Sirt3 Protects Against Thoracic Aortic Dissection Formation by Reducing Reactive Oxygen Species, Vascular Inflammation, and Apoptosis of Smooth Muscle Cells

Figure 1. Sirt3 deficiency is associated with the development of TAD in murine model. (A,B) Representative Western blot analysis of Sirt3 expression in continuous Ang II (1,000 ng/kg/min) and BAPN (1 g/kg per day) infusion. Data are shown as the mean ± SEM and are representative of six independent experiments, unpaired two-tailed t-test, ***P < 0.001. (C) Representative Western blot analysis of Sirt3 knockout efficiency using dissecting aortic aneurysm tissues. (D,E) Representative figures of the aortas and aorta diameters of WT and Sirt3 KO mice with continuous Ang II and BAPN infusion. Data are shown as the mean ± SEM and are representative of 10 independent experiments, unpaired two-tailed t-test, ***P < 0.001. (F) Survival curves in indicated groups, *P < 0.05. (G) Incidence of Ang II and BAPN-induced TAD (n = 16, 16). (H) Representative macroscopic images of dissecting aortic aneurysm sections stained with hematoxylin and eosin (HE) and Elastin van Gieson (EVG) from WT and Sirt3 KO mice with continuous Ang II and BAPN infusion. Scale bars: 100 μm.

Figure 2. Continuous infusion of Ang II and BAPN in Sirt3 KO mice induces activation of NF-kB, apoptosis, ROS production, and overexpression of MMPs in vivo. WT and Sirt3 KO mice with continuous Ang II (1,000 ng/kg/min) and BAPN (1 g/kg per day) infusion for 28 days. (A,B) Elastin degradation grading of aortas by EVG staining. n = 6. Data are shown as the mean ± SEM and are representative of six independent experiments, unpaired two-tailed t-test, ***P < 0.001. (C,D) Apoptosis level of aortas by TUNEL assay using fluorescence microscopy. n = 6. (E–J) Protein expression in the homogenates of aortas by Western blot. n = 6. Data are shown as the mean ± SEM and are representative of six independent experiments, unpaired two-tailed t-test, ***P < 0.001. (K,L) Representative Western blot analysis of acetylated IDH2 and IDH2 in tissues from WT and Sirt3 KO mice with continuous Ang II (1,000 ng/kg/min) and BAPN (1 g/kg per day) infusion. Data are shown as the mean ± SEM and are representative of six independent experiments, unpaired two-tailed t-test, ***P < 0.001. (M) Representative images of MMP-2 and MMP-9 activity were obtained by a gelatin zymogram using dissecting aortic aneurysm tissue from…

Figure 3. Silencing Sirt3 leads to increased Ang II-induced inflammation-related protein expression, ROS production, and apoptosis in HASMCs in vitro. Control siRNA and siSirt3 were transfected into HASMCs with Ang II (100 nM) treatment. (A) Western blot analysis verifies the transfection efficiency of sirt3 interfering RNA. (B,C) ROS production by DCFH-DA staining and measuring DCF levels using a BioTek Synergy H4 fluorescence microplate reader. n = 6. Data are shown as the mean ± SEM and are representative of six independent experiments, one-way ANOVA with Newman–Keuls multiple-comparison test, ***P < 0.001. (D–K) Protein expression of phosphorylation of p65, p65, phosphorylation of p38, p38, TNF-α, IL-1β, cleaved-caspase-3, caspase-3, Ac-IDH2, IDH2, and GAPDH by Western blot. Data are shown as the mean ± SEM and are representative of six independent experiments, one-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01. (L) Representative images of MMP-2 and MMP-9 activity were obtained by a gelatin zymogram in HASMCs with or without Sirt3 interfere.

Figure 4. Sirt3 overexpression reduces Ang II and BAPN-induced TAD formation. (A,C) Representative figures of the aortas and aorta diameters of WT and Sirt3OE mice with continuous Ang II (1,000 ng/kg/min) and BAPN (1 g/kg per day) infusion for 28 days. Data are shown as the mean ± SEM and are representative of 10 independent experiments, unpaired two-tailed t-test, ***P < 0.001. (B) Incidence of Ang II and BAPN-induced TAD (n = 20, 20). (D) Representative macroscopic images of dissecting aortic aneurysm sections stained with HE and EVG from WT and Sirt3OE mice with continuous Ang II and BAPN infusion. Scale bars: 100 μm.

Figure 5. Sirt3 overexpression ameliorates Ang II-induced phosphorylation of p65, overexpression of MMP-2 and MMP-9, ROS production, and apoptosis in HASMCs in vitro. pcDNA3.1 and Sirt3 were transfected into HASMCs with Ang II (100 nM) treatment. (A) Western blot analysis confirmed the transfection efficiency of the SIRT3-overexpressing plasmid. (B,C) ROS production by DCFH-DA staining and measuring DCF levels using a BioTek Synergy H4 fluorescence microplate reader. n = 6. Data are shown as the mean ± SEM and are representative of six independent experiments, one-way ANOVA with Newman–Keuls multiple comparison test, ***P < 0.001. (D–K) Protein expression of phosphorylation of p65, p65, phosphorylation of p38, p38, MMP-2/9, TNF-α, IL-1β, cleaved-caspase-3, caspase-3, Ac-IDH2, IDH2, and GAPDH by Western blot. Data are shown as the mean ± SEM and are representative of six independent experiments, one-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01. (L) Representative images of MMP-2 and MMP-9 activity were obtained by a gelatin zymogram in HASMCs with or without Sirt3 overexpression.