Retroviral vector-mediated transfer and expression of human tissue plasminogen activator cDNA in bovine brain endothelial cells

OBJECTIVES: Gene transfer of thrombolytic enzymes to vascular endothelial c ells may influence the kinetics of intravascular thrombosis. This study def ines the potential for gene transfer of tissue plasminogen activator (tPA) into bovine brain endothelial cells (BBEC). METHODS: The retroviral vectors derived from murine leukemia virus (MuLV) w ere used to transfer human tPA cDNA to BBEC. The tPA activity, tPA antigen and tPA inhibitor 1 (PAI-1) antigen were determined in the supernatant of t ransduced (BBEC/tPA) cell cultures by an immunoassay. RESULTS: The tPA antigen and enzymatic activity in cell culture supernatant s of BBEC/tPA transduced cells were 75 ng/ml and 14 IU/ml after 4 days, tha t was 25 and 28-fold higher compared to the respective values in control ce lls. The PAI-1 antigen was not affected by tPA cDNA transfer. The Western b lot assay of cell lysates confirmed that the majority of tPA in BBEC/tPA tr ansduced cells was in the form of free tPA. While the maximal transduction efficiency of BBEC with an amphotropic MuLV vector was about 15%, a MuLV ps eudotyped with vesicular stomatitis virus G glycoprotein envelope achieved high >90% maximal transduction efficiency. CONCLUSIONS: The fibrinolytic activity of brain endothelial cells can be en hanced by transferring human tPA cDNA. These findings provide an initial st ep in implementation of future studies that investigate the use of this tec hnology as an adjunctive treatment for cerebrovascular disease.