High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system.
BACKGROUND
Gene therapy strategies for the treatment of vascular disease such as the prevention of post-angioplasty restenosis require efficient, non-toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex.


METHODS
Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP-1.


RESULTS
The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four-fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP-1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h.


CONCLUSIONS
This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo.
Molecular Immunology Unit, Institute of Child Health, University College London, London, UK.
Richard
R
Parkes
Qing-Hai
QH
Meng
K Elena
KE
Siapati
Jean R
JR
McEwan
Stephen L
SL
Hart
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage; D004730:Endothelium, Vascular / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D013552:Swine; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D014162:Transfection / Q000592:standards*
D019715:Tissue Inhibitor of Metalloproteinase-1; D004247:DNA