Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells.
OBJECTIVE
The efficient introduction of regulatory genes into vascular smooth muscle cells (SMCs) is one of the most promising options for gene therapy of cardiovascular diseases. Cationic liposome-mediated gene transfer may become a favorable transfection technique with regard to patient's safety for in vivo administration. However, this method until now has its limitation in a low transfection efficiency. Therefore, the present study was designed to improve cationic liposome-mediated transfection of rabbit vascular SMCs in vitro and in vivo, in order to enhance transfection efficiency and present an optimized system which may offer a potential therapeutic benefit for in vivo application.


RESULTS
Optimized lipofection of rabbit SMCs with the mammalian expression vector pE-N1 and the reporter gene green fluorescent protein resulted in a mean transfection efficiency of about 50%. The unique transfection of rabbit SMCs in vitro and in vivo with the inducible isoform of human nitric oxide synthase (NOSII), using the same vector, resulted in a successful transient transcription and translation of a functionally active human NOSII in rabbit SMC, persisting 5-6 days. We could further demonstrate that the transfection procedure and the transgene product did neither induce necrosis nor apoptosis under the conditions chosen and did not result in the induction of endogenous NOSII of transfected SMCs.


CONCLUSIONS
These findings indicate potential therapeutic relevance for this nonviral gene transfer system for in vivo gene therapy for cardiovascular diseases.
Veit
K
K
Department of Medicine II, Johannes Gutenberg-University, Mainz, Germany.
Boissel
J P
JP
Buerke
M
M
Grosser
T
T
Meyer
J
J
Darius
H
H
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002448:Cell Adhesion; D002455:Cell Division; D003327:Coronary Disease / Q000201:enzymology / Q000628:therapy*; D046508:Culture Techniques; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D008070:Lipopolysaccharides; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008070:Lipopolysaccharides; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II