Adenovirus-mediated transfer of tissue-type plasminogen activator gene to human endothelial cells.
BACKGROUND
Seeding of vascular grafts with genetically engineered human endothelial cells (hECs) secreting antithrombogenic or fibrinolytic agents has considerable clinical potential.


METHODS
An adenoviral vector was used to transfer the human tissue-type plasminogen activator (htPA) gene to hECs, and the ability of the transduced hECs to secrete htPA was examined. Cultured hECs on plates were incubated with various concentrations of recombinant adenoviruses containing the htPA or LacZ gene for various times to determine the optimal transfer conditions. Transduced hECs were seeded onto fibronectin-coated expanded polytetrafluoroethylene grafts (4 mm in diameter), some of which were exposed to pulsatile flow in vitro.


RESULTS
Effective transduction of the htPA gene into hECs (htPAhECs) was achieved with viral soup at a multiplicity of infection of 30 after incubation for 1 day, which yielded 4.8 +/- 0.20 x 10(3) ng/10(6) cells/6 hr htPA antigen on plates (n = 3), 2.2 +/- 2.0 x 10(3) ng/10(6) cells/6 hr on grafts (n = 6), and 6.8 +/- 1.7 x 10(2) ng/10(6) cells/6 hr on perfused grafts (n = 6). The retention of htPAhECs by perfused grafts was 84.0% +/- 3.0%, comparable with the noninfected (82.1% +/- 8.0%) and mock-infected (94.2% +/- 0.4%) hEC values.


CONCLUSIONS
By adenoviral vector-mediated gene transfer, 10(2-3)-fold enhancement of htPA secretion was demonstrated, which did not affect cell retention by grafts.
Sugawara
Y
Y
Second Department of Surgery, Faculty of Medicine, University of Tokyo, Japan.
Sakata
Y
Y
Minowada
S
S
Hamada
H
H
Yoshida
Y
Y
Sato
O
O
Deguchi
J
J
Kimura
H
H
Namba
T
T
Makuuchi
M
M
Miyata
T
T
D000256:Adenoviridae / Q000235:genetics*; D000328:Adult; D001483:Base Sequence; D002452:Cell Count; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D005337:Fibrin / Q000032:analysis; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques / Q000592:standards; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D011138:Polytetrafluoroethylene; D019736:Prostheses and Implants; D011673:Pulsatile Flow; D012501:Saphenous Vein / Q000166:cytology; D012967:Sodium Dodecyl Sulfate; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D012967:Sodium Dodecyl Sulfate; D005337:Fibrin; D011138:Polytetrafluoroethylene; D010959:Tissue Plasminogen Activator