D Ekhterae1, J C Stanley. 1. Jobst Research Laboratories, Department of Surgery, Ann Arbor, MI, USA.
Abstract
PURPOSE: Enhancement of the fibrinolytic activity of vascular cells by tissue plasminogen activator (tPA) gene transfer has considerable clinical potential. However, it is unknown whether greater constitutive expression of the tPA gene might increase plasminogen activator inhibitor-1 (PAI-1) secretion, which could negate expected increases in fibrinolytic activity that accompany greater tPA protein production. The objective of this investigation was to determine whether transduction of human endothelial cells (EC) and vascular smooth muscle cells (SMC) with a retroviral vector containing the human tPA gene would increase tPA production and what effect this would have on endogenous PAI-1 secretion and subsequent fibrinolytic activity. METHODS: Cultivated human EC and SMC either were transduced with a murine leukemia retroviral vector (MFG) containing the human tPA gene and, in the case of controls, the lacZ gene, or they were exposed to media alone. On days 14 and 28 after transduction, supernatent tPA antigen and PAI-1 antigen levels were measured by ELISA, and supernatent tPA activity was quantitated with a spectrolyse tPA/PAI assay. RESULTS: Southern and Northern blot analyses documented integration and transcription of the tPA gene in both EC and SMC. Greater tPA antigen production occurred in MFG-tPA-transduced EC and SMC compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). The tPA activity increased in transduced human saphenous vein EC (up to 5.1-fold) and human iliac artery EC (up to 4.7-fold), but no increased tPA activity occurred in transduced SMC, compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). PAI-1 antigen was unchanged in transduced SMC but decreased in MFG-tPA-transduced EC (p < 0.05). PAI-1 mRNA was unchanged in the transduced EC and SMC compared with nontransduced cells, suggesting that posttranslational events may have caused the changes in EC PAI-1. CONCLUSIONS: This investigation demonstrated that MFG-mediated tPA gene transfer into human EC resulted in a significant increase in tPA activity. Enhancement of adult human EC fibrinolytic activity by transfer of the human tPA gene has not been previously reported and represents a necessary finding in the development of this gene therapy technology for the prevention of thrombotic complications of vascular disease.
PURPOSE: Enhancement of the fibrinolytic activity of vascular cells by tissue plasminogen activator (tPA) gene transfer has considerable clinical potential. However, it is unknown whether greater constitutive expression of the tPA gene might increase plasminogen activator inhibitor-1 (PAI-1) secretion, which could negate expected increases in fibrinolytic activity that accompany greater tPA protein production. The objective of this investigation was to determine whether transduction of human endothelial cells (EC) and vascular smooth muscle cells (SMC) with a retroviral vector containing the humantPA gene would increase tPA production and what effect this would have on endogenous PAI-1 secretion and subsequent fibrinolytic activity. METHODS: Cultivated human EC and SMC either were transduced with a murineleukemia retroviral vector (MFG) containing the humantPA gene and, in the case of controls, the lacZ gene, or they were exposed to media alone. On days 14 and 28 after transduction, supernatent tPA antigen and PAI-1 antigen levels were measured by ELISA, and supernatent tPA activity was quantitated with a spectrolyse tPA/PAI assay. RESULTS: Southern and Northern blot analyses documented integration and transcription of the tPA gene in both EC and SMC. Greater tPA antigen production occurred in MFG-tPA-transduced EC and SMC compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). The tPA activity increased in transduced human saphenous vein EC (up to 5.1-fold) and human iliac artery EC (up to 4.7-fold), but no increased tPA activity occurred in transduced SMC, compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). PAI-1 antigen was unchanged in transduced SMC but decreased in MFG-tPA-transduced EC (p < 0.05). PAI-1 mRNA was unchanged in the transduced EC and SMC compared with nontransduced cells, suggesting that posttranslational events may have caused the changes in EC PAI-1. CONCLUSIONS: This investigation demonstrated that MFG-mediated tPA gene transfer into human EC resulted in a significant increase in tPA activity. Enhancement of adult human EC fibrinolytic activity by transfer of the humantPA gene has not been previously reported and represents a necessary finding in the development of this gene therapy technology for the prevention of thrombotic complications of vascular disease.