腺病毒介导的碱性成纤维细胞生长因子对慢性缺血冬眠心肌的保护作用

目的探讨重组腺病毒介导的碱性成纤维细胞生长因子(bFGF)对慢性缺血冬眠心肌的保护作用。方法制备猪慢性缺血冬眠心肌模型,并随机分为3组,分别心内注射腺病毒介导的bFGF(AdbFGF组)、空病毒(空病毒组)和生理盐水(空白对照组),每组7头猪,观察心肌间质胶原容积分数、心肌细胞核密度、核长度、心肌细胞直径以及心肌细胞增殖活性和凋亡的改变。结果与空病毒组和空白对照组比较,AdbFGF组冬眠心肌处心肌细胞直径和核长度明显增加,而心肌细胞核密度明显降低。而且心肌细胞的增殖活性增高,凋亡减少。结论bFGF一方面诱导心肌细胞增殖分裂,促进冬眠心肌细胞肥大;另一方面抑制细胞凋亡,减少冬眠心肌的损伤。     

血管生成素相关蛋白2对大鼠缺血心肌促血管生长作用的初步观察

目的观察和评价血管生成素相关蛋白2(angiopoietin-related protein 2,ARP2)基因移植于大鼠急性缺血心肌后的促血管新生作用,探讨其促血管新生机制。方法建立急性心肌缺血大鼠模型,将42只大鼠随机分为3组:缺血区域心肌内注射ARP2重组腺病毒组(Ⅰ组,n=14);注射ARP2空腺病毒组(Ⅱ组,n=14)和注射磷酸缓冲溶液组(Ⅲ组,n=14),注射后28天行心脏彩色超声检查、心肌声学造影,并取注射点心肌切片作常规病理HE染色、VB染色、Ⅷ因子相关抗原免疫学检测。结果Ⅰ组大鼠心脏功能及心肌灌注较Ⅱ、Ⅲ组明显改善,免疫组织化学检测显示Ⅰ组心肌高度表达ARP2蛋白,心肌纤维间毛细血管数目明显增多。结论ARP2能通过促进大鼠缺血心肌血管新生,从而改善心脏功能。     

重组腺相关病毒2型载体-血管内皮生长因子165改善小型猪慢性心肌缺血的研究

目的应用重组腺相关病毒2型载体(recombinant adenovirus-associated virus2,rAAV2)介导血管内皮生长因子165(vascular endothelial growth factor,VEGF165)基因转染,观察其促进小型猪慢性缺血心肌血管生成并改善心肌血流灌注和心功能的有效性。方法小型猪左冠状动脉回旋支(LCX)放置血管缩窄环,建立慢性心肌缺血模型。5周后行心电图、冠状动脉造影和心脏核磁共振成像检查确认LCX闭塞或相应心肌的缺血。动物随机分为实验组和对照组,每组8只,分别在心肌内直接注射rAAV2-VEGF165(1×1012virus genome)或磷酸盐缓冲液。治疗后3个月和6个月,观察心肌VEGF mRNA和蛋白的表达;6个月后,观察心肌毛细血管和小动脉密度,行冠状动脉造影进行LCX血流分级,应用心脏核磁共振成像观察心肌灌注及左心室功能。结果放置血管缩窄环后5周,所有动物均出现LCX完全/次全闭塞或LCX支配区域的心肌缺血。基因治疗后3个月,实验组心肌VEGF mRNA和蛋白表达显著高于对照组;6个月时,实验组VEGF表达水平较3个月时下降。基因治疗后6个月,VEGF组心肌毛细血管密度和小动脉密度[分别为(1404.06±250.48)/mm2和(167.81±36.29)/mm2]均高于对照组[分别为(976.88±344.79)/mm2和(116.56±34.48)/mm2](P<0.05);潘生丁负荷后心肌灌注成像显示VEGF组心肌灌注明显优于对照组(P<0.05),且较治疗前有改善(P<0.05);两组左心室功能在治疗前后均无明显变化。结论在小型猪慢性心肌缺血模型中,心肌内注射rAAV2-VEGF165后外源VEGF基因的表达至少可持续3个月;rAAV2-VEGF165能够促进缺血心肌毛细血管和小动脉生成并改善心肌灌注。     

