Redox-sensitive up-regulation of eNOS by purple grape juice in endothelial cells: role of PI3-kinase/Akt, p38 MAPK, JNK, FoxO1 and FoxO3a.

Mahmoud Alhosin,Jong Hun Kim,Christian Bronner,Socorro Vanesca Frota Madeira,Valérie B Schini-Kerth,Eric Anselm,Sherzad Rashid,Demetrios Vavvas

doi:10.1371/journal.pone.0057883

Abstract

The vascular protective effect of grape-derived polyphenols has been attributable, in part, to their direct action on blood vessels by stimulating the endothelial formation of nitric oxide (NO). The aim of the present study was to determine whether Concord grape juice (CGJ), which contains high levels of polyphenols, stimulates the expression of endothelial NO synthase (eNOS) in porcine coronary artery endothelial cells and, if so, to determine the signaling pathway involved. CGJ dose- and time-dependently increased eNOS mRNA and protein levels and this effect is associated with an increased formation of NO in endothelial cells. The stimulatory effect of CGJ on eNOS mRNA is not associated with an increased eNOS mRNA stability and inhibited by antioxidants such as MnTMPyP, PEG-catalase, and catalase, and by wortmannin (an inhibitor of PI3-kinase), SB 203580 (an inhibitor of p38 MAPK), and SP 600125 (an inhibitor of JNK). Moreover, CGJ induced the formation of reactive oxygen species (ROS) in endothelial cells and this effect is inhibited by MnTMPyP, PEG-catalase, and catalase. The CGJ-induced the phosphorylation of p38 MAPK and JNK kinases is abolished by MnTMPyP. CGJ induced phosphorylation of transcription factors FoxO1 and FoxO3a, which regulate negatively eNOS expression, and this effect is prevented by MnTMPyP, PEG-catalase, wortmannin, SB203580 and SP600125. Moreover, chromatin immunoprecipitation assay indicated that the FoxO3a protein is associated with the eNOS promoter in control cells and that CGJ induced its dissociation. Thus, the present study indicates that CGJ up-regulates the expression of eNOS mRNA and protein leading to an increased formation of NO in endothelial cells. The stimulatory effect of CGJ is a redox-sensitive event involving PI3-kinase/Akt, p38 MAPK and JNK pathways, and the inactivation of the FoxO transcription factors, FoxO1 and FoxO3a, thereby preventing their repression of the eNOS gene.

Highlights

Several epidemiological studies have suggested that regular intake of polyphenolic rich meals including vegetables, fruits and beverages such as red wine and green tea, is associated with beneficial effects on the cardiovascular system [1,2,3]
To determine the effect of long-term treatment of endothelial cells with Concord grape juice (CGJ), endothelial NO synthase (eNOS) mRNA levels were determined as a function of concentration and time using RT-PCR As shown in Fig. 1A, treating endothelial cells with CGJ for 8 hours induced a concentration-dependent increase of the eNOS mRNA level, which reached significance at 11 mg/l
Western blot analysis was performed to verify that the increased eNOS mRNA level induced by CGJ leads to an increased eNOS protein level

Summary

Introduction

Several epidemiological studies have suggested that regular intake of polyphenolic rich meals including vegetables, fruits and beverages such as red wine and green tea, is associated with beneficial effects on the cardiovascular system [1,2,3]. Vascular protection might be due to the direct action of polyphenols on blood vessels by stimulating the formation of nitric oxide (NO), which is a potent vasodilator and inhibitor of platelet activation, in endothelial cells [3,10,11,12]. Red wine polyphenols have been shown to cause the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of eNOS at Ser 1177 and the formation of NO [10,12]. Previous studies have shown that ingestion of purple grape juice has protective effects on the vascular system by improving flowmediated vasodilatation, platelet function and platelet-dependent inflammatory responses in patients with coronary artery disease [5,13,14], and by reducing blood pressure in moderately hypertensive patients [15]. The signaling pathway leading to eNOS activation in response to grape juice is initiated by the intracellular formation of reactive oxygen species (ROS), in particular superoxide anions, which activate the Src/PI3-kinase/Akt pathway leading to the phosphorylation of eNOS at Ser 1177 [12]

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Conclusion