Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells

Abstract

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted H(2)O(2)-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular H(2)O(2) levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Fig. 1.

Resveratrol (for 48 h) significantly decreases steady-state mitochondrial O₂⁻ production (A; assessed by MitoSox fluorescence) in cultured coronary arterial endothelial cells (CAECs). Overexpression of silent information regulator 2/sirtuin 1 (SIRT1) mimics the effects resveratrol. In contrast, resveratrol treatment after SIRT1 small-interfering RNA (siRNA) pretreatment fails to decrease mitochondrial reactive oxygen species generation (B). AU, arbitrary units. *P < 0.05 vs. untreated.

Fig. 2.

A: in CAECs, high glucose (30 mM) induces mitochondrial oxidative stress, as shown by the significant increases in the mean fluorescence intensity of oxidized MitoSox. Resveratrol treatment (for 48 h) significantly attenuates mitochondrial O₂⁻ production. Mannitol was used for osmotic control (n = 4 in each group). *P < 0.05 vs. baseline; #P < 0.05 vs. no resveratrol. B: representative fluorescent image showing CAECs transfected with mitochondria-targeted H₂O₂-sensitive HyPer-Mito fluorescent sensor. C: high glucose (30 mM) significantly increases HyPer-Mito fluorescence, which was prevented by resveratrol pretreatment. Resveratrol is ineffective in siRNA-treated cells that lack the ability to express SIRT1. *P < 0.05 vs. baseline; #P < 0.05 vs. no resveratrol. D: results from Amplex red/horseradish peroxidase assays. In CAECs, high glucose significantly increases H₂O₂ production, as shown by the significant increases in resorufin fluorescence. Resveratrol treatment significantly attenuates cellular H₂O₂ levels. The effect of resveratrol is blunted in cells in which SIRT1 was downregulated by siRNA. *P < 0.05 vs. untreated; #P < 0.05 vs. no resveratrol.

Fig. 3.

A: original Western blot and densitometric results show that resveratrol (RES) induces MnSOD expression in CAECs. Knockdown of SIRT1 (siRNA) prevents the effect of resveratrol, whereas SIRT1 overexpression (Overexp) substantially augments expression. Data are means ± SE. *P < 0.05; #P < 0.05 vs. untreated. B: summary data for HPLC coulometric analysis of glutathione (GSH) content in homogenates of CAECs. Resveratrol in concentration-dependent manner elicits significant increases in cellular GSH content. C: resveratrol increases cellular content of GSH vs. control, whereas knockdown of SIRT1 using siRNA blocks resveratrol-enhancing effect. By contract, overexpression of SIRT1 amplifies endogenous GSH levels. Data are means ± SE. *P < 0. 05 vs. control; #P < 0.05 vs. no resveratrol.