内皮型一氧化氮合酶基因转染对移植人工血管内膜增生的作用

目的：平滑肌细胞增殖移行和血小板激活导致血栓形成是移植血管再狭窄的主要原因，一氧化氮可以抑制上述生物反应，但内皮型一氧化氮合酶基因转染是否抑制种植了平滑肌细胞的人工血管内膜增生还未得到证实。实验拟进一步观察内皮型一氧化氮合酶基因转染对种植平滑肌细胞的人工血管内膜增生的影响。 方法：实验于2006-04／2007—05在西安交通大学医学院中心实验室及分子生物学实验室完成。①实验材料；1月龄新西兰大白兔1只，用来获取平滑肌细胞。成年新西兰大白兔18只，随机数字表法分成3组，每组6只。正常细胞组移植未种植细胞的人工血管；LacZ转染组移植种植转染lacZ的平滑肌细胞的人工血管，内皮型一氧化氮合酶转染组移植种植内皮型一氧化氮合酶的平滑肌细胞的人工血管。②实验方法：构建含有报道基因lacZ和内皮型一氧化氮合酶基因的假型反转录病毒载体小鼠白血病病毒，疱疹性口炎病毒G糖蛋白，并转染平滑肌细胞。将转染了基因的细胞种植在人工血管上，并用血管旁路移植的方法植入兔腹主动脉。③实验评估：测定转染内皮型一氧化氮合酶基因及LacZ基因细胞培养上清中一氧化氮含量。血管植入30，100d后X—gal染色及苏木精一伊红染色观察人工血管上的平滑肌细胞，同时显微镜下测量每段血管内膜增生的厚度。 结果：纳入成年新西兰大白兔18只，均进入结果分析。①内皮型一氧化氮合酶转染组一氧化氮含量明显高于未转染的正常细胞组（P〈0．05）。平滑肌细胞转染lacZ基因后经X—gal染色，倒置显微镜下可见转染了基因的细胞被染成蓝色。②血管植入30d，与正常细胞组比较，LacZ转染组和内皮型一氧化氮合酶转染组内膜厚度差异无显著性（P〉0．05）；100d后，内皮型一氧化氮合酶转染组内膜厚度与正常细胞组?     

内皮型一氧化氮合酶基因转染对移植人工血管内膜增生的作用

目的:平滑肌细胞增殖移行和血小板激活导致血栓形成是移植血管再狭窄的主要原因,一氧化氮可以抑制上述生物反应,但内皮型一氧化氮合酶基因转染是否抑制种植了平滑肌细胞的人工血管内膜增生还未得到证实.实验拟进一步观察内皮型一氧化氮合酶基因转染对种植平滑肌细胞的人工血管内膜增生的影响.方法:实验于2006-04/2007-05在西安交通大学医学院中心实验室及分子生物学实验室完成.①实验材料:1月龄新西兰大白兔1只,用来获取平滑肌细胞.成年新西兰大白兔18只,随机数字表法分成3组,每组6只.正常细胞组移植未种植细胞的人工血管;LacZ转染组移植种植转染lacZ的平滑肌细胞的人工血管,内皮型一氧化氮合酶转染组移植种植内皮型一氧化氮合酶的平滑肌细胞的人工血管.②实验方法:构建含有报道基因lacZ和内皮型一氧化氮合酶基因的假型反转录病毒载体小鼠白血病病毒/疱疹性口炎病毒G糖蛋白,并转染平滑肌细胞.将转染了基因的细胞种植在人工血管上,并用血管旁路移植的方法植入兔腹主动脉.③实验评估:测定转染内皮型一氧化氮合酶基因及LacZ基因细胞培养上清中一氧化氮含量.血管植入30,100 d 后X-gal染色及苏木精-伊红染色观察人工血管上的平滑肌细胞,同时显微镜下测量每段血管内膜增生的厚度.结果:纳入成年新西兰大白兔18只,均进入结果分析.①内皮型一氧化氮合酶转染组一氧化氮含量明显高于未转染的正常细胞组(P ＜ 0.05).平滑肌细胞转染lacZ基因后经X-gal染色,倒置显微镜下可见转染了基因的细胞被染成蓝色.②血管植入30 d,与正常细胞组比较,LacZ转染组和内皮型一氧化氮合酶转染组内膜厚度差异无显著性(P ＞ 0.05);100 d后,内皮型一氧化氮合酶转染组内膜厚度与正常细胞组无明显差异,与LacZ转染组相比较,差异显著(P ＜ 0.05).结论:内皮型一氧化氮合酶基因转染抑制了种植平滑肌细胞的人工血管内膜增生.     

