for vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2000; 20: 1512-1520.
57. Bhardwaj S, Roy H, Gruchala M, et al. Angiogenic responses of vascular endothelial growth factors in periadventitial tissue. Hum Gene Ther. 2003; 14: 1451-1462.
58. Ozawa CR, Banfi A, Glazer NL, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest. 2004; 113: 516-527.
59. Dor Y, Djonov V, Abramovitch R, et al. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J. 2002; 21:1939-1947
60. Rissanen TT, Markkanen JE, Gruchala M, et al. VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res, 2003; 92(10):1098-1106.
61. Sirois MG,Edelman ER. VEGF effect on vascular permeability is mediated by synthesis of platelet activating factor. Am J Physiol. 1997, 272(6 pt 2): H2746-H2756.
62. Mannucci PM. von Willebrand factor: a marker of endothelial damage? Arterioscler Thromb Vasc Biol. 1998; 18: 1359-1362.
63. De Meyer GRY, Hoylaerts MF, Kockx MM, et al. Intimal deposition of functional von Willebrand factor in atherogenesis. Arterioscler Thromb Vasc Biol. 1999; 19: 2524-2534.
64. Kumamoto M, Nakashima Y, Sueishi K. Intimal neovascularization in human coronary atherosclerosis: its origin and pathophysiological significance. Hum Pathol. 1995; 26: 450-456.
65. O'Brien ER, Garvin MR, Dev R, et al. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol. 1994; 145: 883-894.
66. Muhlhauser J, Merrill MJ, Pili R, et al. VEGF 165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res. 1995; 77(6): 1077-1086.
67. Moulton KS, Vakili K, Zurakowski D, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003; 100: 4736-4741.
68. Davies MJ, Richardson PD, Woolf N, et al. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage and smooth muscle cell component. Br Heart. 1993; 69:377-381.
69. Halayko AJ, Solway J. Molecular mechanisms of phenotypic plasticity in smooth muscle cells. J Appl Physiol. 2001; 90: 358-368.
70. Regan CP, Adam PJ, Madsen CS, et al: Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury. J Clin Invest. 2000; 106: 1139-1147.
71. Feil S, Hofmann F, Feil R. SM22alpha modulates vascular smooth muscle cell phenotype during atherogenesis. Circ Res. 2004; 94(7): 863-865.
72. Faries PL, Rohan DI, Wyers MC, et al. Vascular smooth muscle cells derived from atherosclerotic human arteries exhibit greater adhesion, migration, and proliferation than venous cells. J Surg Res, 2002; 104: 22-28.
73. Lavigne MC, Ramwell PW, Clarke R. Growth and phenol-typic characterization of porcine coronary artery smooth muscle cells. In Vitro Cell Dev Biol Anim. 1999; 35: 136-143.
74. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801-809.
75. Glukhova MA, Kabakov AE, Frid MG, et al. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988; 85(24): 9542-9546..
76. von der Thusen JH, van Berkel TJ, Biessen EA. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
77. Lucerna M, Zernecke A, de Nooijer R, et al. Vascular endothelial growth factor-A induces plaque expansion in ApoE knock-out mice by promoting de novo leukocyte recruitment. Blood. 2007; 109(1):122-129.
78. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917): 868-874.
79. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95(9): 858-866.
80. Groszek E, Grundy SM. The possible role of the arterial microcirculation in the pathogenesis of aterosclerosis. J Chronic Dis,1998:33(11-12): 679-684.
81. de Boer OJ, van der Wal AC, Teeling P, et al. Leukocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role of neovascularization? Cardiovasa Res. 1999; 41: 443-449.
82. O'Brien KD, McDonald TO, Chait A, et al. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation. 1996; 93: 672-682.
83. Kaartinen M, Penttila A, Kovanen PT. Mast cells accompany microvessels in human coronary atheromas: implications for intimal neovascularization and hemorrhage. Atherosclerosis. 1996; 123: 123-131
84. Couffinhal T, Kearney M, Witzenbichler B, et al. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997; 150(5): 1673-1685.
85. Newby A. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 2005; 85(1): 1-31.
86. Loftus IM, Naylor AR, Goodall S, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques. A potential role in acute plaque disruption. Stroke. 2000; 31: 40-47.
