Department of Cardiology, Medical College of Ewha Womans University, Mokdong Hospital, Seoul, Korea.
Gene Ther. 2010 Dec;17(12):1442-52. doi: 10.1038/gt.2010.101. Epub 2010 Jul 29.
Hepatocyte growth factor (HGF) has been shown to induce angiogenesis in vivo and has potential as a candidate gene for 'therapeutic angiogenesis'. In vivo, two isoforms of HGF, HGF₇₂₃ and HGF₇₂₈, consisting of 723 and 728 amino acids, are generated through alternative splicing between exons 4 and 5, but the biological effects of their coexpression have not yet been elucidated. In this study, we generated a series of genomic-complementary DNA (cDNA) hybrids of the HGF gene by inserting various truncated intron 4 into the junction of exons 4 and 5 of HGF cDNA and analyzed the biological activities of these hybrid constructs. We showed that: (1) the hybrid called HGF-X7, which contained 1502 base pairs of intron 4, could drive a higher level of HGF expression than other hybrid constructs and cDNAs of each isoform alone; (2) the pCK vector was most efficient for the gene expression of HGF-X7; (3) coexpression of both isoforms of HGF could more efficiently induce the migration of human umbilical vein endothelial cell (HUVEC) and of the mouse myoblast cell line C₂C₁₂ myoblasts than a single isoform of HGF and human vascular endothelial growth factor (VEGF)₁₆₅ at a given concentration; (4) intramuscular administration of pCK-HGF-X7 resulted in transient and localized HGF expression in the injected muscle without an increase in the HGF protein levels in other tissues including serum; and (5) intramuscular injection of pCK-HGF-X7 could more efficiently increase the number of angiographically recognizable collateral vessels, as well as improve an intra-arterial Doppler wire-measured blood flow in the rabbit model of hindlimb ischemia when compared with the identical vector encoding VEGF₁₆₅ gene. These results showed that transfer of the genomic-cDNA hybrid of the HGF gene could be used as a potential therapeutic approach to human vascular diseases.
肝细胞生长因子（HGF）已被证明可在体内诱导血管生成，并有望成为“治疗性血管生成”的候选基因。在体内，HGF 通过外显子 4 和 5 之间的选择性剪接产生两种同工型 HGF₇₂₃和 HGF₇₂₈，分别由 723 和 728 个氨基酸组成，但它们的共表达的生物学效应尚未阐明。在这项研究中，我们通过将各种截短的内含子 4 插入 HGF cDNA 的外显子 4 和 5 的连接处，生成了一系列 HGF 基因的基因组-cDNA 杂种，并分析了这些杂种构建体的生物学活性。我们表明：（1）含有 1502 个碱基对内含子 4 的杂种 HGF-X7 可以驱动比其他杂种构建体和每种同工型的 cDNA 更高水平的 HGF 表达；（2）pCK 载体最有利于 HGF-X7 的基因表达；（3）与单独的同工型 HGF 相比，两种同工型 HGF 的共表达可以更有效地诱导人脐静脉内皮细胞（HUVEC）和小鼠成肌细胞系 C₂C₁₂ 成肌细胞的迁移，并且在给定浓度下，人血管内皮生长因子（VEGF）₁₆₅也更有效；（4）pCK-HGF-X7 肌内给药导致注射肌肉中 HGF 的短暂和局部表达，而包括血清在内的其他组织中的 HGF 蛋白水平没有增加；（5）与编码 VEGF₁₆₅ 基因的相同载体相比，pCK-HGF-X7 的肌内注射可以更有效地增加兔后肢缺血模型中血管造影可识别的侧支血管数量，并改善经皮动脉内多普勒导丝测量的血流。这些结果表明，HGF 基因的基因组-cDNA 杂种的转移可以作为治疗人类血管疾病的潜在方法。