转染Angiopoietin-1基因对急性心梗后左室功能的影响

目的探讨重组腺病毒载体介导的血管形成素1(Ang1)基因转染对兔急性心梗后左室功能影响。方法构建含有Ang1的腺病毒粘粒载体,通过转染293细胞进行同源重组制备重组腺病毒颗粒。64只雄性新西兰大白兔行高位冠状动脉结扎,4只用于急性心梗后循环中VEGF含量的检测,其余60只随机均分为实验组、单纯培养基组(DMEM)与LacZ重组腺病毒组,分别于冠状动脉结扎后心肌内直接注射Ang1重组腺病毒,DMEM与LacZ重组腺病毒。并于转染后第3、14、28d应用超声心动图观察心功能情况,第3、7、14、28d应用RT PCR方法测定重组腺病毒基因在转染心肌中的表达,第14、28d用免疫组化方法观察缺血心肌内血管生长情况。结果获得的Ang1重组腺病毒滴度为5.6×1011pfu/L。重组腺病毒直接注射转染兔缺血心肌后能有效表达目的基因,RT PCR测定提示Ang1基因转染后第3d心肌中即有表达,7、14d仍呈阳性表达,28d未测出。心肌梗死后Ang1组心功能从术后第3d至第28d得到明显改善,其幅度显著好于对照组。心肌梗死后左室心肌收缩期截面积与舒张期截面积均明显增加,尤以术后第3d为著,随时间延长均有所缩小,至28d后Ang1组缩小幅度明显高于其他两组。转染14、28d后Ang1组新生毛细血管密度及αSMA阳性的小动脉性血管密度均明显高于对照组(P<0.01)。结论腺病毒介导的Ang1基因在兔缺血心肌中能有效地促进新生血管形成,并能改善缺血心肌的功能。     

激光心肌血运重建术与血管内皮生长因子基因治疗慢性缺血性心脏病的对比研究

目的:探索激光生物效应和血管内皮生长因子(VEGF)基因治疗促进缺血区血管再生的作用。方法:心肌缺血模型猪分别进行激光心肌血运重建术(TMLR)和PcDNA3·1VEGF165基因治疗,6周后行冠状动脉造影、单光子发射型计算机断层扫描(SPECT)、逆转录-聚合酶链反应(RT-PCR)病理学检查。结果:TMLR、基因治疗组冠状动脉造影显示侧支循环明显改善,SPECT显示缺血侧壁灌注显著改善,RT-PCR显示TMLR组VEGF基因有中度表达,基因治疗组有高度或中度表达,心肌血管面积、血管周长、血管数目明显增加(P<0·05);TMLR组血管管径≤25μm数目比基因组明显增加(P<0·05)。结论:TMLR或VEGF基因治疗可以增加心肌血管密度并改善缺血心肌的灌注;TMLR疗效优于VEGF心肌直接注射。     

经冠状动脉注射骨髓单个核细胞治疗缺血性心肌损伤的实验研究

目的　证实骨髓干细胞心肌内移植治疗对心脏功能的改善和促血管新生作用。方法　小型猪冠状动脉结扎制备心肌梗死模型 ,然后经冠状动脉内注射骨髓单个核细胞 ,术后 3周用超声心动图以及左心室造影检测心功能的变化 ,核素心肌显像观察心肌灌注的情况 ,冠状动脉造影观察侧支循环的形成 ,免疫组化计数血管密度。结果　心肌梗死小型猪冠状动脉注射骨髓单个核细胞后 ,左心室造影显示左室 dP dtmax与对照组比较增高。核素心肌显像显示冠状动脉注射骨髓单个核细胞后心肌灌注显著改善。移植治疗后冠状动脉造影显示有侧支循环形成 ,血管密度计数比对照组增加了5 2 2 % (5 6 6± 11 7 mm2 vs 37 2± 8 4 mm2 ,P <0 0 5 )。结论　骨髓单个核细胞心肌内移植可能通过促进血管新生改善心脏收缩功能。     

