Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling

Figures

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  Fig. 1. VSMC-derived endogenous SO2 inhibits vascular remodeling and hypertension in vivo. (A) Comparison of endogenous SO2 levels in the aortic tissues AAT1f/f and VSMC-AAT1−/− mice (n = 6). SO2 was measured by HPLC-FD as described in the Materials and Methods. (B) Representative immunofluorescence staining of collagen I/III in aortas of AAT1f/f and VSMC-AAT1−/− mice. E, endothelium; M, media; and A, adventitia. Scale bar, 20 μm. (C) Comparison of the systolic blood pressure (SBP) of AAT1f/f and VSMC-AAT1−/− mice monitored by 24-h telemetry analysis (n = 4). Data represent mean values ± SEM. *P < 0.05. (D) Comparison of the SBP of AAT1f/f and VSMC-AAT1−/− mice measured by noninvasive tail-cuff method (n = 22). (E) Comparison of endogenous SO2 levels in the aortic tissues of wild type (WT) and TgAAT1 mice pretreated with or without Ang II (n = 10). (F) Representative immunofluorescence staining of aortic collagen I/III in WT or TgAAT1 mice. Mice were infused with Ang II or saline for two weeks. Scale bar, 20 μm. (G) Hart's staining of thoracic aorta (upper panel) and quantification (lower panels) the medial thickness and medial surface in WT and TgAAT1 mice infused with Ang II or saline…

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  Fig. 2. SO2 promotes dynamic changes in the sulfenylome of VSMCs. (A) Plausible mechanism of SO2-mediated formation of peroxymonosulfite. Sulfur atom is highlighted in red color. (B) Scheme of chemoselective labeling of protein sulfenic acid by BTD. (C) Schematic workflow for globally and site-specifically profiling protein sulfenylation using the BTD-based SulfenQ method. After 1 h pre-incubation with Ang II, VSMCs were treated with or without 100 μM SO2 donors (SO32‾: HSO3‾ = 2.5:1) for 2 h at 37 °C. Cell lysates were harvested, labeled with 5 mM BTD for 2 h at 37 °C, and then subjected to tryptic digestion. Equal amounts of peptides were reacted with light (for SO2) and heavy (for control) tagged azido-biotin with a photocleavable linker (Az-UV-biotin), respectively. The UV-cleavable biotinylated peptides were captured by streptavidin, and photoleased for LC-MS/MS analysis. (D) Rank order of the determined RSO2/control (RL/H) values of BTD-adducted peptides from VSMCs treated with or without exogenous SO2. GO (E) and KEGG (F) annotations enriched for protein targets of SO2-dependent dynamic sulfenylation. (For interpretation of the references to color in this figure legend, the reader…

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  Fig. 3. Smad3C64 is a functional target of SO2-mediated sulfenylation. (A) Left panel: Representative MS/MS spectrum of the peptide containing Smad3C64 labeled by BTD-derived modification. Diagnostic fragment ion (DFI) generated from the modified peptide via C–S cleavage is shown in pink color. Right panel: Representative extracted ion chromatogram showing for the change in BTD-labeled cysteine of Smad3 protein from VSMCs treated with or without SO2, with the profiles for light and heavy-labeled peptides in red and blue, respectively. (B) C64 of Smad3 is evolutionarily conserved in vertebrates and is adjacent to a lysine residue that is conserved as well. (C) DAz-2-labeled sulfenylation of Smad3 in Smad3-deficient VSMCs transfected with the wild-type (Smad3WT) or C64S mutated Smad3 (Smad3C64S) plasmid. Cells were treated with or without SO2 donors, harvested and lysed for DAz-2-based labeling and detection. For C, D, G, J, cell/tissue lysates were labeled with DAz-2, ligated with phosphine-biotin, captured with NeutrAvidin, and subjected to western blotting against Smad3. (D) DAz-2-labeled sulfenylation of Smad3 in vehicle- or AAT1-overexpressing VSMCs. Cells pre-stimulated with or…

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  Fig. 4. Smad3C64 is required for SO2-mediated vascular remodeling and hypertension. (A) Western blots (upper panel) and quantification (lower panel) of Smad3 phosphorylation in the aortic tissues of wild type and TgAAT1 mice pre-stimulated with Ang II (n = 6). (B) SO2 inhibits DNA binding activity of wild-type Smad3 but not its C64S mutant. Nuclear proteins were extracted, and the protein-DNA complexes were measured by electrophoretic mobility shift assay. (C) Western blots (upper panel) and quantification (lower panel) of collagen I/III in Smad3-deficient VSMCs transfected with Smad3 WT or C64S plasmid. Cells were pre-incubated with or without Ang II for 1 h, followed by adding SO2 donors or vehicle. Data are from three independent experiments. (D) Representative immunofluorescence staining of phosphorylated Smad3 (p-Smad3), collagen I/III in the aortas of Smad3C64S/WT and Smad3WT/WT mice. During the two weeks of Ang II or saline infusion, the mice were treated daily with SO2 donors or saline. Scale bar, 20 μm. E, endothelium; M, media; and A, adventitia. (E) Hart's staining of thoracic aorta (upper panel) and quantification of the medial thickness (lower panel), n = 5–6. Scale bar, 500…