High-level expression of recombinant human tPA in cultivated canine endothelial cells under varying conditions of retroviral gene transfer.
Successful genetic transduction of endothelial cells (EC) provides a theoretic means of increasing luminal secretion of tissue-type plasminogen activator (tPA) and lessening arterial and venous thrombotic processes. To identify the duration and number of retroviral exposures for an optimal tPA expression, enzymatically derived adult canine jugular venous EC were subjected to different exposure regimens using an amphotropic murine retroviral vector, MFG, containing the human tPA gene. Human tPA antigen secretion and its functional activity were determined at 2 days (subconfluent cells) and 14 days (confluent cells) after retroviral exposure. High-level secretion of human tPA was detected among transduced EC in all experimental groups. No secretion of human tPA occurred in control EC exposed to media alone. At 2 days after transduction, no significant differences in tPA secretion rates occurred among the different exposure regimens. At 14 days, the 12-hour X two-exposure group exhibited higher tPA secretion rates than all other exposure regimens (analysis of variance, p < 0.05). All exposure groups at 14 days exhibited significantly higher tPA secretion compared with those at 2 days (analysis of variance, p < 0.05). The presence of retroviral sequences in the genome of transduced EC was confirmed by Southern blot analysis. At 14 days, increased EC numbers were observed in vector-exposed wells compared with controls. Human tPA functional activity paralleled tPA antigen secretion. Genetically modified canine EC are capable of high levels of constitutive expression of human tPA after relatively short exposures to a retroviral vector containing the reporter gene. Increased cell number of tPA-transduced EC in culture suggests that tPA also may have other biologically important effects. These results support the efficacy of MFG-tPA gene transfer as a means of genetically modifying EC fibrinolytic activity and establishes the potential of this technology in vivo.
Podrazik
R M
RM
Department of Surgery, University of Michigan Medical Center, Ann Arbor 48109-0329.
Whitehill
T A
TA
Ekhterae
D
D
Williams
W D
WD
Messina
L M
LM
Stanley
J C
JC
D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D004285:Dogs; D004730:Endothelium, Vascular / Q000378:metabolism*; D005822:Genetic Vectors; D007150:Immunohistochemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator