Human Endothelial Nitric Oxide Synthase Gene Delivery Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia

R. S. Smith, Kuei-Fu Lin, J. Agata, L. Chao, J. Chao
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引用次数: 103

Abstract

Objective—Endothelium-derived NO has been shown to mediate the mitogenic effect of vascular endothelial growth factor on cultured microvascular endothelium. To evaluate the role of endothelial NO synthase (eNOS) in angiogenesis in the ischemic hindlimb, we engineered an adenovirus containing human eNOS cDNA. Methods and Results—After gene transfer, expression of eNOS in cultured cells was detected by increased intracellular cGMP and nitrate/nitrite levels and NO synthase activity. Adenovirus containing either the eNOS or luciferase gene was injected into the adductor muscle of rat hindlimbs immediately after femoral artery removal. Human eNOS protein was detected throughout the course of the experiment by immunostaining. Significant increases in blood perfusion were monitored by laser Doppler imaging from 2 to 4 weeks after gene delivery in the ischemic hindlimb of rats receiving eNOS compared with control rats receiving the reporter gene. An increase in regional blood flow was also detected after eNOS gene transfer by a fluorescent microsphere assay. eNOS gene delivery in the ischemic hindlimb resulted in significant increases in intracellular cGMP levels and in capillary density identified by anti–CD-31 immunostaining. Angiogenesis was further confirmed in mice after eNOS gene transfer by increased hemoglobin content in Matrigel implants. Conclusions—Taken together, these results indicate that eNOS enhances angiogenesis and raises the potential of eNOS gene transfer for modulation of vascular insufficiency.
人内皮型一氧化氮合酶基因在大鼠后肢缺血模型中促进血管生成
目的:内皮源性一氧化氮介导血管内皮生长因子对培养的微血管内皮细胞的有丝分裂作用。为了评估内皮NO合成酶(eNOS)在缺血后肢血管生成中的作用,我们设计了一种含有人eNOS cDNA的腺病毒。方法与结果-基因转移后,通过细胞内cGMP、硝酸盐/亚硝酸盐水平和NO合成酶活性的升高检测eNOS在培养细胞中的表达。将含有eNOS或荧光素酶基因的腺病毒在股动脉切除后立即注射到大鼠后肢内收肌中。在整个实验过程中,通过免疫染色检测人eNOS蛋白。在基因传递后2 ~ 4周,激光多普勒成像监测到,与接受报告基因的对照组大鼠相比,eNOS大鼠缺血后肢的血流灌注明显增加。荧光微球法检测eNOS基因转移后,局部血流量增加。通过抗cd -31免疫染色检测,eNOS基因在缺血后肢中的传递导致细胞内cGMP水平和毛细血管密度显著增加。eNOS基因转染后,Matrigel植入物中血红蛋白含量增加,进一步证实了小鼠血管生成。综上所述,这些结果表明eNOS促进血管生成,并提高eNOS基因转移调节血管功能不全的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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