Retroviral Vector-Mediated Transfer and Expression of Human Tissue Plasminogen Activator cDNA in Bovine Brain Endothelial Cells

Retroviral Vector-Mediated Transfer and Expression of Human Tissue Plasminogen Activator cDNA in... AbstractOBJECTIVESGene transfer of thrombolytic enzymes to vascular endothelial cells may influence the kinetics of intravascular thrombosis. This study defines the potential for gene transfer of tissue plasminogen activator (tPA) into bovine brain endothelial cells (BBEC).METHODSThe retroviral vectors derived from murine leukemia virus (MuLV) were 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 transduced (BBEC/tPA) cell cultures by an immunoassayRESULTSThe tPA antigen and enzymatic activity in cell culture supernatants of BBEC/tPA transduced cells were > ng/ml and 14 lU/ml after 4 days, that was 25 and 28-fold higher compared to the respective values in control cells. The PAI-1 antigen was not affected by tPA cDNA transfer. The Western blot assay of cell lysates confirmed that the majority of tPA in BBEC/tPA transduced 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 pseudotyped with vesicular stomatitis virus G glycoprotein envelope achieved high > 90% maximal transduction efficiency.CONCLUSIONSThe fibrinolytic activity of brain endothelial cells can be enhanced by transferring human tPA cDNA. These findings provide an initial step in implementation of future studies that investigate the use of this technology as an adjunctive treatment for cerebrovascular disease. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neurosurgery Oxford University Press

Retroviral Vector-Mediated Transfer and Expression of Human Tissue Plasminogen Activator cDNA in Bovine Brain Endothelial Cells

Retroviral Vector-Mediated Transfer and Expression of Human Tissue Plasminogen Activator cDNA in Bovine Brain Endothelial Cells

Retroviral Vector-Mediated Transfer and Expression of Human Tissue Plasminogen Activator cD N A in Bovine Brain Endothelial Cells Hong Yu, Ph.D., Ying Wang, B.S., Darwin Eton, M.D., Monique Stins, Ph.D., Liang Wang, M.D., Ph.D., Michael L.J. Apuzzo, M.D., Fred A. Weaver, M.D., J. Gordon McComb, M.D., Martin H. Weiss, M.D., Berislav V. Zlokovic, M.D., Ph.D. Departments of Surgery (HY, Y W , DE, FAW ) and Neurological Surgery (MS, LW, ML)A, )GM, M H W , BVZ) and Division of Neurosurgery, Children's Hospital Los Angeles (JGM, BVZ), University of Southern California, School of Medicine, Los Angeles, Caiifornia O BJEC T IV ES: Gene transfer of thrombolytic enzymes to vascular endothelial cells may influence the kinetics of intravascular thrombosis. This study defines the potential for gene transfer of tissue plasminogen activator (tPA) into bovine brain endothelial cells (BBEC ). M ETH O D S: The retroviral vectors derived from murine leukemia virus (M uLV ) were used to transfer human tPA cD N A to BBEC. The tPA activity, tPA antigen and tPA inhibitor 1 (PAI-1) antigen were determined in the supernatant of transduced (BBEC/tPA) cell cultures by an immunoassay. RESULTS: The tPA antigen and enzymatic activity in cell culture supernatants of BBEC/tPA transduced cells were > ng/ml and 14 lU/ml after 4 days, that was 25 and 28-fold higher compared to the respective values in control cells. The PAI-1 antigen was not affected by tPA cD N A transfer. The W estern blot assay of cell lysates confirmed that the majority of tPA in BBEC/tPA transduced cells was in the form of free tPA. W h ile the maximal transduction efficiency of BBEC with an amphotropic M uLV vector was about 1 5 % , a M uLV pseudotyped with vesicular stomatitis virus G glycoprotein envelope achieved high > 9 0 % maximal transduction efficiency. C O N C L U S IO N S : The fibrinolytic activity of brain endothelial cells can be enhanced by transferring human tPA cD N A . These findings provide an...
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Publisher
Oxford University Press
Copyright
© Published by Oxford University Press.
ISSN
0148-396X
eISSN
1524-4040
D.O.I.
10.1097/00006123-199910000-00072
Publisher site
See Article on Publisher Site

Abstract

AbstractOBJECTIVESGene transfer of thrombolytic enzymes to vascular endothelial cells may influence the kinetics of intravascular thrombosis. This study defines the potential for gene transfer of tissue plasminogen activator (tPA) into bovine brain endothelial cells (BBEC).METHODSThe retroviral vectors derived from murine leukemia virus (MuLV) were 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 transduced (BBEC/tPA) cell cultures by an immunoassayRESULTSThe tPA antigen and enzymatic activity in cell culture supernatants of BBEC/tPA transduced cells were > ng/ml and 14 lU/ml after 4 days, that was 25 and 28-fold higher compared to the respective values in control cells. The PAI-1 antigen was not affected by tPA cDNA transfer. The Western blot assay of cell lysates confirmed that the majority of tPA in BBEC/tPA transduced 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 pseudotyped with vesicular stomatitis virus G glycoprotein envelope achieved high > 90% maximal transduction efficiency.CONCLUSIONSThe fibrinolytic activity of brain endothelial cells can be enhanced by transferring human tPA cDNA. These findings provide an initial step in implementation of future studies that investigate the use of this technology as an adjunctive treatment for cerebrovascular disease.

Journal

NeurosurgeryOxford University Press

Published: Oct 1, 1999

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