87. Glagov S, Weisenberg E, Zarins CK, et al. Compensatory enlargement of human atherosclerotic coronary arteries . N Engl J Med, 1987; 316: 371-375.
88. Pasterkamp G, Fitzgerald PF, de Kleijn DP. Atherosclerotic expansive remodeled plaques: a wolf in sheep's clothing. J Vasc Res, 2002; 39: 514-523.
89. Ivan E, Khatri JJ, Johnson C, et al. Expansive arterial remodelling is associated with increased neointimal macrophage foam cell content: the murine mode macrophage-rich carotid artery lesions. Circulation. 2002; 105: 2686-2691.
90. Godin D, Ivan E, Johnson C, et al. Remodeling of carotid artery is associated with increased expression of matrix metalloproteinases in mouse blood flow cessation model. Circulation. 2000; 102: 2861-2866,
91. Fryer J A, Myers PC, Appleberg M. Carotid intraplaque hemorrhage: the significance of neovascularity. J Vasc Surg. 1987; 6: 341-349.
92. Shen H, Clauss M, Ryan J, et al. Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes. Blood. 1993; 81:2767-2773.
93. MuroharaT, Horowitz JR, Silver M, et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacylin. Circulation, 1998; 97: 99-107.
94. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 1995; 146: 1029-1039.
95. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev. 1992; 13: 18-32.
96. Milei J, Parodi JC, Alonso GF, et al. Carotid rupture and intraplaque hemorrhage: immunophenotype and role of cells involved. Am Heart J. 1998; 136: 1096-1105.
97. McCarthy MJ, Loftus IM, Thompson MM, Jones L, London NJ, Bell PR, Naylor AR, Brindle NP. Angiogenesis and the atherosclerotic carotid plaque: an association between symptomatology and plaque morphology. J Vasc Surg. 1999; 30: 261-268.
98. Mofidi R, Crotty TB, McCarthy P, Sheehan SJ, Mehigan D, Keaveny TV. Association between plaque instability, angiogenesis and symptomatic carotid occlusive disease. Br J Surg. 2001; 88: 945-950.
99. Fleiner M, Kummer M, Mirlacher M, Sauter G, Cathomas G, Krapf R, Biedermann BC. Arterial neovascularization and inflammation in vulnerable patients: early and late signs of symptomatic atherosclerosis. Circulation. 2004; 110: 2843-2850.
100. Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation. 2004; 110: 2032-2038.
101. Felton CV, Crook D, Davies MJ, Oliver MR. Relation of plaque lipid composition and morphology to the stability of human aortic plaques. Arterioscler Thromb Vasc Biol 1997;17:1337-1345.
102. Takaya N, Yuan C, Chu B, et al. Presence of intraplaque hemorrhage stimulates progression of carotid atherosclerotic plaques: a high-resolution magnetic resonance imaging study. Circulation. 2005; 111: 2768-2775.
103. Ishikawa K, Sugawara D, Wang X, et al. Heme oxygenase-1 inhibits atherosclerotic lesion formation in ldl-receptor knockout mice. Circ Res 2001; 88: 506-512.
104. Belotti D, Paganoni P, Manenti L, et al. Matrix metalloproteinases (MMP9 and MMP2) induce the release of vascular endothelial growth factor (VEGF) by ovarian carcinoma cells: implications for ascites formation. Cancer Res. 2003; 63: 5224-5229.
105. Cullen P, Baetta R, Bellosta S,et al. Rupture of the atherosclerotic plaque: does a good animal model exist? Arterioscler Thromb Vasc Biol. 2003, 23: 535-542.
106. der Thusen JH, van Vlijmen BJ, Hoeben RC, et al. Induction of atherosclerotic plaque rupture in apolipoprotein E-/- mice after adenovirus-mediated transfer of p53. Circulation. 2002; 105: 2064-2070.
107. Caligiuri G, Groyer E, Khallou-Laschet J, et al. Reduced immunoregulatory CD31+ T cells in the blood of atherosclerotic mice with plaque thrombosis. Arterioscler Thromb Vasc Biol. 2005; 25: 1659-1664.
108. Sasaki T, Kuzuya M, Nakamura K, et al. A simple method of plaque rupture induction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2006; 26: 1304-1309.