肝细胞生长因子治疗心肌梗死后心力衰竭的实验研究

目的研究经冠状动脉转染携带人肝细胞生长因子(hepatocytegrowthfactor,HGF)的腺病毒(adenovirus,Ad5)对猪心肌梗死后心力衰竭的改善作用。方法12只苏中幼猪,随机分成转染Ad5-HGF组(治疗组)和转染未携带肝细胞生长因子的腺病毒载体(null-Ad5)组(对照组),每组6只。结扎前降支制成心肌梗死模型,手术后第4周及第7周时行冠状动脉造影和门控心肌灌注显像,评价侧支循环形成程度(Rentorp法)、心肌灌注及心功能的变化;用ELISA法检测肝细胞生长因子蛋白表达;以免疫组织化学法检测基因导入后平滑肌肌动蛋白抗体阳性(SMA )有灌注功能血管生成情况。结果经冠状动脉转染Ad5-HGF后心肌高度表达人HGF蛋白;治疗组心肌血流灌注及左心室射血分数(LVEF)较对照组明显改善;治疗组侧支循环形成明显增多,每平方毫米功能血管的数量明显高于对照组。结论经冠状动脉转染Ad5-HGF能使心肌高度表达HGF蛋白。HGF对梗死心肌的侧支循环形成、心肌灌注及心功能均有改善作用,能促进功能血管数增多。     

猪慢性缺血心肌内注射PcDNA_(3.1)VEGF_(165)对心电稳定性的影响

探讨猪慢性缺血心肌内注射PcDNA3 .1VEGF165(血管内皮生长因子 )后 ,在心肌血供改善的基础上 ,缺血心肌的电稳定性能否随缺血的改善而有所改善。用Ameroid收缩环制备猪慢性心肌缺血模型 ,在其右室心尖部及流出道行心内程序电刺激 ,检测室性心动过速 (VT)、心室颤动 (VF)的诱发率 ,并测定左室室壁运动功能和心肌血管密度 ,比较VEGF基因治疗组与病理对照组间的差异。结果 :在对照组 10例中 ,2例诱发出单形性持续性VT ,2例诱发出多形性VT(持续 10s左右转为VF) ,2例诱发出VF ,恶性心律失常诱发率 6 0 %。治疗组 12例中 ,1例诱发出VF ,1例诱发出多形性VT(持续数秒后转为VF) ,恶性心律失常诱发率 17%。基因治疗组缺血区室壁运动和心肌血管密度明显优于病理对照组 ,两组间存在显著差异。结论 :慢性缺血心肌内注射PcDNA3 .1VEGF165基因不仅可增加心肌供血、改善心肌功能 ,而且可能在此基础上提高心电稳定性 ,减少冠心病猝死的发生     

经冠状动脉注射骨髓单个核细胞影响心肌细胞凋亡的实验研究

目的观察经冠状动脉注射方式移植骨髓单个核细胞对心脏功能的改善和心肌细胞凋亡的影响。方法结扎小型猪冠状动脉制备心肌梗死模型，然后经冠状动脉注射骨髓单个核细胞，术后3周用超声心动图以及左心室造影检测心功能．核素心肌显像观察心肌灌注，冠状动脉造影观察侧支循环形成．用TUNEL检测心肌细胞凋亡。结果把骨髓单个核细胞通过冠状动脉注射到猪心肌梗死模型，显示左心室dp／dtmax较对照组增高，心肌灌注显著改善。冠状动脉有侧支循环形成，缺血心肌细胞；较对照组减少了53．6％结论骨髓单个核细胞心肌内移植可改善心脏收缩功能，可能与移植细胞抑制心肌细胞凋亡有关。     

