A novel ex vivo angiogenesis assay based on electroporation-mediated delivery of naked plasmid DNA to skeletal muscle.
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 the tibialis anterior muscle of BALB/c mice, followed by in vivo electroporation 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 the CTGF gene alone induced weak sprouting activity, it appeared to inhibit the angiogenic activity of the VEGF165 gene during cotreatment. This attenuating activity of CTGF on VEGF was reproduced in vivo in 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 novel ex vivo assay 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.
Cardiac and Vascular Center, Department of Medicine, and Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-ku, Seoul 135-710, South Korea.
Hyung-Suk
HS
Jang
Hyun-Joong
HJ
Kim
Jeong-Min
JM
Kim
Young-Sam
YS
Lee
Koung Li
KL
Kim
Jeong-A
JA
Kim
Jae-Young
JY
Lee
Wonhee
W
Suh
Jin-Ho
JH
Choi
Eun-Seok
ES
Jeon
Jonghoe
J
Byun
Duk-Kyung
DK
Kim
D000818:Animals; D002196:Capillaries / Q000378:metabolism; D002465:Cell Movement; D003094:Collagen / Q000378:metabolism / Q000494:pharmacology; D055513:Connective Tissue Growth Factor; D003470:Culture Media / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004338:Drug Combinations; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D017874:Immediate-Early Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007511:Ischemia; D007797:Laminin / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000378:metabolism*; D009389:Neovascularization, Pathologic; D010957:Plasmids / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
C526318:CCN2 protein, mouse; D003470:Culture Media; D018076:DNA, Complementary; D004338:Drug Combinations; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D011509:Proteoglycans; D042461:Vascular Endothelial Growth Factor A; C059485:matrigel; D055513:Connective Tissue Growth Factor; D003094:Collagen; D004247:DNA