109. Reddick RL, Zhang SH, Maeda N. Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice. Atherosclerosis. 1998,140(2): 297-305
110. Leclercq A, Houard X, Philippe M, et al. Involvement of intraplaque hemorrhage in atherothrombosis evolution via neutrophil protease enrichment. J Leukoc Biol. 2007 Sep 7; [Epub ahead of print]
111. Kuzuya M, Satake S, Esaki T, et al. Induction of angiogenesis by smooth muscle cell-derived factor: possible role in neovascularization in atherosclerotic plaque. J Cell Physiol. 1995, 164(3):658-667.
112. Leong-Poi H, Christiansen J, Heppner P, et al. Assessment of endogenous and therapeutic arterio genesis by contrast ultrasound molecular imaging of integrin expression. Circulation. 2005; 111: 3248-3254.
1. VanderLaan PA, Reardon CA, Getz GS. Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators. Arterioscler Thromb Vasc Biol. 2004; 24(1): 12-22.
2. Fry DL. Certain histological and chemical responses of the vascular interface to acutely induced mechanical stress in the aorta of the dog. Circ Res.1969;24:93-108.
3. Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma and arterial wall shear: observation, correlation and proposal of a shear dependent mass transfer mechanism for atherogenesis. Proc R Soc Lond B Biol Sci. 1971; 177: 109-159.
4. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA. 1999,282: 2035-2042.
5. Chiu JJ, Wang DL, Chien S. Effects of disturbed flow on endothelial cells. J . Biomech Eng. 1998, 120(1): 2-8.
6. Nerem RM, Alexander RW, Chappell DC et al. The study of the influence of flow . on vascular endothelial biology. Am J Med Sci. 1998, 316(3): 169-175
7. Taylor CA, Hughes TJ, Zarins CK. Finite element modeling of three-dimensional pulsatile flow in the abdominal aorta: relevance to atherosclerosis. Ann Biomed Eng. 1998; 26(6): 975-987.
8. Weinbaum S, Chien S. Lipid transport aspects of atherogenesis. J Biomech Eng. 1993,115(4B): 602-610.
9. Matharu NM, Rainger GE, Vohra R, et al. Effects of disturbed flow on endothelial cell function: Pathogenic implications of modified leukocyte recruitment. Biorheology. 2006; 43(1):31-44.
10. Plump AS, Smith JD, Hayek T, et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E deficient mice created by homologous recombination in ES Cells. Cell. 1992; 71(2): 343-353.
11. Zhang SH, Reddick RL, Piedrahita JA, et al. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258(5081): 468-471.
12. Nakashima Y, Plump AS, Raines EW, Breslow JL, et, al. ApoE Deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994; 14(1): 133-140.
13. Rekhter MD, Hicks GW, Brammer DW, et al. Animal model that mimics atherosclerotic plaque rupture. Circ Res. 1998; 83(7):705-713.
14. Johnson JL, Jackson CL. Atherosclerotic plaque rupture in the apolipoprotein E knockout mouse. Atherosclerosis. 2001; 154(2): 399-406.
15. Majesky MW. Mouse model for atherosclerotic plaque rupture. Circulation. 2002; 105(17): 2010-2011.
16. Cullen P, Baetta R, Bellosta S, et al. Rupture of the atherosclerotic plaque: does a good animal model exist? Arterioscler Thromb Vasc Biol. 2003; 23(4): 535-542.
17. Breslow JI. Mouse models of atherosclerosis. Science. 1996, 272(5262): 685-688.
18. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1995; 92:1355-1374.
19. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb. 1994; 14(1): 141-147.
20 von der Thusen JH, van Berkel TJ, Biessen EA. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
21. Gan LM, Gronros J, Hagg U, et al. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis. 2007; 190(2): 313-320.
22. Zhou YQ, Foster FS, Nieman BJ, et al. Comprehensive transthoracic cardiac imaging in mice using ultrasound biomicroscopy with anatomical confirmation by magnetic resonance imaging. Physiol Genomics. 2004; 18: 232-244.
23. Weinberg PD, Ross Ethier C. Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40(7): 1594-1598.
24. Nederkoorn PJ, Mali WP, Eikelboom BC, et al. Preoperative diagnosis of carotid artery stenosis: accuracy of noninvasive testing. Stroke. 2002; 33(8):2003-2008.