骨髓间充质干细胞移植对心肌缺血/再灌注大鼠心肌胶原与血管新生的影响

目的通过心肌缺血/再灌注模型观察大鼠骨髓间充质干细胞心肌移植对心肌细胞外胶原、小血管新生和心功能变化的影响,并探讨其机制。方法分离、原代培养Wistar大鼠骨髓间充质干细胞,建立缺血/再灌注动物模型,分为假手术组、心肌梗死 骨髓间充质干细胞移植组、心肌梗死对照组,观察骨髓间充质干细胞移植后动物模型血流动力学指标、心肌梗死面积和心肌细胞形态、心肌间质、血管密度、心室膨展指数、心肌细胞横截面积和心肌胶原变化。结果(1)骨髓间充质干细胞心肌移植可缩小60min缺血/再灌注大鼠28d心肌梗死面积;(2)骨髓间充质干细胞移植可减轻心肌梗死动物左心室重塑,但心功能改善作用不明显;(3)骨髓间充质干细胞移植发挥细胞外基质抑制、促血管生成作用,心肌间质胶原含量、胶原分数下降,心肌毛细血管、小动脉密度增加。结论骨髓间充质干细胞移植可改善心肌梗死后心室重塑、促进血管新生和降低心肌胶原作用。     

Effect of angiopoietin-related protein 2 on coronary angiogenesis and myocardial function in a porcine model of acute myocardial ischemia

Our previous studies have suggested that angiopoietin-related protein 2 (Arp2) may improve rat cardiac function after acute myocardial infarction (AMI) by accelerating angiogenesis.We want to study the efficacy of the adenoviral vector-mediated gene transfer of Arp2 (Ad.Arp2) in inducing angiogenesis and in improving the myocardial perfusion and function in a porcine acute myocardial ischemic model.Methods The minipigs underwent ligation of the proximal circumflex coronary artery (LCx) and were randomly assigned to treatment with Ad.Arp2,adenoviral vectors with no transgene (Ad.Null) or PBS.Four weeks later,the animals were evaluated using echocardiography,cardiac perfusion imaging and pathologic observation.Results Four weeks after treatment,the Arp2 protein was revealed in the myocardium of Ad.Arp2 animals,but was not found in the Ad.Null or PBS animals.Also,a significant revival of myocardial perfusion was found in the ischemic area in Ad.Arp2-treated animals,whose global and regional myocardial function was greatly improved.The quantitation of new capillaries was much greater in the Ad.Arp2 group than in the Ad.Null or PBS groups.Conclusion Treatment with Ad.ARP2 offers the obvious advantage of greatly improving the blood supply and the heart function.(J Geriatr Cardiol 2008;5:230-234)     

腺病毒siRNA抑制RhoA基因并联合TNF-α诱导肝癌细胞凋亡的研究

目的建立RhoA基因的腺病毒siRNA系统,并联合TNF-α诱导肝癌细胞凋亡,分析RhoA在肿瘤细胞中的功能和作用。方法用已构建的RhoA干扰质粒构建RhoA的腺病毒siRNA系统,筛选重组病毒并感染HepG2细胞,应用Western blot和RT-PCR检测RhoA蛋白表达和基因表达水平。感染Ad-siRNA-RhoA腺病毒的肝癌细胞用TNF-α诱导后进行MTT以及细胞内DNA片段化检测。结果成功构建RhoA基因的siRNA腺病毒系统。用重组病毒感染肝癌细胞,RhoA蛋白表达抑制率为76.48%;RhoA基因的mRNA转录水平降低74.46%。MTT检测显示,感染腺病毒Ad-U6-control对照组以及感染腺病毒Ad-siRNA-RhoA实验组的细胞A值差异无统计学意义(F=5.41,P>0.01),即利用siRNA抑制肝癌细胞中RhoA表达,肿瘤细胞凋亡不明显。TUNEL检测显示,RhoA腺病毒siRNA联合TNF-α致肿瘤细胞凋亡作用显著。结论构建的腺病毒siRNA载体系统能抑制目的基因RhoA的表达,联合TNF-α能抑制肿瘤细胞生长和增殖并诱导肿瘤细胞凋亡,为肿瘤基因功能的基础研究和肿瘤基因治疗打下了实验基础。     

Effect of long-term exercise on regional myocardial function and coronary collateral development after gradual coronary artery occlusion in pigs

