Deletion of Superoxide Dismutase 1 Blunted Inflammatory Aortic Remodeling in Hypertensive Mice under Angiotensin II Infusion

Figure 1. The efficiency of superoxide dismutase (SOD1) deletion and effect of AngII infusion on systolic blood pressure. (A) Immunohistochemistry (upper part) and Western blot analysis (lower part) of aortas from WT mice and SOD1−/− mice using anti-SOD1 antibodies. (Scale bars = 100 µm.) SOD activity in red blood cells (B) and serum (C). Red blood cells (RBCs) do not have mitochondria so SOD activity would be that of cytosol SOD (SOD1). (n = 22~26 per group. *** p < 0.001 n.s.: not significant). (D) Saline or AngII (3.2 mg/kg per day) was chronically infused using osmotic mini-pumps. Systolic blood pressure was measured by the tail-cuff method (n = 12 per group). Error bars represent SEM. WT: wild type; SOD1−/−: superoxide dismutase 1 deficient; AngII: angiotensin II.

Figure 2. Microscopic appearance of aortas and quantitative analysis. (A) Hematoxylin–eosin (HE), Elastica van Gieson (EVG), and Masson’s staining of aortas. WT mice with saline (upper left), SOD1−/− mice with saline (upper right), WT mice with AngII (lower left), SOD1−/− mice with AngII (lower right). (Scale bar = 100 μm.) (B) Quantification of aorta wall thickness and (C) medial areas measured by NIH Image software (n = 10–12 per group. ** p < 0.01 *** p < 0.001) Error bars represent SEM. HE: Hematoxylin–Eosin Stain; EVG: Elastica van Gieson stain; Masson’s: Masson’s trichrome stain.

Figure 3. Extracellular and intracellular H2O2 levels in blood and aortas in vivo. (A) Derivatives of reactive oxygen metabolites (d-ROMs), which detect hydro-peroxide (R–OOH), were measured as an extracellular H2O2 indicator (n = 12 per group. *** p < 0.001). (B) Oxidized glutathione (GSSG)/reduced glutathione (GSH) ratios were measured in RBCs as an indicator of intracellular H2O2 (n = 8 per group. * p < 0.05 ** p < 0.01 n.s.: not significant). (C) GSSG/GSH ratios in aortas (n = 10~11 per group. * p < 0.05). Error bars represent SEM. ROS: reactive oxygen species; WT: wild type; SOD1−/−: superoxide dismutase 1 deficient; Ang II: angiotensin II; d-ROMs: derivatives of reactive oxygen metabolites; RBC: red blood cell; GSSG: oxidized glutathione; GSH: reduced glutathione; DHE: Dihydroethidium.

Figure 4. The protein expressions of aortas from WT and SOD1−/− mice with saline or AngII. (A) Representative results of Western blot analyses of phosphorylated STAT3, phosphorylated Akt, phosphorylated ERK1/2, and SOD1 in aortas from WT and SOD1−/− mice, with saline or AngII. (B) Quantitative analyses by densitometry of immunoblots expressed as ratios of phosphorylated STAT3 to STAT3. (n = 8 per group. * p < 0.05 n.s.: not significant) Error bars represent SEM. STAT3: signal transducer and activator of transcription 3; ERK: extracellular signal-regulated kinase.

Figure 5. Serum IL-6 concentration and mRNA expression in aortas. (A) Serum IL-6 levels were analyzed by the ELISA test (n = 9–10 per group. * p < 0.05 ** p < 0.01 n.s.: not significant). (B–E) The local aortic IL-6 (B), IL-1β (C), MCP-1 (D) and NOX2 (E) levels were detected as mRNA expressions, which were compensated with an internal control: 18S (n = 4–8 per group; * p < 0.05, ** p < 0.01 n.s.: not significant). Error bars represent SEM. IL-6: interleukin 6; IL-1β: interleukin 1β; MCP-1: Monocyte Chemotactic Protein-1; NOX2: NADPH Oxidase 2.

Figure 6. Rat vascular smooth muscle cells (VSMCs) incubated with AngII. (A) Time course of STAT3 phosphorylation of VSMCs due to AngII. VSMCs were stimulated with 100 nM AngII for the indicated times. The top panels are representative immunoblots for Ang II-induced phosphorylation of STAT3. The bottom panels represent averaged data quantified by densitometry of immunoblots, expressed as fold increases in phosphorylation, in which the phosphorylation for the control was defined as 1.0 (n = 4 per group. * p < 0.05) (B) The expression of phosphorylated STAT3 of VSMCs induced with AngII. The expression of SOD1 was downregulated with SOD1 siRNA (6 nM). The bottom panels represent quantification of densitometry, expressed as fold increases in phosphorylation, in which the phosphorylation for “scramble siRNA control’’ was defined as 1.0 (n = 7 per group. * p < 0.05 ** p < 0.01 n.s.: not significant). (C) The SOD1 activity was pharmacologically inhibited by 0.1 mM DETC (copper-chelating compound). DETC was added to VSMC culture fluid 4 h before AngII. The bottom panels represent averaged data quantified by densitometry of immunoblots (n = 9 per group. * p < 0.05 ** p < 0.01 n.s.: not…

Figure 7. The cytosolic H2O2 produced by SOD1 facilitated IL-6 synthesis in VSMCs and its secretion from them, which had been induced by AngII. H2O2 facilitated the activation of STAT3 by IL-6. These mechanisms were responsible for the inflammatory remodeling of aortas. AT-1R: angiotensin II receptor 1; NOX: NADPH oxidase.