25. Hunink MG, Polak JF, Barlan MM, et al. Detection and quantification of carotid artery stenosis: efficacy of various Doppler velocity parameters. Am J Roentgenol. 1993; 160(3): 619-625.
26 Gnasso A, Carallo C, Irace C, et al. Association between intima-media thickness and wall shear stress in common carotid arteries in healthy male subjects. Circulation. 1996; 94: 3257-3262.
27. Geary RL, Kohler TR, Vergel S, et al. Time course of flow-induced smooth muscle cell proliferation and intimal thickening in endothelialized baboon vascular grafts. Circ Res. 1994; 74(1): 14-23.
28. Castier Y, Brandes RP, Leseche G, et al. p47phox-dependent NADPH oxidase regulates flow-Induced vascular remodeling. Circ Res. 2005; 97(6):533-540.
29. Nicolaides AN, Shifrin EG, Bradbury A, et al. Angiographic and duplex grading of internal carotid stenosis: can we overcome the confusion? J Endovasc Surg. 1996; 3: 158-165.
. 30. Wolinsky H, Glagov S. Nature of species differences in the medial distribution of aortic vasa vasorum in mammals. Circ Res. 1967; 20(4): 409-421.
31. Booth RF, Martin JF, Honey AC, et al. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis. 1989; 76(2-3): 257-268.
32. Akishita M, Ouchi Y, Miyoshi H, et al. Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells. Atherosclerosis. 1997; 130(1-2):l-10.
33. Scott TM, Honey AC, Martin JF, et al. Perivascular innervation is lost in experimental atherosclerosis. Cardioscience. 1992;3(3):145-153.
34. De Meyer GR, Van Put DJ, Kockx MM, et al. Possible mechanisms of collar-induced intimal thickening. Arterioscler Thromb Vasc Biol. 1997; 17(10): 1924-1930.
35. Van Put DJM, Van Osselaer N, De Meyer GRY, et al. Role of polymorphonuclear leukocytes in collar-induced intimal thickening in the rabbit carotid artery. Arterioscler Thromb Vasc Biol. 1999; 18 :915-921.
36. Lubbers J, de Vries MP, Veldman AE, et al. Influence of a downstream narrowing on the flow profile in a tube. J Biomech. 2006; 39(1):70-77.
37. Stroud JS, Berger SA, Saloner D. Influence of stenosis morphology on flow through severely stenotic vessels: implications for plaque rupture. J Biomech. 2000; 33(4): 443-455.
38. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990; 66: 1045-1066.
39. Ku DN, Giddens DP, Zarins CK, et al. Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis. 1985; 5(3): 293-302.
40. Glagov S, Zarins C, Giddens DP, et al. Hemodynamics and atherosclerosis: insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med. 1988;112(10):1018-1031.
41 Buchanan JR Jr, Kleinstreuer C, Truskey GA, et al. Relation between non-uniform hemodynamics and sites of altered permeability and lesion growth at the rabbit aorto-celiac junction. Atherosclerosis. 1999; 143(1): 27-40.
42. Feintuch A, Ruengsakuirach P, Lin A, et al. Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am J Physiol Heart Circ Physiol. 2007; 292(2): H884-H892.
43. Suo J, Ferrara DE, Sorescu D, et al. Hemodynamic shear stresses in mouse aortas: implications for atherogenesis.Arterioscler Thromb Vasc Biol.2007;27(2):346-351.
44. Gronros J, Wikstrom J, Hagg U, et al. Proximal to middle left coronary artery flow velocity ratio, as assessed using color Doppler echocardiography, predicts coronary artery atherosclerosis in mice. Arterioscler Thromb Vasc Biol, 2006; 26(5): 1126-1131.
45. Womersley JR. Oscillatory flow in arteries: the constrained elastic tube as a model of arterial flow and pulse transmission. Phys Med Biol. 1957; 2(2):178-187.
46. Cheng C, Tempel D, van Haperen R, et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress, Circulation. 2006; 113(23): 2744-2753.
47. Cheng C, van Haperen R, de Waard M, et al. Shear stress affects the intracellular distribution of eNOS: direct demonstration by a novel in vivo technique. Blood. 2005; 106: 3691-3698.