转染组织纤溶酶原激活物及内皮型一氧化氮合酶基因的内皮细胞和平滑肌细胞共同种植于人工血管对内膜增生的影响

背景：前期研究表明,双层细胞种植能有效提高内皮细胞保留率,将组织纤溶酶原激活物基因转染到内皮细胞中能提高其细胞溶解纤维蛋白的能力。目的：采用双细胞种植及修饰内皮的组织纤溶酶原激活物基因提高内皮细胞保留率和抗栓能力,通过内皮型一氧化氮合酶基因调控平滑肌细胞的增殖,观察对小口径人工血管通畅率的影响。方法：以4种不同组合细胞（内皮细胞＋平滑肌细胞SMC,内皮细胞/组织纤溶酶原激活物＋平滑肌细胞,内皮细胞＋平滑肌细胞/内皮型一氧化氮合酶,内皮细胞/组织纤溶酶原激活物＋平滑肌细胞/内皮型一氧化氮合酶）种植在PTFE管腔面。将新西兰大白兔随机分成4组,4种人工血管经旁路分别移植在4组兔的腹主动脉上。结果与结论：移植后60d,种植内皮细胞＋平滑肌细胞组和内皮细胞＋平滑肌细胞/内皮型一氧化氮合酶组内膜厚度无明显差异（P〉0.05）。与内皮细胞/组织纤溶酶原激活物＋平滑肌细胞/内皮型一氧化氮合酶组相比,未转染内皮型一氧化氮合酶组（内皮细胞/组织纤溶酶原激活物＋平滑肌细胞）内膜明显增厚（P〈0.05）;未转染组织纤溶酶原激活物组（内皮细胞＋平滑肌细胞/内皮型一氧化氮合酶）内膜较薄（P〈0.05）。提示组织纤溶酶原激活物基因转染可以促进血管内膜增生导致血管狭窄,但同时转入内皮型一氧化氮合酶基因可以抑制组织纤溶酶原激活物的促进平滑肌细胞增殖及内膜增生的作用。     

转染组织纤溶酶原激活物及内皮型一氧化氮合酶基因的内皮细胞和平滑肌细胞共同种植于人工血管对内膜增生的影响

背景:前期研究表明,双层细胞种植能有效提高内皮细胞保留率,将组织纤溶酶原激活物基因转染到内皮细胞中能提高其细胞溶解纤维蛋白的能力.目的:采用双细胞种植及修饰内皮的组织纤溶酶原激活物基因提高内皮细胞保留率和抗栓能力,通过内皮型一氧化氮合酶基因调控平滑肌细胞的增殖,观察对小口径人工血管通畅率的影响.方法:以4种不同组合细胞(内皮细胞+平滑肌细胞SMC,内皮细胞/组织纤溶酶原激活物+平滑肌细胞,内皮细胞+平滑肌细胞/内皮型一氧化氮合酶,内皮细胞/组织纤溶酶原激活物+平滑肌细胞/内皮型一氧化氮合酶)种植在PTFE管腔面.将新西兰大白兔随机分成4组,4种人工血管经旁路分别移植在4组兔的腹主动脉上.结果与结论:移植后60 d,种植内皮细胞+平滑肌细胞组和内皮细胞+平滑肌细胞/内皮型一氧化氮合酶组内膜厚度无明显差异(P > 0.05).与内皮细胞/组织纤溶酶原激活物+平滑肌细胞/内皮型一氧化氮合酶组相比,未转染内皮型一氧化氮合酶组(内皮细胞/组织纤溶酶原激活物+平滑肌细胞)内膜明显增厚(P < 0.05);未转染组织纤溶酶原激活物组(内皮细胞+平滑肌细胞/内皮型一氧化氮合酶)内膜较薄(P < 0.05).提示组织纤溶酶原激活物基因转染可以促进血管内膜增生导致血管狭窄,但同时转入内皮型一氧化氮合酶基因可以抑制组织纤溶酶原激活物的促进平滑肌细胞增殖及内膜增生的作用.     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响

