Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer.
In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t-PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.
Dichek
D A
DA
Molecular Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Nussbaum
O
O
Degen
S J
SJ
Anderson
W F
WF
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D003470:Culture Media / Q000032:analysis; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D005342:Fibrinolysis / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics*; D015849:Plasminogen Inactivators / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D012756:Sheep / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection / Q000235:genetics / Q000502:physiology*
D003470:Culture Media; D004279:DNA, Viral; D015849:Plasminogen Inactivators; D010959:Tissue Plasminogen Activator