Variations in transfection efficiency of VEGF165 and VEGF121-cDNA: its effects on proliferation and migration of human endothelial cells.
Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF121 and VEGF165 isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51-0.95 pg/mL/micrograms protein in umbilical artery, through 2.32-2.39 pg/mL/micrograms protein in coronary artery, to 5.45-7.52 pg/mL/micrograms protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 micrograms VEGF121-cDNA /in the wells)- an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.
Djurovic
Srdjan
S
Institute of Medical Genetics, University of Oslo, Norway.
Kristansen
Heidi
H
Sletten
Marit
M
Halvorsen
Kathrine
K
Berg
Kåre
K
D002455:Cell Division; D002465:Cell Movement; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A