Resveratrol induces mitochondrial biogenesis in endothelial cells

Abstract

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Fig. 1.

When compared with untreated controls (A), resveratrol (Res; B; 10 μM for 48 h) significantly increased the number of mitochondria in cultured coronary arterial endothelial cells (CAECs). Relative mitochondrial mass was estimated by using Mitotracker red staining. The cell body was visualized by differential interference contrast and by green calcein fluorescence (blue: nuclei). Only cells with intact calcium staining were analyzed. Summary data for mitochondrial surface area are shown in C. D: Mitotracker Fluorescent intensities in CAECs were measured to assess mitochondrial biogenesis. Knockdown of sirtuin 1 (SIRT1) [small interfering RNA (siRNA)] or N^ω-nitro-l-arginine methyl ester (l-NAME) prevented resveratrol-induced mitochondrial biogenesis. E: resveratrol increased mitochondrial DNA (mtDNA) content in CAECs, which was prevented by l-NAME or SIRT1 siRNA. Data are means ± SE. *P < 0.05; #P < 0.05 vs. resveratrol only. au, Arbitrary units.

Fig. 2.

Treatment of CAECs with resveratrol (Res; 10 μmol/l) upregulated SIRT1 protein expression (A; Western blotting) and activity (B; data from fluorimetric SIRT1 activity assay). C: time course for siRNA knockdown of SIRT1 in CAECs (Western blotting). OD, optical density; AFU, arbitrary fluorescence units; c, control; sc, scrambled siRNA control. *P < 0.05 vs. control.

Fig. 3.

A: effect of resveratrol on mRNA expression of endothelial nitric oxide synthase (eNOS) in CAECs. Knockdown of SIRT1 (siRNA) prevented resveratrol-induced upregulation of eNOS. *P < 0.05 vs. control; #P < 0.05 vs. resveratrol only. B: representative Western blot showing that in vitro resveratrol treatment elicited a concentration-dependent induction of eNOS protein. *P < 0.05. Bar graphs are densitometric data. C–F: effect of resveratrol on mtDNA copy number (C) and mRNA expression of proliferator-activated receptor-coactivator-1α (PGC-1α; D), nuclear respiratory factor-1 (Nrf1; E), and mitochondrial transcription factor A (Tfam; F) in cultured aortic segments of eNOS^−/− mice (n = 5; differences are not significant).

Fig. 4.

In CAECs resveratrol (Res; 10 μM for 48 h) significantly upregulated protein expression of porin (A) and complexes I (B), II (C), III (D), IV (E), and V (F; Western blotting). l-NAME prevented resveratrol-induced mitochondrial biogenesis. Data are means ± SE. *P < 0.05 vs. untreated; #P < 0.05 vs. resveratrol only.

Fig. 5.

Effect of resveratrol (Res) on mRNA expression of PGC-1α (A), Nrf-1 (B), and Tfam (C) in CAECs. Knockdown of SIRT1 (siRNA) prevented resveratrol-induced upregulation of mitochondrial biogenesis factors. *P < 0.05 vs. control; #P < 0.05 vs. resveratrol only; n = 5 in each group.

Fig. 6.

In aortas of type 2 diabetic db/db mice, relative mtDNA content (A) was significantly decreased compared with vessels of control mice. In aortas of db/db mice, expression of PGC-1α and Nrf-1 was downregulated (B). Resveratrol (Res) treatment significantly increased vascular mtDNA content (A) and induced the expression of mitochondrial biogenesis factors (B and C) in aortas of db/db mice, eliminating the difference between the 2 groups. Data are means ± SE. *P < 0.05 vs. no resveratrol; #P < 0.05 vs. control mice. wt, Wild-type.