© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Estrogen receptor-
gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration
Department of Anatomy and Cell Biology, and The Cardiovascular Center, The University of Iowa College of Medicine, Iowa City, IA 52242, USA
* Corresponding author. Tel.: +1-319-335-7718; fax: +1-319-335-71980 ramesh-bhalla{at}uiowa.edu
Objectives: It has been suggested that estrogen may improve endothelial cell function to delay the onset of atherosclerosis in pre-menopausal females, though its mechanism of action is not fully understood. We examined the hypothesis that human estrogen receptor-
(ER) gene transfection improves the endothelial cell function. Methods: A replication deficient adenoviral vector was used to transfect the ER gene into bovine aortic endothelial cells (BAEC) and a GFP gene containing vector was used as control. Expression of the eNOS gene was determined by Northern blot analysis and enzyme activity assay; cell migration was assayed using a Transwell apparatus; and tyrosine phosphorylation of FAK was estimated by Western blot analysis. Results: ER gene transfection of endothelial cells produced a 2–3-fold increase in eNOS mRNA and protein levels as well as a significant increase (P<0.05) in NOS activity as measured by citrulline assay and nitrite accumulation in the media in response to bradykinin stimulation. Treatment of cells with estrogen blocking agent ICI 182780 inhibited eNOS induction in response to ER transfection. ER gene transfection significantly inhibited (P<0.05) bFGF-induced chemotactic migration of endothelial cells but increased cell attachment to fibronectin, laminin, and type I and IV collagens. ER gene transfer also inhibited bFGF-stimulated tyrosine phosphorylation of FAK. Conclusion: Our results suggest that the atheroprotective effects of estrogen may in part be mediated by ER-induced upregulation of eNOS gene expression and maintenance of endothelial cell function and integrity.
KEYWORDS Ad5/RSV, Replication deficient adenovirus sero type 5 construct containing Rous Sarcoma Virus promoter sequence; BAEC, bovine aortic endothelial cells; eNOS, endothelial nitric oxide synthase; FAK, focal adhesion kinase; GFP, green fluorescent protein; ER, human estrogen receptor ; LNA, nitro L-arginine; MEM, minimal essential medium; NO, nitric oxide; PBS, phosphate buffered saline
1 Present address: Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Thorn 1319, 75 Francis St, MA, 02115, USA.
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