Urokinase expression in transduced endothelial cells

Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cell...

Full description

Saved in:

Bibliographic Details
Published in:	The American surgeon Vol. 62; no. 8; p. 619
Main Authors:	Townsend, L E, Glover, J L, Trevor, K T, Ellmers, B R, Bendick, P J, Ordona, M
Format:	Journal Article
Language:	English
Published:	United States 01-08-1996
Subjects:	Animals Cells, Cultured Culture Media, Conditioned Dogs Endothelium, Vascular - metabolism Endothelium, Vascular - pathology Gene Expression Regulation, Enzymologic Genetic Vectors Humans Immunohistochemistry Mice Retroviridae - genetics Spectrophotometry Transfection Urokinase-Type Plasminogen Activator - analysis Urokinase-Type Plasminogen Activator - biosynthesis Urokinase-Type Plasminogen Activator - genetics
Online Access:	Get more information
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cells were enzymatically harvested from canine external jugular vein in nine animals and grown to confluence in culture. One-third of these cells served as controls, and the remaining two-thirds were transduced via incubation with an LXSN-type retroviral vector carrying the urokinase gene and a neomycin resistance gene. Successfully transduced cells were selected by incubation with 400 micrograms/mL G418 and pure cultures grown to confluence. Supernatants from confluent control and experimental cell cultures after 48 hours in defined, serum-free medium were assayed for human urokinase concentration and overall enzyme activity. ELISA quantitation of concentration using mouse antihuman urokinase antibody showed 0.15 +/- 0.11 ng/mL/hr/10(6) cells in the transduced cell supernatant; no measurable concentration was found in the control cells. (P < 0.01) Overall (human plus canine) enzyme activity of urokinase was determined using an indirect spectrophotometric assay based on plasminogen activation (ploug U/mL). Transduced cells showed activities of 0.12 at 10 days and 0.45 at confluence; control cell activity was 0.0 and 0.15, respectively. (P < 0.05) These data show that endothelial cells can be transduced with a urokinase expressing gene that increases the release of this thrombolytic agent. Lining small diameter prosthetic grafts with these cells may improve their thromboresistance and long-term patency.
ISSN:	0003-1348