Adenoviral-mediated transfer of tissue plasminogen activator gene into brain capillary endothelial cells in vitro

Tissue plasminogen activator (tPA) has a critical role in fibrinolysis, con verting plasminogen into active protease plasmin. Because intravenous tPA h as only limited effectiveness as acute stroke therapy, enhancement of endog enous tPA represents a potential alternative to stroke treatment. Adenovira l-mediated gene transfer was used to enhance production of tPA in bovine br ain capillary endothelial cells (BEC). Antigen and activity levels of tPA a nd plasminogen activator inhibitor-1 (PAI-1) in media from BEC infected wit h AdCMVtPA were analyzed. Conditioned media were analyzed for thrombomoduli n, the integral membrane antithrombotic molecule that co-activates protein C. BEC infected with AdCMVtPA demonstrated enhanced expression of tPA antig en (40.2 +/-0.4 ng/mL vs 1.1 +/-1.5 ng/mL [p <0.001] and 0.3 +/-0.5 ng/mL [ p <0.0001], respectively) and increased tPA enzymatic activity (27.4 +/-5.7 IU/mL vs 8.3 +/-1.7 IU/mL [p <0.05] and 13.3 +/-3.2 IU/mL [p <0.05], respe ctively) compared to BEC infected with the control adenovirus (Ad/327) or u ninfected BEC. There was a moderate increase in PAI-1 protein 4 days after transfection with AdCMVtPA, and the integral membrane protein thrombomoduli n was released into media by transfected BEC. These results demonstrate tha t adenoviral-mediated delivery in vitro of the human tPA gene resulted in h igh levels of expression of tPA in BEC. Transient overexpression of tPA by gene transfer might be a useful strategy to protect against thrombotic occl usion during the period of risk of acute stroke.