Epigallocatechin Gallate (EGCG), a Green Tea Polyphenol, Reduces Expression of Endothelin-1 (ET-1) in Vascular Endothelial Cells by a FOXO-1-Dependent Mechanism.

Consumption of green tea is associated with decreased cardiovascular morbidity and mortality. Tissue and circulating levels of the potent vasoconstrictor ET-1 are elevated in patients with diabetes. We hypothesized that EGCG, a bioactive polyphenol in green tea, may reduce expression of ET-1 in vascular endothelial cells. In bovine aortic endothelial cells (BAEC) in primary culture, acute treatment of cells with EGCG increased phosphorylation of Akt (Ser[sup 473]) and FOXO-1 (Akt phosphorylation site Thr[sup 24]) in a time- and dose-dependent manner (maximal increases at 30 min and 50 µM as determined by immunoblotting with phospho-specific antibodies). Chronic EGCG treatment (8 h, l0 µM) also caused phosphorylation of Akt and FOXO-1 (immunoblotting) with an increase in the amount of FOXO-1 localized to the cytoplasm (immunohistochemistry). Treatment of BAEC with EGCG (8 h, l0 µM) reduced ET-1 mRNA by 60% (quantitative real time PCR, p < 0.001). Moreover, ET-l protein secreted into conditioned media was also reduced 70% (ELISA, p < 0.0001) and intracellular ET-1 protein levels were reduced 65% (ELISA, p <0.005). Treatment of human aortic endothelial cells (HAEC) with EGCG (50 µM, 1 h) reduced ET- 1 promoter activity (assessed by chromatin immunoprecipitation with FOXO-1 antibodies; p < 0.05). We examined ET-l promoter activity by transfecting BAEC with a luciferase reporter containing the human ET-1 promoter. Co-transfection of BAEC with the reporter and human FOXO-1 increased ET-1 promoter activity 2.5-fold when compared with cells co-transfected with reporter and empty vector (p < 0.05). When we truncated or mutated a putative FOXO-1 binding sequence in our reporter (TGTTTAC to TGTcTAC), co-transfection of BAEC with these mutants and either wild-type or constitutively active FOXO-1 did not increase luciferase activity. Finally, results from EMSA assays demonstrate that recombinant FOXO-1 binds to the human ET-1 promoter. Thus, our data suggest that EGCG reduces ET-1 expression and secretion in endothelial cells using a mechanism involving phosphorylation of FOXO-1 and dissociation of FOXO-1 from the ET-1 promoter. We conclude that the ET-1 promoter is a novel target for FOXO-1 and that beneficial effects of green tea consumption on cardiovascular health may be mediated, in part, by actions of EGCG to reduce ET-1 expression and secretion. [ABSTRACT FROM AUTHOR]