Korean Circ J.  2003 Apr;33(4):321-332. 10.4070/kcj.2003.33.4.321.

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

• Kim HJ
• Jang HS
• Kim JM
• Lee YS
• Kim KR
• Kim JA
• Jeon ES
• Byun JH
• Kim DK

• KMID: 2093869
• DOI: http://doi.org/10.4070/kcj.2003.33.4.321

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.

Keyword

Angiogenesis assay; Skeletal muscle; Electroporation; Vascular endothelial growth factor (VEGF); Connective tissue growth factor (CTGF)

MeSH Terms

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
Connective Tissue Growth Factor
DNA
Vascular Endothelial Growth Factor A