Estrogen receptor-alpha gene transfer into bovine aortic endothelial cellsinduces eNOS gene expression and inhibits cell migration

Objectives: It has been suggested that estrogen may improve endothelial cel l function to delay the onset of atherosclerosis in pre-menopausal females, though its mechanism of action is not fully understood. We examined the hy pothesis that human estrogen receptor-alpha (ER alpha) gene transfection im proves the endothelial cell function. Methods: A replication deficient aden oviral vector was used to transfect the ER alpha gene into bovine aortic en dothelial 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 apparat us; and tyrosine phosphorylation of FAK was estimated by Western blot analy sis. Results: ER alpha gene transfection of endothelial cells produced a 2- 3-fold increase in eNOS mRNA and protein levels as well as a significant in crease (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 alpha transfection. ER alpha gene transfection significan tly inhibited (P<0.05 bFGF-induced chemotactic migration of endothelial cel ls but increased cell attachment to fibronectin, laminin, and type I and IV collagens. ER alpha gene transfer also inhibited bFGF-stimulated tyrosine phosphorylation of FAK. Conclusion: Our results suggest that the atheroprot ective effects of estrogen may in part be mediated by ER alpha-induced upre gulation of eNOS gene expression and maintenance of endothelial cell functi on and integrity. (C) 1999 Elsevier Science B.V. All rights reserved.