Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells.
The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 isoforms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, beta-galactosidase gene, was about 50% in COS-7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121, and pSG5-VEGF165 plasmids can be used for therapeutic application.
Dulak
J
J
Department of Clinical Biochemistry, Jagiellonian University, Medical Faculty, Kraków, Poland.
Józkowicz
A
A
Ratajska
A
A
Szuba
A
A
Cooke
J P
JP
Dembińska-Kieć
A
A
D000818:Animals; D019556:COS Cells; D002411:Cation Exchange Resins / Q000378:metabolism; D002455:Cell Division; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D050356:Lipid Metabolism; D008055:Lipids; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D002411:Cation Exchange Resins; D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors; D008055:Lipids; C086724:Lipofectamine; D008222:Lymphokines; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D001616:beta-Galactosidase