Adenoviral-Mediated Transfer of Tissue Plasminogen Activator Gene into Brain Capillary Endothelial Cells In Vitro

Abstract

Tissue plasminogen activator (tPA) has a critical role in fibrinolysis, converting plasminogen into active protease plasmin. Because intravenous tPA has only limited effectiveness as acute stroke therapy, enhancement of endogenous tPA represents a potential alternative to stroke treatment. Adenoviral-mediated gene transfer was used to enhance production of tPA in bovine brain capillary endothelial cells (BEC). Antigen and activity levels of tPA and plasminogen activator inhibitor-1 (PAI-1) in media from BEC infected with AdCMVtPA were analyzed. Conditioned media were analyzed for thrombomodulin, the integral membrane antithrombotic molecule that co-activates protein C. BEC infected with AdCMVtPA demonstrated enhanced expression of tPA antigen (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], respectively) compared to BEC infected with the control adenovirus (AdI327) or uninfected BEC. There was a moderate increase in PAI-1 protein 4 days after transfection with AdCMVtPA, and the integral membrane protein throm bomodulin was released into media by transfected BEC. These results demonstrate that aden oviral-mediated delivery in vitro of the human tPA gene resulted in high levels of expression of tPA in BEC. Transient overexpression of tPA by gene transfer might be a useful strategy to protect against thrombotic occlusion during the period of risk of acute stroke.