The effect of myocardial ischemia, induced by long-term exercise, on regional myocardial function and coronary collateral development was examined in pigs after gradual occlusion of the left circumflex coronary artery (LCx) with an ameroid occluder. Thirty days after surgery, regional myocardial function and blood flow were assessed during exercise in 22 pigs separated into exercise (n = 12) and sedentary groups (n = 10). The exercise group trained on a treadmill for 25 +/- 1 days, 30-50 min/day, at heart rates of 210-220 beats/min. After 5 weeks, another exercise test was performed. In the exercise group, after training, we observed an improvement in systolic wall thickening, expressed as a percentage of rest, in the collateral-dependent LCx region from 64 +/- 8% to 87 +/- 6% (p less than 0.01) at moderate exercise levels (220 beats/min) and from 45 +/- 7% to 73 +/- 7% (p less than 0.01) at severe exercise levels (265 beats/min). Transmural myocardial blood flow in the LCx region expressed as a ratio of flow in the nonoccluded region of the left ventricle also increased significantly (p less than 0.01) during severe exercise after 5 weeks. The sedentary group showed an improvement in systolic wall thickening in the LCx region during moderate exercise compared with the initial exercise test (p less than 0.05) but no significant change in systolic wall thickening or myocardial blood flow ratios during severe exercise after 5 weeks. We conclude that long-term exercise after gradual LCx coronary artery occlusion in pigs improves myocardial function and coronary collateral reserve in collateral-dependent myocardium during exercise.     

Chronic reduction of myocardial ischemia does not attenuate coronary collateral development in miniswine.

BACKGROUND. Myocardial ischemia is considered to be a possible stimulus for development of the coronary collateral circulation. We therefore hypothesized that chronic reduction of myocardial oxygen demand to lessen ischemia would attenuate coronary collateral development over an 8-week period using left circumflex coronary artery (LCx) ameroid-induced constriction in pigs. METHODS AND RESULTS. Collateral development was assessed by myocardial blood flow (radioactive microspheres) and left ventricular regional function (sonomicrometer dimension gauges). beta-Adrenoceptor blockade with propranolol (160 or 320 mg b.i.d.p.o.) was initiated in 15 animals 1 day after surgery. Compared with 16 untreated animals, beta-adrenoceptor antagonism was documented in the treated group by 1) pharmacological stimulation with isoproterenol, 2) physiological stimulation during graded treadmill exercise, and 3) repeated long-term biotelemetry recordings of oxygen demand (heart rate and blood pressure) and regional myocardial function. In addition to pharmacological and physiological verification of beta-blockade, biotelemetry showed that, compared with the untreated animals, propranolol significantly reduced the daily number, individual duration, and severity of events representing myocardial dysfunction. This suggests that in the beta-blocked group, little if any ischemia was present throughout the first 5 weeks when collateral growth occurs. Transmural myocardial blood flow (expressed as a ratio of flow in the LCx region to the nonoccluded region of the left ventricle) and systolic wall thickening in the LCx region were determined at rest and during treadmill exercise (240 beats per minute) 31-38 days (5 weeks) and 60-67 days (8 weeks) after surgery. Propranolol was withdrawn 3 days before flow and function determinations and was resumed immediately after testing. Blood flow ratios at 5 weeks decreased similarly from rest to exercise in the untreated (0.83 +/- 0.04 to 0.60 +/- 0.05, p less than 0.05) and beta-blockade group (0.82 +/- 0.09 to 0.57 +/- 0.10, p less than 0.05). Systolic wall thickening from rest to exercise was attenuated to the same degree in the untreated (59 +/- 6% to 38 +/- 6%, p less than 0.05) and beta-blockade group (50 +/- 8% to 30 +/- 5%, p less than 0.05). Similar flow and function responses were observed in both groups at 8 weeks. CONCLUSIONS. We conclude that growth and development of the coronary collateral circulation measured functionally during exercise at 90% of maximal heart rate is unrelated to the extent and duration of myocardial ischemia in this model.     