48. Penn MS, Koelle MR, Schwartz SM, et al. Visualization and quantification of transmural concentration profiles of macromolecules across the arterial wall. Circ Res. 1990; 67: 11-22.
49. Rangaswamy S, Penn MS, Saidel GM, et al. Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo. Circ Res. 1997; 80: 37-44.
50. Himburg HA, Grzybowski DM, Hazel AL, et al. Spatial comparison between wall shear stress measures and porcine arterial endothelial permeability. Am J Physiol Heart Circ Physiol. 2004; 286: H1916-H1922.
51. Nielsen LB. Transfer of low density lipoprotein into the arterial wall and the risk of atherosclerosis. Arteriosclerosis. 1996; 123:1-15.
52. Schwenke DC. Selective increase in cholesterol at atherosclerosis-susceptible aortic sites after short-term cholesterol feeding. Arterioscler Thromb Vasc Biol. 1995; 15(11): 1928-1937.
53. Aviram M. The contribution of the macrophage receptor for oxidized LDL to its cellular uptake. Biochem Biophys Res Commu. 1991; 179(1):359-365.
54. Han J, Nicholson AC. Lipoproteins modulate expression of the macrophage scavenger receptor. Am JPathol. 1998; 152(6):1647-1654.
55. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917): 868-874.
56. Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999; 340(2): 115-126.
57. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362(6423): 801-809.
58. Chiu JJ, Lee PL, Chen CN, et al. Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-alpha in endothelial cells. Arterioscler Thromb Vasc Biol. 2004; 24(1): 73-79.
59. Chappell DC, Varner SE, Nerem RM, et al. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res. 1998; 82(5): 532-539.
60. Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95(9): 858-866.
61. Cheng C, Tempel D, van Haperen R, et al. Shear stress-induced changes in atherosclerotic plaque composition are modulated by Chemokines. J Clin Invest. 2007; 117(3): 616-626.
62. Iiyama K, Hajra L, Iiyama M, et al. Patterns of vascular cell adhesion molecule-1 and intercellular molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res. 1999; 85: 199-207.
63. Topper JN, Gimbrone MA Jr. Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype.Mol Med Today.999;5: 40-46.
64. Resnick N, Gimbrone MA Jr. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 1995; 9(10): 874-882.
65. Korenaga R, Ando J, Kosaki K, et al. Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells. Am J Physiol. 1997; 273(5 Pt 1): C1506-C1515.
66. Ben Driss A, Benessiano J, Poitevin P, et al. Arterial expansive remodeling induced by high flow rates. Am J Physiol. 1997; 272(2 Pt 2): H851-H858.
67. Yilmaz A, Lipfert B, Cicha I, et al. Accumulation of immune cells and high expression of Chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques. Exp Mol Pathol. 2007; 82(3):245-55.
68. Bentzon JF, Weile C, Sondergaard CS, et al. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice. Arterioscler Thromb Vasc Biol. 2006; 26(12): 2696-2702.
69. Lardenoye JH, Delsing DJ, de Vries MR, et al. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87(3): 248-253.
1. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med 1991; 325: 445-53.
2. European Carotid Trial Collaborative Group. MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70-99%) or mild (0-29%) carotid stenosis. Lancet 1991; 337:1235-43.
3. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study. Endarterectomy for asymptomatic carotid artery stenosis. JAMA 1995; 273: 1421- 28.
4. Chassin MR. Appropriate use of carotid endarterectomy. N Engl J Med 1998;339: 1468-71.
5. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke 2000;31:774-81.
6. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation 2003; 108:1664-72.
7. Hollander M, Bots ML, del Sol AI, et al. Carotid plaques increase the risk of stroke and subtypes of cerebral infarction in asymptomatic elderly: the Rotterdam study. Circulation 2002; 105:2872-77.
8. Bamford J, Sandercock P, Dennis M, et al. Classification and natural history of clinically identifiable subtypes of cerebral infarction. Lancet 1991 ;337:1521-26.
9. Gray-Weale AC, Graham JC, Burnett JR, et al. Carotid artery atheroma: comparison of preoperative B-mode ultrasound appearance with carotid endarterectomy specimen pathology. J Cardiovasc Surg 1988; 29:676-81.
10. Gronholdt ML, Nordestgaard BG, Schroeder TV, et al. Ultrasonic echolucent carotid plaques predict future stroke. Circulation 2001; 104:68-73.