背景：一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。目的：观察内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。方法：建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。结果与结论：AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组（P〈0.01）。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响

背景:一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。目的:观察内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。方法:建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。结果与结论:AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组(P<0.01)。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

人内皮型一氧化氮合成酶基因转染桡动脉的表达

目的:桡动脉作为血管桥材料在冠状动脉旁路移植术中具有重要作用,但血管桥痉挛一直是困扰桡动脉移植的难题.实验通过腺病毒载体介导的人内皮型一氧化氮合成酶基因(AdCMVeNOS)转染人桡动脉,体外培养后检测人内皮型一氧化氮合成酶基因的表达,观察其对内膜增生的影响.方法:①实验于2005-03104在解放军沈阳军区总医院完成.实验用Enos-Ad由该院麻醉科张铁铮主任惠赠.桡动脉来自15例冠状动脉旁路移植手术患者剩余桡动脉.将桡动脉横切成5 mm血管环,采用随机数字表法分为3组,对照组、空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组,每组8份.空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组桡动脉血管环分别置于空载腺病毒液和AdCMVeNOS溶液中1 h,取出后体外培养14 d.应用多聚寡核苷酸探针和高敏感标记技术检测外源性基因内皮型一氧化氮合酶mRNA的表达:免疫组织化学染色方法检测内皮型一氧化氮合酶蛋白的表达:苏木精-伊红染色及弹性纤维染色后,应用计算机图像分析系统检测桡动脉内膜、中膜增生情况.结果:①3组培养的桡动脉在14 d后有新内膜形成和显著的中膜增厚,内皮型一氧化氮合酶基因转染组内膜厚度及内膜/中膜厚度比值低于对照组(P＜0.01),空载腺病毒液感染组与对照组差异无显著性(P＞0.05).②培养14d后,内皮型一氧化氮合酶基因转染组内膜和中膜可检测到内皮型一氧化氮合酶mRNA和蛋白质表达,对照组及空载腺病毒液感染组未见明显表达.结论:腺病毒载体介导的人内皮型一氧化氮合成酶基因成功转染体外培养桡动脉中并有效表达,内皮型一氧化氮合酶基因具有防治体外培养桡动脉内膜增生的作用.     

内皮型一氧化氮合酶与血管结构及功能的相关性

目的：应用内部核糖体进入位点构建携带内皮型一氧化氮合酶和血管内皮生长因子的双基因表达载体，研究外源性基因内皮型一氧化氮合酶和血管内皮生长因子在人脐静脉内皮细胞ECV304中的转染表达，探讨内皮型一氧化氮合酶与血管结构及功能的相关性。方法：实验于2003-12／2004-12在南京鼓楼医院科研部完成。将Phcmvspla—enos中内皮型一氧化氮合酶基因和PcDNA—vegf中血管内皮生长因子基因定向克隆到真核表达载体PcDNA—ires中，构建重组质粒PcDNA—enos—ires-lvegf，经酶切，聚合酶链反应扩增和部分DNA测序分析证实后，转染人脐静脉内皮细胞，硝酸还原酶法检测转染后细胞中一氧化氮和一氧化氮合酶活性，免疫组化和荧光双标记及western blotting检测转染后细胞蛋白水平的表达，Trizol提取总RNA，反转录一聚合酶链反应测定转染后细胞mRNA水平的表达。结果：成功构建携带内皮型一氧化氮合酶和血管内皮生长因子的双基因表达载体PcDNA-enos-ires-vegf,双基因内皮型一氧化氮合酶和血管内皮生长因子在人脐静脉内皮细胞基因组中获得整合及表达。重组基因PcDNA-enos-ires-vegf组与对照组PcDNA-enos在一氧化氮含量和一氧化氮合成酶活性的表达差异无显著性意义。PcDNA-enos-ires-vegf转染细胞后转染效率为30％-40％。结论：双基因表达载体PcDNA-enos-ires-vegf的成功构建和双基因内皮型一氧化氮合酶和血管内皮生长因子在人脐静脉内皮细胞基因组中表达为研究内皮型一氧化氮合酶在血管结构与功能上所起上所起的作用奠定了基础。     

