hVEGF121 transfection and animal model for in vivo angiogenesis / 대한내과학회지

BACKGROUND: This study was designed to prove the superiority of ACP vector containing ITR, and to establish animal model quantifying angiogenesis in vivo. METHODS: hVEGF121, therapeutic gene, was inserted to various vectors (pcDNA3.1, pcDNA 3.2, pActin, pDesm, pACP vector), and these vectors were transfected to various cells using FuGENE6. We cultured for 48hrs, and then quantified amounts of hVEGF121 of supernatants by ELISA. The long-term transfection was assessed for 14 days. Optimal condition of transfection was evaluated by change of the ratio of DNA to FuGENE6, amount of DNA, and confluence of cells. ACP-hVEGF121 was transfected to C2C12 and these transfected C2C12 cells were mixed with Matrigel, and then injected to C3H mouse subcutaneously. Seven days later, hemoglobin assay and pathology of Matrigel were reviewed for angiogenesis. RESULTS: The level of hVEGF121 gene expression using pACP vector was significantly higher than those of others. In 2 weeks culture study, pACP vector showed the highest gene expression and produced VEGF until 2 weeks. The highest gene expression was obtained when the concentration of DNA was 7 microgram, the confluence was up to 80% and the ratio of DNA to FuGENE6 was 1:3. The hemoglobin level in Matrigel of VEGF group was significantly higher than the one of the control group, and active angiogenesis was noted in the VEGF group. CONCLUSIONS: pACP vector might be an efficient vector for angiogenic gene delivery, and animal model using Matrigel and transfected C2C12 cell could be a useful tool for quantitative angiogenesis assay.

A Simple, Quantitative Method for Assessing Angiogenic Genes Using Skeletal Muscle by Electroporation-Mediated Naked DNA Delivery

BACKGROUND AND OBJECTIVES: For the development of an arteriogenic gene therapy in peripheral artery occlusive disease, we developed a novel angiogenesis assay, with electroporation-mediated naked DNA delivery to the skeletal muscle. MATERIALS AND METHODS: The levels of the expression CAT were compared between pJDK and pcDNA3.1, in HeLa and C2C12 cell lines, and skeletal muscle. The well known angiogenic gene, pJDK-hVEGF165, was injected, intramuscularly, into the tibialis anterior muscle of Balb/C mice, which was followed by electroporation. Two days later, the anterior tibialis muscles were divided into halves, embedded, and cultured in growth factor-reduced Matrigel. The capillary network area formed by the newly sprouting tube-like structures was calculated. For validation of this ex vivo assay, the connective tissue growth factor gene (pJDK-CTGF) was tested both by this new assay, and by the mice-hind limb ischemia model, with Laser Doppler imaging. RESULTS: The pJDK showed a significantly higher level of CAT expression than the pcDNA3.1. From the pJDK-hVEGF165 injected explants, endothelial cell migration and tube-like formation occurred on day 2, and the capillary network formation peaked on day 7. The capillary network formation in the pJDK-hVEGF165 group was markedly increased to that in the pJDK group. From the skeletal muscle assay, the pJDK-CTGF showed no angiogenic activity or attenuated VEGF-induced capillary network formation. The LDI flux ratio, on day 10 in the mice-hind limb ischemia model, for the mice treated with the pJDK-CTGF and pJDK-hVEGF165 was significantly lower than that of the mice treated with the pJDK-hVEGF165 alone. CONCLUSION: The skeletal muscle ex vivo assay, using an electroporation-mediated naked DNA delivery, is a simple, quantitative and reproducible method for assessing angiogenic genes. CTGF could be an anti-angiogenic factor due to its inhibition of VEGF.