Role of SIRT3 in Angiotensin II-induced human umbilical vein endothelial cells dysfunction

Abstract

Background: SIRT3, a member of the sirtuin family of NAD(+)-dependent deacetylases, resides primarily in the mitochondria and has been shown to deacetylate several metabolic and respiratory enzymes that regulate important mitochondrial functions. Previous researches show an important role of SIRT3 in regulating the production of reactive oxygen species (ROS), and highlight the ability of SIRT3 to protect cells from oxidative damage. A key substance of renin-angiotensin-aldosterone system (RAAS), Angiotensin II (AngII) can induce cells dysfunction by increasing the production of ROS. In this paper, we focus on the role of SIRT3 in AngII-induced human umbilical vein endothelial cells (HUVECs) dysfunction.

Methods: To study the influence of AngII on SIRT3 expression, HUVECs were treated with AngII of 10(-7), 10(-6), 10(-5) mol/L for 24 h. SIRT3 expression was detected by wester-blotting analysis and RT-PCR. In addition, to research the role of SIRT3 in AngII-induced HUVECs,we used SIRT3 siRNA to knock down SIRT3 expression in HUVECs. Cells pretreated with negative control siRNA or SIRT3 siRNA were exposed to AngII for 24 h, and endothelial nitric oxide synthase (eNOS) expression, eNOS activity, total level of nitric oxide (NO) and ROS generation of each group were detected.

Results: Here we show that AngII treatment could increase generation of ROS, and decrease eNOS activity and total level of NO, while upregulated eNOS expression as a compensatory mechanism. The stimulation of AngII upregulated the expression of SIRT3 in HUVECs. SIRT3 siRNA worsen the AngII-induced effects above, besides, downregulated eNOS protein expression.

Conclusion: These data suggest that SIRT3 plays a role of protection in AngII-induced HUVECs dysfunction via regulation of ROS generation.

Fig. 1

Dose-dependent effects of AngII on SIRT3 expression. Cells were stimulated by AngII of 0, 10⁻⁷,10⁻⁶,10⁻⁵ mol/L for 24 h, SIRT3 protein expression were determined by western blot in each group (a) and Quantification of SIRT3 in each group, β-actin as a reference (b). SIRT3 mRNA expression were determined by RT-PCR (c). Data are the mean ± SEM from three separate experiments. ^* P < 0.05 vs. control, ^# P < 0.05 vs.10⁻⁶ mol/L group

Fig. 2

Screening of SIRT3 siRNA sequences. Cells were transfected by SIRT3 siRNA of different sequences. SIRT3 expression were determined by western blot in each group (a) and Quantification of SIRT3 in each group, β-actin as a reference (b). Data are the mean ± SEM from three separate experiments. ^* P < 0.05 vs. Negative Control (NC)

Fig. 3

Knockdown of SIRT3 worsen the AngII-induced effects on HUVECS. Cells transfected with negative control siRNA or SIRT3 siRNA were then treatment with AngII for 24 h. SIRT3 expression were determined by western blot in each group (a) and Quantification of SIRT3 in each group, β-actin as a reference (c). eNOS expression were determined by western blot in each group (b) and Quantification of eNOS in each group, β-actin as a reference (d). eNOS activity in each group (e), NO concentration in each group (f). Data are the mean ± SEM from three separate experiments. ^* P < 0.05 vs. Negative Control (NC), ^# P < 0.05 vs. AngII group

Fig. 4

The effect of SIRT3 on ROS level in HUVECs treated with AngIIThe effect of SIRT3 on ROS level in HUVECs treated with AngII. Cells pretreated with negative control siRNA or SIRT3 siRNA were exposed to AngII for 24 h. SIRT3 expression were determined by western blot in each group (a) and Quantification of SIRT3 in each group, β-actin as a reference (b). ROS levels in each group (c). Original magnification × 200. Data are the mean ± SEM from three separate experiments. ^* P < 0.05 vs. Negative Control (NC), ^# P < 0.05 vs. AngII group