Resveratrol blocks Akt activation in angiotensin II- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner

Abstract

Aims: Resveratrol (RV), an antioxidant, inhibits angiotensin II (Ang II)-induced hypertrophy and Ang II- or epidermal growth factor (EGF)-induced Akt phosphorylation in rat vascular smooth muscle cells (VSMCs). Both signalling pathways are reported to utilize reactive oxygen species (ROS). The aim of this study was to show whether RV reduces the ROS level in Ang II- or EGF-activated VSMCs and whether reduction of ROS causes the impeded signalling towards Akt in the presence of RV.

Methods and results: We show here that RV reduces intracellular ROS and extracellular H₂O₂ release from VSMCs as measured using 2',7'-dichlorodihydrofluorescein-diacetate and Amplex Red™. Since NADPH oxidases (Nox) 1 and 4 are major ROS sources in VSMCs, we examined their need for Akt phosphorylation in response to Ang II or EGF. Experiments using the blocking peptide gp91ds-tat verified a role for Nox1 in Ang II signalling towards Akt, but excluded a role for Nox1 in the respective EGF signalling. A small interfering RNA-mediated knock-down of Nox4 showed that Nox4 was not required for Ang II- or EGF-induced Akt phosphorylation. Use of the flavoprotein inhibitor diphenyleneiodonium, N-acetyl-cysteine, and non-antioxidant RV derivatives revealed that the antioxidant capacity of RV is not required for the inhibition of Akt phosphorylation, in both rat and human VSMCs.

Conclusion: Thus, although RV acts as an antioxidant, the antihypertrophic response of RV in VSMCs and the signalling downstream of the EGF receptor towards Akt seem to be largely redox independent.

Figure 1

Resveratrol (RV) diminishes intracellular reactive oxygen species (ROS) level and attenuates both basal and angiotensin II (Ang II)- or epidermal growth factor (EGF)-induced extracellular H₂O₂. Quiescent vascular smooth muscle cells (VSMCs) were pre-incubated with dichlorodihydrofluorescein-diacetate (H₂DCF-DA; 20 µM, 15 min) and then incubated with 50 µM RV or vehicle control for 30 min or 10 µM diphenyleneiodonium (DPI) for 1 h and finally stimulated with 100 nM Ang II for 15 min (A) or 100 ng/mL EGF for 10 min (B). ROS production (equivalent to the amount of formed DCF) was analysed by flow cytometry. The average fluorescence signal obtained in the presence of Ang II or EGF was set to 100% (**P < 0.01; one-way ANOVA vs. Ang II or EGF treatment, n = 3). Serum-starved VSMCs were stimulated in Amplex Red™ buffer with Ang II (100 nM; C) or EGF (100 ng/mL; D) for 5–15 min after pre-incubation with RV (50 µM) or vehicle control for 30 min. H₂O₂ production was quantified by fluorescence measurement. Absolute values were correlated to catalase-treated cells used as a negative control. Graphs show the relative extracellular H₂O₂ production expressed as a percentage of vehicle-treated control cells (open square, vehicle-treated cells; filled squares, RV-treated cells; ***P < 0.001; t-test, n = 4).

Figure 2

Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering RNA (siRNA) against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs (A and B) show mean + SEM of Nox4 mRNA (A) or Nox1 mRNA (B) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (***P < 0.001; n = 3). Serum-deprived Nox4 siRNA-treated cells (50 nM, 72 h) were pre-treated with RV (50 µM, 30 min) or vehicle control followed by stimulation with 100 nM Ang II for 10 min (C) or 100 ng/mL EGF for 5 min (D). Akt phosphorylation and tubulin (tub, serving as a loading control) were detected by western blot analysis. One representative blot out of three, which were densitometrically analysed, is shown. The graphs represent the mean densitometric values + SEM of three independent experiments. Akt phosphorylation in response to Ang II or EGF of scrambled control was set to 100% (*P < 0.05, **P < 0.01; one-way ANOVA, n = 3).

Figure 3

Inhibition of Nox1 blunts phosphorylation of Akt and p38 induced by Ang II but does not alter EGF-induced Akt and p38 phosphorylation. Quiescent VSMCs were pre-treated with 100 µM of the gp91ds-tat blocking peptide (gp91ds), 50 µM RV or vehicle control for 30 min, and were then stimulated with 100 nM Ang II for 10 min (A and B) or 100 ng/mL EGF (C and D) for 5 min. Akt (A and C) and p38 phosphorylation (B and D) as well as tubulin (tub, as loading control) were detected by western blot analysis. One representative blot out of three, which were densitometrically analysed, is shown. The graphs depict the mean densitometric values + SEM of three independent experiments. Signals obtained in response to Ang II or EGF were set to 100% (*P < 0.05, **P < 0.01; n.s., not significant; one-way ANOVA vs. Ang II or EGF treatment, n = 3).

Figure 4

DPI and NAC inhibit Ang II-induced phosphorylation of Akt and p38. Serum-deprived cells were pre-treated with 50 µM RV or vehicle control for 30 min, 10 µM DPI for 1 h or 10 mM NAC for 2 h, and then stimulated with 100 nM Ang II for 10 min (A and B), or 100 ng/mL EGF for 5 min (C and D). Lysates were subjected to western blot analysis using antibodies against pAkt (A and C), pp38 (B and D) and tubulin (tub). Representative blots out of three are shown. The graphs depict the mean + SEM of the densitometric analyses. Signals obtained in response to Ang II or EGF were set to 100% (*P < 0.05, **P < 0.01; n.s., not significant; one-way ANOVA vs. Ang II or EGF treatment, n = 3).

Figure 5

Non-antioxidant derivatives of RV, trans-3,5,4′-trimethoxystilbene (RV-3M; A), and trans-3,5-dihydroxy-4′-methoxystilbene (RV-1M; B).

Figure 6

Non-antioxidant trans-3,5-dihydroxy-4′-methoxystilbene (RV-1M) inhibits phosphorylation of Akt after Ang II and EGF stimulation. (A) Starved VSMCs were stimulated in Amplex Red™ buffer with Ang II (100 nM) for 10 min after pre-incubation with RV or RV derivatives (RV, RV-3M, RV-1M; all 50 µM), or vehicle control for 30 min. H₂O₂ production was quantified by fluorescence measurement. The graph shows the relative extracellular H₂O₂ production (**P < 0.01; one-way ANOVA vs. Ang II or EGF treatment, n = 3). (B and C). Serum-deprived cells were pre-treated with RV or RV derivatives, RV-3M or RV-1M, for 30 min (50 µM), followed by stimulation with 100 nM Ang II for 10 min (B), or 100 ng/mL EGF for 5 min (C). Protein levels were detected by western blot using antibodies against pAkt and tubulin. One representative blot out of three per stimulation is shown. Graphs show the mean + SEM of the densitometric signals; Ang II or EGF stimulation is set to 100% (**P < 0.01; one-way ANOVA vs. Ang II or EGF treatment, n = 3).