11. Tegos TJ, Sohail M, Sabetai MM, et al. Echomorphologic and histopathologic characteristics of unstable carotid plaques. Am J Neuroradiol 2000; 21:1937 -44.
12. Gorgan JK, Shaalan WE, Cheng H, et al. B-mode ultrasonographic characterization of carotid atherosclerotic plaques in symptomatic and asymptomatic patients. J Vasc Surg 2005; 42:435-41.
13. Mathiesen EB, Bonaa KH, Joakimsen O. Echo lucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: the Tromso study Circulation 2001; 103: 2171-75.
14 Yamashiro K, Watanabe T, Tanaka R, et al. Clustering of risk factors increases the incidence of echolucent carotid plaque in stroke patients. Cerebrovasc Dis 2006; 22:432-8.
15. Sitzer M, Muller W, Siebler M, et al. Plaque ulceration and lumen thrombus are the main sources of cerebral microemboli in high-grade internal carotid artery stenosis. Stroke 1995; 26:1231-33.
16. Fisher M, Paganini-Hill A, Martin A, et al. Carotid plaque pathology: thrombosis, ulceration, and stroke pathogenesis. Stroke 2005; 36:253-7.
17. Prabhakaran S, Rundek T, Ramas R, et al. Carotid plaque surface irregularity predicts ischemic stroke: The Northern Manhattan Study. Stroke 2006;37: 2696-701.
18. Johnson JM, Ansel AL, Morgan S, et al. Ultrasonographic screening for evaluation and follow-up of carotid artery ulceration: a new basis for assessing risk. Am J Surg 1982; 144:614-18.
19. Jander S, Sitzer M, Schumann R, et al. Inflammation in high-grade carotid stenosis: a possible role for macrophages and T cells in plaque destabilization. Stroke 1998; 29:1625-30.
20. Sluijter JP, Pulskens WP, Schoneveld AH, et al. Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms. Stroke 2006; 37:235-9.
21. Loftus IM, Naylor AR, Bell PR, et al. Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg 2002; 89:680-94.
22. Loftus IM, Naylor R, Goodall S, et al. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke 2000; 31:40-7.
23. Sapienza P, di Marzo L, Borrelli V, et al. Metalloproteinases and their inhibitors are markers of plaque instability. Surgery 2005;137(3): 355-63.
24. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II. Circulation 2003; 108:1772-78.
25. Fujii K, Kobayashi Y, Mintz GS, et al. Intravascular ultrasound assessment of ulcerated ruptured plaques: a comparison of culprit and nonculprit lesions of patients with acute coronary syndromes and lesions in patients without acute coronary syndromes.Circulation 2003;108:2473- 78.
26. Cherian P, Hankey GJ, Eikelboom JW, et al. Endothelial and platelet activation in acute ischemic stroke and its etiological subtypes. Stroke 2003; 34:2132 -37.
27. Inwald DP, McDowall A, Peters MJ, et al. CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res 2003; 92:1041-48.
28. Garlichs CD, Kozina S, Fateh-Moghadam S, et al. Upregulation of CD40-CD40 Ligand (CD154) in patients with acute cerebral ischemia. Stroke 2003; 34:1412-18.
29. Napoli MD, Papa F, Bocola V. Prognostic influence of increased C-reactive protein and fibrinogen levels in ischemic stroke. Stroke 2001;32:133-8.
30. Sabetai MM, Tegos TJ, Nicolaides AN, et al. Reproducibility of computer-quantified carotid plaque echogenicity: can we overcome the subjectivity? Stroke. 2000; 31:2189-96.
1 VanderLaan PA, Reardon CA, Getz GS. Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators. Arterioscler Thromb Vasc Biol. 2004; 24: 12-22.
2 Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking ApoE: evaluation of lesional development and progression. Arterioscler Thromb Vasc Biol. 1994; 14: 141-147.
3 Nakashima Y, Plump AS, Raines EW, et al. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb Vasc Biol. 1994; 14: 133-140.
4 Weinberg PD, Ross Ethier C.Twenty-fold difference in hemodynamic wall shear stress between murine and human aortas. J Biomech. 2007; 40(7): 1594-1598.
