Resveratrol confers endothelial protection via activation of the antioxidant transcription factor Nrf2

Abstract

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. Resveratrol was also shown to confer vasoprotection in animal models of type 2 diabetes and aging. However, the mechanisms by which resveratrol exerts its antioxidative vasculoprotective effects are not completely understood. Using a nuclear factor-E(2)-related factor-2 (Nrf2)/antioxidant response element-driven luciferase reporter gene assay, we found that in cultured coronary arterial endothelial cells, resveratrol, in a dose-dependent manner, significantly increases transcriptional activity of Nrf2. Accordingly, resveratrol significantly upregulates the expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1, gamma-glutamylcysteine synthetase, and heme oxygenase-1. Resveratrol treatment also significantly attenuated high glucose (30 mM)-induced mitochondrial and cellular oxidative stress (assessed by flow cytometry using MitoSox and dihydroethidine staining). The aforementioned effects of resveratrol were significantly attenuated by the small interfering RNA downregulation of Nrf2 or the overexpression of Kelch-like erythroid cell-derived protein 1, which inactivates Nrf2. To test the effects of resveratrol in vivo, we used mice fed a high-fat diet (HFD), which exhibit increased vascular oxidative stress associated with an impaired endothelial function. In HFD-fed Nrf2(+/+) mice, resveratrol treatment attenuates oxidative stress (assessed by the Amplex red assay), improves acetylcholine-induced vasodilation, and inhibits apoptosis (assessed by measuring caspase-3 activity and DNA fragmentation) in branches of the femoral artery. In contrast, the aforementioned endothelial protective effects of resveratrol were diminished in HFD-fed Nrf2(-/-) mice. Taken together, our results indicate that resveratrol both in vitro and in vivo confers endothelial protective effects which are mediated by the activation of Nrf2.

Fig. 1.

A: reporter gene assay showing the effects of resveratrol on nuclear factor-E₂-related factor-2 (Nrf2)/antioxidant response element reporter activity in cultured primary human coronary arterial endothelial cells. Cells were transiently cotransfected with antioxidant response element-driven firefly luciferase and cytomegalovirus-driven renilla luciferase constructs followed by resveratrol (Res) treatment. Cells were then lysed and subjected to luciferase activity assay. After normalization, relative luciferase activity was obtained from 4 to 6 independent transfections. Data are means ± SE. The effect of Res was significant (P < 0.05) at each concentration used. B: effect of Res on mRNA expression of NAD(P)H:quinone-oxidoreductase 1 (Nqo1), γ-glutamylcysteine synthetase (GCLC), and heme oxygenase-1 (Hmox1) in cultured primary human coronary arterial endothelial cells. Data are means ± SE; n = 5 in each group. The effect of Res was significant (P < 0.05) at each concentration used. C: the effects of small interfering RNA (siRNA) downregulation of Nrf2 (siNrf2) or overexpression (Overexp) of Kelch-like erythroid cell-derived protein 1 (Keap-1) on Res (10 μmol/l)-induced mRNA expression of Nqo1, Gclc, and Hmox1 in cultured primary human coronary arterial endothelial cells. Data are means ± SE; n = 5 in each group. *P < 0.05 vs. control; #P < 0.05 vs. Res only.

Fig. 2.

In primary human coronary arterial endothelial cells, high glucose (HG, 30 mmol/l; A) and TNF-α (10 ng/ml; B) induce mitochondrial and cellular oxidative stress, as shown by the significant increases in the mean fluorescence intensity of oxidized MitoSox (A) and dihydroethidine (DHE; B), respectively. Res treatment significantly attenuates both HG-induced mitochondrial oxidative stress (A) and decreases TNF-α-induced cellular O₂^·− levels (B). Both of these effects are prevented by siRNA downregulation of Nrf2 and overexpression of Keap-1. Data are means ± SE; n = 6 in each group. *P < 0.05 vs. baseline; #P < 0.05 vs. no Res.

Fig. 3.

Dilation of skeletal muscle arterioles isolated from Nrf2^+/+ mice (A) and Nrf2^−/− mice (B) in response to increasing concentrations of acetylcholine. Mice were fed a standard diet (SD), high-fat diet (HFD), or HFD enriched with Res. Data are means ± SE; n = 5 in each group. C: results from Amplex red/horseradish peroxidase assay. Data are normalized resorufin fluorescences, representing ROS production by segments of the femoral arteries of Nrf2^+/+ mice and Nrf2^−/− mice, fed a SD, HFD or HFD enriched with Res. AU, arbitrary units. *P < 0.05 vs. untreated; #P < 0.05 vs. no Res.

Fig. 4.

A: in primary human coronary arterial endothelial cells, HG significantly (P < 0. 05 vs. control) increased apoptotic cell death as shown by the increased terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) positivity (flow cytometry). Treatment with Res prevented HG-induced endothelial apoptosis (#P < 0. 05 vs. HG only). By contrast, in endothelial cells with siNrf2, Res failed to inhibit HG-induced endothelial apoptosis. Data are means ± SE; n = 6 for each group. B: DNA fragmentation in femoral arterial branches of Nrf2^+/+ mice and Nrf2^−/− mice fed a SD, HFD, or HFD enriched with Res. Data are means ± SE; n = 6 for each group. *P < 0.05 vs. untreated; #P < 0.05 vs. no Res.