Hiraoka Kazuya, Koike Hiromi, Yamamoto Seiji, Tomita Naruya, Yokoyama Chieko, Tanabe Tadashi, Aikou Takashi, Ogihara Toshio, Kaneda Yasufumi, Morishita Ryuichi
Division of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita 565-0871, Japan.
Circulation. 2003 Nov 25;108(21):2689-96. doi: 10.1161/01.CIR.0000093275.78676.F4. Epub 2003 Oct 20.
Although clinical trials of therapeutic angiogenesis by angiogenic growth factors with intramuscular injection of naked plasmid DNA have been successful, there are still unresolved problems such as low transfection efficiency. From this viewpoint, we performed the following modifications: (1) combination with vasodilation using prostacyclin and (2) changing the agents or volume of naked plasmid DNA in vivo.
First, we examined cotransfection of the VEGF gene with the prostacyclin synthase gene in a mouse hindlimb ischemia model. Cotransfection of the VEGF gene with the prostacyclin synthase gene resulted in a further increase in blood flow and capillary density compared with single VEGF gene. Similar results were obtained with other angiogenic growth factors, such as hepatocyte growth factor (HGF). Alternatively, we changed the injection volume of the solution of plasmid DNA. Luciferase activity was increased in a volume-dependent manner. An increase in injection volume at 1 site rather than separate injections at multiple sites resulted in high transfection efficiency, which suggests that transfection of naked plasmid DNA is mediated by pressure. Interestingly, treatment with hyperbaric oxygen increased the transfection efficiency. Finally, we also examined the effects of different solutions. Saline and PBS, but not water, achieved high transfection efficiency. In addition, sucrose solution but not glucose solution resulted in high luciferase activity.
Overall, angiogenesis might be enhanced by cotransfection of prostacyclin synthase gene or an increase in injection volume and osmotic pressure. These data provide important information for the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.
尽管通过肌内注射裸质粒DNA的血管生成生长因子进行治疗性血管生成的临床试验已取得成功，但仍存在诸如转染效率低等未解决的问题。从这个角度出发，我们进行了以下改进：（1）与使用前列环素的血管舒张相结合；（2）在体内改变裸质粒DNA的制剂或体积。
首先，我们在小鼠后肢缺血模型中检测了VEGF基因与前列环素合酶基因的共转染。与单一VEGF基因相比，VEGF基因与前列环素合酶基因的共转染导致血流量和毛细血管密度进一步增加。使用其他血管生成生长因子，如肝细胞生长因子（HGF），也获得了类似结果。另外，我们改变了质粒DNA溶液的注射体积。荧光素酶活性呈体积依赖性增加。在一个部位增加注射体积而非在多个部位分开注射导致了高转染效率，这表明裸质粒DNA的转染是由压力介导的。有趣的是，高压氧治疗提高了转染效率。最后，我们还检测了不同溶液的效果。生理盐水和磷酸盐缓冲液（PBS）而非水可实现高转染效率。此外，蔗糖溶液而非葡萄糖溶液导致高荧光素酶活性。
总体而言，前列环素合酶基因的共转染或注射体积及渗透压的增加可能会增强血管生成。这些数据为治疗性血管生成治疗外周动脉疾病的临床应用提供了重要信息。