5 Castier Y, Brandes RP, Leseche G, et al. p47phox-dependent NADPH oxidase regulates flow-Induced vascular remodeling. Circ Res. 2005; 97:533-540.
6 Feintuch A, Ruengsakulrach P, Lin A, Zhang J et al. Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am J Physiol Heart Circ Physiol. 2007; 292(2):H884-H892.
7 Suo J, Ferrara DE, Sorescu D, Guldberg RE et al. Hemodynamic shear stresses in mouse aortas: implications for atherogenesis. Arterioscler Thromb Vasc Biol. 2007; 27(2): 346-351.
8 Gan LM, Gronros J, Hagg U, Wikstrom J, et al. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis. 2007; 190(2): 313-320.
9 von der Thusen JH, van Berkel TJ, Biessen EA, et al. Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Circulation. 2001; 103: 1164-1170.
10 Cheng C. Tempel D, van Haperen R, et al. Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress. Circulation. 2006; 113(23): 2744-53.
11 Ben Driss A, Benessiano J, Poitevin P, et al. Arterial expansive remodeling induced by high flow rates. Am J Physiol. 1997;272(Heart Circ Physiol 41):H851-H858.
12 Spielmann RP, Zhen J, Triebel HJ, et al. Magnetic resonance imaging and pulsed Doppler sonography of poststenotic jets: correlation between signal void and flow velocity distribution. Magn Reson Imaging. 1992; 10(6):893-901.
13 Lubbers J, de Vries MP, Veldman AE, et al. Influence of a downstream narrowing on the flow profile in a tube._J Biomech. 2006; 39(1):70-77.
14 Stroud JS, Berger SA, Saloner D. Influence of stenosis morphology on flow through severely stenotic vessels: implications for plaque rupture. J Biomech. 2000; 33(4): 443-455.
15 Chakravarty S, Mandal PK. Two-dimensional blood flow through tapered arteries under stenotic conditions International Journal of Non-Linear Mechanics. 2000; 35(5): 779-793.
16 Zand T, Majno G, Nunnari JJ, et al. Lipid deposition and intimal stress and strain: a study in rats with aortic stenosis. Am J Pathol. 1991;139:101-113.
17 Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res. 1990 ;66:1045-1066.
18 Himburg HA, Grzybowski DM, Hazel AL, et al. Spatial comparison between wall shear stress measures and porcine arterial endothelial permeability. Am J Physiol Heart Circ Physiol. 2004;286:H1916- H1922.
19 Nielsen LB. Transfer of low density lipoprotein into the arterial wall and the risk of atherosclerosis. Arteriosclerosis. 1996; 123:1-15.
20 Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease. Circ Res. 2004; 95:858-866.
21 Cheng C, Tempel D, van Haperen R, et al. Shear stress-induced changes in atherosclerotic plaque composition are modulated by Chemokines. J Clin Invest. 2007; 117(3): 616-626.
22 Zarins CK, Bomberger RA, Glagov S. Local effects of stenoses: increased flow velocity inhibits atherogenesis. Circulation. 1981;64(suppl II): 221-227.
23 Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma and arterial wall shear: observation, correlation and proposal of a shear dependent mass transfer mechanism for atherogenesis. Proc R Soc Lond B Biol Sci. 1971; 177:109-159.
24 Schwenke DC. Selective increase in cholesterol at atherosclerosis-susceptible aortic sites after short-term cholesterol feeding. Arterioscler Thromb Vasc Biol. 1995;15:1928-1937.
25 Yilmaz A, Lipfert B, Cicha I, et al. Accumulation of immune cells and high expression of Chemokines/chemokine receptors in the upstream shoulder of atherosclerotic carotid plaques. Exp Mol Pathol. 2007; 82(3):245-55.
26 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362:801-809.
27 Bentzon JF, Weile C, Sondergaard CS, et al. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in apoE knockout mice. Arterioscler Thromb Vasc Biol. 2006; 26(12): 2696-2702.
28 Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998; 18: 677-685.
29 Booth RFG, Martin JF, Honey AC, et al. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis. 1989;76:257-268.
30 Lardenoye JH, Delsing DJ, de Vries MR, et al. Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice. Circ Res. 2000; 87:248-253.
31 Wang YX, Halks-Miller M, Vergona R, et al. Increased aortic stiffness by pulse wave velocity in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol. 2000;278: H428-H434.