An adenoviral vector cancer vaccine that delivers a tumor-associated antigen/CD40-ligand fusion protein to dendritic cells

To develop a method to overcome the anergy that exists in tumor hosts to cancer, we have designed an adenoviral vector for the in vivo activation and tumor antigen loading of dendritic cells. This adenoviral vector encodes a fusion protein composed of an amino-terminal tumor-associated antigen fragment fused to the CD40 ligand (CD40L). Subcutaneous injection of an adenoviral vector encoding a fusion protein of the human papillomavirus E7 foreign antigen linked to the CD40L generates CD8+ T cell-dependent immunoresistance to the growth of the E7-positive syngeneic TC-1 cancer cells in C57BL/6 mice for up to 1 year. We also studied the s.c. injection of a vector carrying the gene for the human MUC-1 (hMUC-1) self-antigen fused to the CD40L. When this vector was injected into hMUC-1.Tg mice, which are transgenic for the hMUC-1 antigen, the growth of syngeneic hMUC-1-positive LL1/LL2hMUC-1 mouse cancer cells was suppressed in 100% of the injected animals. The hMUC-1.Tg mice are anergic to the hMUC-1 antigen before the injection of the vector. These experimental results show that it is possible to use vector injection to activate a long-lasting cellular immune response against self-antigens in anergic animals. The vector-mediated in vivo activation, and tumor-associated antigen loading of dendritic cells does not require additional cytokine boosting to induce the immune response against the tumor cells. This vector strategy may therefore be of use in the development of immunotherapy for the many carcinomas in which the hMUC-1 antigen is overexpressed.     

Alterations in transmural blood flow and body surface ST segment abnormalities produced by ischemia in the circumflex and left anterior descending coronary arterial beds of the dog

Previous studies have documented a quantitative relation between alterations in transmural myocardial blood flow and body surface electrocardiographic distributions during rapid atrial pacing after chronic occlusion of the left circumflex coronary artery (LCx). Because other studies have described functional differences between the left anterior descending (LAD) and the LCx perfusion beds, we tested the hypothesis that these two territories exhibit quantitative differences in their responses to demand-dependent myocardial ischemia. To do so, 25 sedated dogs were studied 3 weeks after implantation of an ameroid constrictor around the proximal LCx (15 dogs, group I) or the LAD (group II). Oxygen demand was increased by rapid atrial pacing at rates of 90 to 210 beats/min, myocardial blood flow was measured by serial injections of radiolabeled microspheres, and the electrocardiographic consequences were evaluated by isopotential body surface mapping. Endocardial flows and the endocardial/epicardial flow ratio fell to significantly lower levels during atrial pacing in the ischemic LAD bed than in the LCx perfusion zone. Electrocardiographic patterns indicative of subendocardial ischemia also developed with lesser abnormalities in endocardial/epicardial ratios as determined by logistic regression models, in the LAD than in the LCx bed. Thus the LAD bed is more susceptible to ischemia than the LCx region because of differences in collateral blood flow patterns. In addition, the intensity of the surface electrocardiographic potentials during ischemia was significantly greater, as measured by linear regression, after LAD than after LCx obstruction. These data thus demonstrate significant differences between the two cardiac regions as electrocardiographic potential sources during ischemia.     

Structural stability of neoangiogenic intramyocardial microvessels supports functional recovery in chronic ischemic myocardium

We hypothesize that combining angiopoietin-1 (ANG-1) or ANG-2 with vascular endothelial growth factor (VEGF) improves myocardial perfusion and contractile function by modulating vascular adaptation of neoangiogenic microvessels in a chronic ischemic swine model. Four weeks after occlusion of the left circumflex coronary artery (LCx), animals were injected with AdVEGF₁₆₅ (n = 6), AdVEGF₁₆₅+AdANG-1 (n = 6), AdVEGF₁₆₅+AdANG-2 (n = 6) or control vector (n = 5) into the left ventricular posterolateral wall. Regional perfusion by fluorescent microspheres and segmental myocardial tissue velocity by tissue Doppler imaging (TDI) were assessed at baseline, 4 weeks post occlusion and 4 weeks post therapy. Despite similar vascular growth following VEGF+ANG-1 and VEGF+ANG-2 treatments, transmural myocardial contractility improved only when VEGF was paired with ANG-1. In contrast, regional systolic function deteriorated uniformly across subepicardial, mid-myocardial and subendocardial segments in VEGF and VEGF+ANG-2 treated groups. Contractile improvement was associated with enhanced vascular stability through augmented arteriole formation, tight structural integration between VE-cadherin and β-catenin at endothelial junctions and improved cross-talk between endothelium and myocardium. Structural stability of developing intramyocardial microvessels contributes to systolic function during ischemic neovascularization. Coordinated regulation of angiogenic revascularization that supports vascular stability is a key aspect in improving therapeutic outcomes in ischemic myocardium.     

