A novel ex vivo angiogenesis assay based on electroporation-mediated delivery of naked plasmid DNA to skeletal muscle.
A novel ex vivo angiogenesis assay based on electroporation-mediated delivery of naked plasmid DNA to skeletal muscle.
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一种基于电穿孔介导的裸质粒 DNA 向骨骼肌递送的新型离体血管生成测定。
DOI：
10.1016/j.carpath.2004.03.062
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
D. Kim
中科院分区：
文献类型：
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作者：
H. Jang;Hyun;Jeong‐Min Kim;Young;Koung Li Kim;Jeong;Jae‐Young Lee;W. Suh;Jin‐Ho Choi;E. Jeon;J. Byun;D. Kim
基于基因转移的血管生成检测将是非常有用的血管生成基因治疗。一个简单的，可重复的，和定量的测定来测试血管生成基因将提供更准确的预测比传统的肽为基础的测定。在这里，我们已经开发了一种半定量血管生成检测利用基因转移到骨骼肌，这是缺血性肢体疾病的靶组织。为了便于快速和干净的分析，裸质粒DNA载体结合电穿孔程序用于基因转移。将编码血管内皮生长因子cDNA的质粒载体（pJDK-VEGF 165）注射入BALB/c小鼠胫骨前肌，然后进行体内电穿孔和在生长因子减少的Matrigel中进行组织块培养，早在第2天就观察到出芽细胞的向外迁移。细胞很快形成毛细血管网络，在第7天达到峰值，并持续到第14天。血管性血友病因子、血小板内皮细胞粘附分子和波形蛋白阳性，提示它们是内皮细胞。对照载体（pJDK）注射组几乎没有（如果有的话）毛细血管的发芽或形成。与发芽和网络形成的区域一致，在外植体培养物的条件培养基中分泌的VEGF的量增加。结缔组织生长因子（CTGF）的血管生成潜力进行了检查，使用新的分析。而CTGF基因单独诱导弱发芽活动，它似乎抑制血管生成活性的VEGF 165基因在共同处理。CTGF对VEGF的这种减弱活性在小鼠后肢缺血模型中重现。在用pJDK-CTGF和pJDK-VEGF 165两者治疗的小鼠组中，在股动脉切除后10天，通过激光多普勒成像测量的血流量显著低于pJDK-VEGF 165治疗组。这些结果与最近的报道一致，表明CTGF抑制VEGF。这证实了该novelex体内测定法在评估感兴趣基因的血管生成能力中的有用性。总之，这种新的基因为基础的血管生成检测应广泛适用于血管生成或抗血管生成基因的研究，因为它可以很容易地预测特定基因及其组合的血管生成潜力。
An angiogenesis assay based on gene transfer would be extremely useful for angiogenic gene therapy. A simple, reproducible, and quantitative assay to test angiogenic genes would provide more accurate predictions than conventional peptide-based assays. Here, we have developed a semiquantitative angiogenesis assay utilizing gene transfer into skeletal muscle, which is a target tissue for ischemic limb diseases. To facilitate quick and clean analysis, a naked plasmid DNA vector combined with an electroporation procedure was used for gene transfer. When the plasmid vector encoding vascular endothelial growth factor cDNA (pJDK-VEGF165) was injected into thetibialis anteriormuscle of BALB/c mice, followed byin vivoelectroporation and explant culture in growth factor-reduced Matrigel, the outward migration of sprouting cells was observed as early as day 2. The cells soon formed capillary networks, which peaked at day 7 and persisted until day 14. The capillary-like structures were positive for von Willebrand factor, platelet endothelial cell adhesion molecule, and vimentin, suggesting they were endothelial cells. There was little, if any, sprouting or formation of capillaries from the control vector (pJDK)-injected group. Consistent with the region of sprouting and network formation, the amount of secreted VEGF increased in the conditioned medium of explant cultures. The angiogenic potential of connective tissue growth factor (CTGF) was examined using the new assay. Whereas theCTGFgene alone induced weak sprouting activity, it appeared to inhibit the angiogenic activity of theVEGF165gene during cotreatment. This attenuating activity of CTGF on VEGF was reproducedin vivoin a murine model of hindlimb ischemia. In a group of mice treated with both pJDK-CTGF and pJDK-VEGF165, the blood flow measured by laser Doppler imaging was significantly lower than that of the pJDK-VEGF165-treated group 10 days after femoral artery excision. These results are consistent with recent reports that suggest that CTGF inhibits VEGF. This confirms the usefulness of this novelex vivoassay in assessing the angiogenic capacity of genes of interest. In summary, this new gene-based angiogenesis assay should be widely applicable in the study of angiogenic or antiangiogenic genes because it can readily predict the angiogenic potential of specific genes and their combinations.