A Simple, Quantitative Method for Assessing Angiogenic Genes Using Skeletal Muscle by Electroporation-Mediated Naked DNA Delivery.
10.4070/kcj.2003.33.4.321
- Author:
Hyun Joong KIM
1
;
Hyung Suk JANG
;
Jeong Min KIM
;
Yong Sam LEE
;
Kyung Ree KIM
;
Jeong A KIM
;
Eun Seok JEON
;
Jong Hoe BYUN
;
Duk Kyung KIM
Author Information
1. Department of Medicine, Sungkyunkwan University School of Medicine, Seoul, Korea.
- Publication Type:Original Article
- Keywords:
Angiogenesis assay;
Skeletal muscle;
Electroporation;
Vascular endothelial growth factor (VEGF);
Connective tissue growth factor (CTGF)
- MeSH:
Animals;
Arteries;
Capillaries;
Cats;
Cell Line;
Connective Tissue Growth Factor;
DNA*;
Electroporation;
Endothelial Cells;
Extremities;
Genetic Therapy;
Ischemia;
Mice;
Muscle, Skeletal*;
Muscles;
Vascular Endothelial Growth Factor A
- From:Korean Circulation Journal
2003;33(4):321-332
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
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.
