[Therapeutic angiogenesis using genetic transfection. An in vitro quantitative and functional study after gene code...

Vascular endothelial growth factor (VEGF) gene transfer is a new therapeutic approach in clinical situations where insufficient angiogenesis may impair processes that require vessel formation, such as myocardial ischemia or peripheral vascular disease. The feasibility of this strategy was addressed in vitro by (i) optimisation of DNA transfection into cultured cells using a cationic liposome, (ii) study of the transgene production and it's angiogenic properties. An expression plasmid encoding VEGF165 was used to transfect endothelial cells (EAhy-926) in vitro. Different amounts of the cationic liposome were complexed to the plasmid DNA in order to achieve increasing ratios (1/1, 2/1, 3/1, 4/1, 5/1). Maximal gene expression was obtained when the cationic liposome was mixed to plasmid DNA in a 3/1 ratio. Using this optimised liposome/plasmid DNA formulation, VEGF expression measured by ELISA, reached a maximum of 60 ng/mL 24 hours after gene transfer then rapidly decreased. Conditioned media from transfected cells (CMVEGF) significantly increased the proliferation rate of endothelial cells. The maximal mitogenic effect was observed when the cell media was supplemented with 25% of CMVEGF, corresponding to 10 ng/mL of recombinant VEGF. Addition of the VEGF165 peptide into the cell media showed a similar mitogenic effect. The angiogenic property of the transgene was assessed by the demonstration of its ability to stimulate capillary tubes formation in a tridimensional biologic gel.