双细胞种植改善人工血管内皮细胞的保留率

背景: 前期研究表明,在人工血管内壁种植内皮细胞,使其早期快速内皮化,可以改善移植血管的通畅性,但种植的单层内皮细胞往往黏附力差,在血流的冲击下容易脱落.目的: 采用血管内皮细胞和平滑肌细胞双层种植方法,提高人工血管腔面内皮细胞的保存率.方法: 采用标准酶分离技术和细胞培养技术分离培养兔内皮细胞.采用组织贴块法分离培养兔平滑肌细胞.并分别以lacZ和GFP基因转染内皮细胞及平滑肌细胞.双层细胞种植组在聚四氟乙烯人工血管管腔面先后种植平滑肌细胞和内皮细胞,以单纯种植血管内皮细胞为对照.在体外将受试的人工血管接入作者设计的灌流模型下灌注2 h,计算比较灌注前后受试标本内皮细胞密度.体内实验,将人工血管植入兔子体内7 d,分别测定内皮细胞保留率.结果与结论: 灌注2 h后,单纯内皮细胞种植组细胞保留率为0.39±0.04,而双层细胞种植组内皮细胞保留率为0.73±0.07,双层细胞种植组细胞保存率明显高于单纯内皮细胞种植组(P<0.01).人工血管植入兔体内7 d,单纯内皮细胞种植组内皮细胞保留率为0.63±0.10,双层细胞种植组内皮细胞保留率为0 95±0.06,两组间差异有显著性意义(P<0.05).结果证实在内皮细胞和人工血管壁之间种植平滑肌细胞在体外和体内均可提高内皮细胞的保存率.     

腺病毒转染一氧化氮合酶基因抑制大鼠静脉桥血管内膜增生

目的探讨转染一氧化氮合酶（NOS）基因对自体移植静脉内膜增生的影响.方法制作20只自体颈静脉腹主动脉移植Wistar大鼠模型,实验组、对照组各10只.移植前,实验组血管行腺病毒介导的eNOS溶液浸泡,对照组仅行空载腺病毒溶液浸泡.术后2周取出移植血管,利用病理学、免疫组织化学、RT-PCR检测移植血管内膜厚度、管腔狭窄度、内膜血管平滑肌细胞（VSMC）数及PCNA阳性表达、血管eNOS mRNA表达情况.结果实验组移植血管内膜厚度、管腔狭窄度、VSMC数及PCNA阳性表达均较对照组明显减小或减少,而eNOS mRNA表达则明显增加（均P＜0.01）.结论移植血管转染NOS基因可有效抑制移植静脉内膜的增生.     

Gene therapy to prevent occlusion of venous bypass grafts

Revascularization with vein grafts is standard surgical therapy for occlusive arterial diseases. Autologous saphenous vein grafts are important conduits for repairing blocked coronary arteries and are used in the majority of vein graft procedures. Up to 50% of saphenous vein grafts will be occluded during the first decade after surgery. Vein graft occlusion occurs as a result of neointimal hyperplasia, which takes place in response to hemodynamic changes and vessel wall injury, and is characterized by the migration and proliferation of vascular smooth muscle cells. Intimal hyperplasia is further complicated by the concomitant development of atherosclerosis and thrombosis. In the absence of effective pharmacological interventions for the treatment and prevention of occlusive vein graft disease, gene therapy has emerged as a potential therapeutic alternative. Gene therapy could improve vein graft patency by reducing early thrombosis, neointimal hyperplasia and atherosclerosis. In this review we will summarize the emerging applications of gene therapy as a therapeutic tool in occlusive vein graft disease.     

