Inhibition of vascular endothelial growth factor activity by transfection with the soluble FLT-1 gene.
Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-1 (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.
Chen
H
H
Department of Cardiology, Jichi Medical School, Tochigi, Japan.
Ikeda
U
U
Shimpo
M
M
Maeda
Y
Y
Shibuya
M
M
Ozawa
K
K
Shimada
K
K
D015153:Blotting, Western; D002460:Cell Line; D017077:Culture Media, Conditioned; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D017978:Receptors, Growth Factor / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D004247:DNA; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1