Catheter-based plasmid-mediated transfer of genes into ischemic myocardium using the pCOR plasmid

BACKGROUND: Direct transfer of genes holds promise for the sustained delivery of therapeutic proteins to treat cardiovascular diseases. This can be accomplished by several approaches, including use of adenoviral vectors and naked plasmid DNA vectors. We previously demonstrated achieval of effective delivery of genes into the myocardium with a left ventricular-guided catheter-based approach using an adenoviral vector. OBJECTIVE: To evaluate the levels and duration of expression of genes induced after injection of a specific plasmid vector, using the same delivery platform as that in our previous work. METHODS: The pCOR plasmids are narrow-host-range plasmid vectors designed for nonviral gene therapy. We tested the ability of the pCOR plasmid vector to express its transgene after injection into the myocardium of pigs with chronic experimental ischemia using a catheter-based transendocardial delivery system. Four animals were subjected to transendocardial injections of the luciferase reporter pCOR gene into ischemic and nonischemic zones using the Biosense intramyocardial injection catheter. Injections (1 mg per animal, 50 micrograms per injection site) were performed at 20 sites in ischemic and nonischemic zones. Measurements of luciferase activity were performed 3 and 7 days thereafter. RESULTS: We observed high levels of expression of luciferase gene in ischemic and nonischemic regions (on days 3 and 7, respectively, in ischemic zone 58,237 and 33,709 pg; in nonischemic zone 39,928 and 46,036 pg). Control noninjected samples from the left and right ventricles contained no detectable luciferase activity. CONCLUSIONS: With a catheter-based approach, the pCOR plasmid was successfully used to deliver genes into designated myocardial regions, and provides sustained expression of protein for at least 7 days, of roughly similar magnitudes in ischemic and nonischemic myocardium.     

Could plasmid‐mediated gene transfer into the myocardium be augmented by left ventricular guided laser myocardial injury?

Early studies have indicated no correlation between the amount of mechanical injury and the level of myocardial gene expression following direct plasmid vector injection. Recently, however, evidence suggests that combined laser myocardial injury and plasmid-based gene delivery exert synergistic effects on gene expression and activity. The purpose of the study was to determine whether laser-induced myocardial injury followed by transendocardial gene transfer increases gene expression compared to gene transfer alone. We assessed the ability of a plasmid vector to express its transgene after injection into porcine ischemic myocardium with and without preceding laser myocardial injury. Thirteen animals had transendocardial injections of the luciferase reporter gene in a plamid vector using a catheter-based injection system. Injections (0.5 mg per animal, 50 microg per injection site) were divided into 10 sites in the ischemic territory. Eight animals underwent transendocardial laser injury of the ischemic region (2 Joule per pulse x 10 sites) prior to gene delivery. In five animals, gene injection sites were dispersed between laser channels, and in three animals laser and gene delivery were applied in close proximity (< 5 mm) or at the same location. Luciferase activity was measured at 3 and 7 days. Luciferase expression in ischemic zones was markedly elevated at day 3 and 7, and similar whether animals were pretreated using laser injury followed by gene transfer compared to gene transfer alone. Neither same-spot injection nor dispersed gene delivery were associated with augmented gene expression compared to gene transfer alone. Using the above-described catheter-based approach to combine localized laser injury and injection of naked DNA into ischemic myocardium, laser injury did not augment gene expression above levels present with gene transfer alone.