Dominant negative c-jun gene transfer inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in rats

We previously reported that activator protein-1 (AP-1), containing c-Jun, is rapidly activated in balloon-injured artery. Therefore, we examined the role of c-Jun in vascular smooth muscle cell (SMC) proliferation, by using in vitro and in vivo gene transfer techniques. (1) Serum (2%) stimulation significantly increased AP-1 DNA binding activity in aortic SMCs, followed by the increase in both 3H-thymidine incorporation and cell number. Aortic SMCs were infected with recombinant adenovirus containing TAM67, a dominant negative c-Jun lacking transactivation domain of wild c-Jun (Ad-DN-c-Jun), to specifically inhibit AP-1. Ad-DN-c-Jun significantly inhibited serum-induced SMC proliferation, by inhibiting the entrance of SMC into S phase. (2) The effect of DN-c-Jun was examined on balloon injury-induced intimal hyperplasia in rats. Before balloon injury, DN-c-Jun was transfected into rat carotid artery using the hemagglutinating virus of Japan-liposome method. In vivo transfection of DN-c-Jun significantly inhibited vascular SMC proliferation in the intima and the media and subsequently prevented intimal thickening at 14 days after balloon injury. We obtained the first evidence that DN-c-Jun gene transfer prevented vascular SMC proliferation in vitro and in vivo, and c-Jun was involved in balloon injury-induced intimal hyperplasia. Thus, AP-1 seems to be the new therapeutic target for treatment of vascular diseases.     

Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes.

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.     

DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.     

Mechanisms in the inhibition of neointimal hyperplasia with triflavin in a rat model of balloon angioplasty

RGD-containing peptides are able to inhibit the binding of ligands to certain beta3 integrins, such as alpha(IIb)beta3 and alpha(v)beta3, both of which are involved in neointimal hyperplasia. The present study was designed to elucidate the detailed mechanisms involved in the inhibition of neointimal hyperplasia with triflavin in a rat model of balloon angioplasty. Triflavin (0.25 mg x kg(-1) x d(-1)), an RGD-containing disintegrin, time dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, electron micrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. PDGF-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, and triflavin significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic guanosine monophosphate levels compared with levels found in sham-operated rats, and these were not significantly changed with infusion of triflavin (0.25 mg x kg(-1) x d(-1)). Furthermore, the plasma level of TXB2 obviously increased after angioplasty, and triflavin markedly suppressed the elevation of plasma TXB2 concentration. The results indicate that triflavin effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms. First, triflavin binds to alpha(IIb)beta3 integrin on platelet membranes, resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of TXA2 formation and PDGF-BB release from platelets. Second, triflavin may also bind to alpha(v)beta3 integrin on SMCs, thus subsequently inhibiting cell migration and proliferation. These results provide new insights into the mechanisms of neointimal hyperplasia and have significant implications for disintegrin therapy for the treatment of restenosis and atherosclerosis.     

Cell cycle inhibition preserves endothelial function in genetically engineered rabbit vein grafts.

We have recently shown that ex vivo gene therapy of rabbit autologous vein grafts with antisense oligodeoxynucleotides (AS ODN) blocking cell cycle regulatory gene expression inhibits not only neointimal hyperplasia, but also diet-induced, accelerated graft atherosclerosis. We observed that these grafts remained free of macrophage invasion and foam cell deposition. Since endothelial dysfunction plays an important role in vascular disease, the current study examined the effect of this genetic engineering strategy on graft endothelial function and its potential relationship to the engineered vessels' resistance to atherosclerosis. Rabbit vein grafts transfected with AS ODN against proliferating cell nuclear antigen (PCNA) and cell division cycle 2 (cdc2) kinase elaborated significantly more nitric oxide and exhibited greater vasorelaxation to both calcium ionophore and acetylcholine than did untreated or control ODN-treated grafts. This preservation of endothelial function was associated with a reduction in superoxide radical generation, vascular cell adhesion molecule-1 (VCAM-1) expression, and monocyte binding activity in grafts in both normal and hypercholesterolemic rabbits. Our data demonstrate that AS ODN arrest of vascular cell cycle progression results in the preservation of normal endothelial phenotype and function, thereby influencing the biology of the vessel wall towards a reduction of its susceptibility to occlusive disease.