Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Impaired BK-β1-mediated channel activation, reduced BK-β1 expression, and increased MuRF1 expression in diabetic vessels and in HG-cultured human coronary SMCs. A, inside-out single-channel BK currents were elicited at +60 mV in freshly isolated coronary SMCs from control and diabetic mice before and after exposure to DHS-1 (0.1 μm). DHS-1 robustly increased BK channel NPo in control SMCs, but its effects were diminished in diabetic SMCs. Dashed lines represent the channel closed state (c). B, left panel, the cell lysates of control and diabetic mouse aortas were blotted against anti-ubiquitin antibody. Right panel, the immunoprecipitates (IPs) of anti-BK-β1 antibodies were blotted against anti-ubiquitin (Ub) antibody, showing a significant increase of ubiquitinated BK-β1 protein in diabetic mice. Ctrl, control. C and D, a marked reduction of BK-β1 expression in the aortas of diabetic mice compared with those of control mice and in human coronary SMCs after a 2-week culture with 22 mm glucose (HG) compared with that with 5 mm glucose (NG) culture. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Activation Assay, Expressing, Cell Culture, Isolation, Control, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of BK-β1 expression by MuRF1. A, after a 2-week culture in NG or HG, BK-β1 protein expression in human coronary SMCs was determined under the following conditions: NG culture alone, HG culture after a 48-h transfection with control siRNA (0.1 μm), and HG culture after a 48-h transfection with MuRF1 siRNA (0.1 μm). MuRF1 siRNA suppressed MuRF1 expression under HG culture conditions to that of NG culture conditions. Ctrl, control. B, FLAG-BK-β1 or FLAG-BK-α stably expressed in HEK293 cells 48 h after cotransfection with Myc-MuRF1 WT cDNA or Myc-MuRF1ΔR mutant cDNA. The BK-β1 protein level was halved in cells with MuRF1 WT transfection, but BK-α expression remained unchanged. N.S., not significant. C and D, coimmunoprecipitation experiment shows attenuated BK-β1 protein ubiquitination in the pulldown complexes with anti-HA-ubiquitin (anti-HA-Ub) antibodies or with anti-FLAG antibodies in HEK293 cells after a 24-h transfection with MuRF1ΔR compared with those with MuRF1 WT. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Transfection, Control, Stable Transfection, Cotransfection, Mutagenesis, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Reduced BK-β1 expression and BK-β1-mediated channel activation in vascular SMCs by MuRF1 expression. A, fluorescence microscopy images illustrate GPF expression in endothelially denudated mouse coronary artery 12 h after transduction with Ad-GFP but not in a negative control artery. Immunoblot analyses show increased MuRF1 and decreased BK-β1 expression in the aortas of control (Ctrl) mice after a 12-h transduction with Ad-MuRF1 compared with controls with Ad-GFP transduction. B, inside-out single BK currents were elicited from +60 mV at baseline, after exposure to 0.1 μm DHS-1, and after washout (W/O). DHS-1 had no effect on mouse coronary SMCs 12 h after transduction with Ad-MuRF1. Dashed lines represent the channel closed state (c). Group data with statistical analysis are shown in the bar graphs. N.S., not significant. C, effects of Ad-MuRF1 transduction on dose-dependent, NS1619-mediated (10−9-10−5 m) dilation of endothelium-denuded coronary arteries from non-diabetic mice. Transduction with Ad-MuRF1 (12 h) attenuated the NS1619-induced vasodilation, but the NS1619 response was preserved in Ad-MuRF1-treated coronaries by incubation with MG132 (10 μm, 12 h). Control vessels received Ad-GFP transduction. *, p < 0.05, Ad-MuRF1 versus Ad-GFP; +, p < 0.05 Ad-MuRF1+MG132 versus Ad-GFP.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Fluorescence, Microscopy, Transduction, Negative Control, Western Blot, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Enhanced BK-β1 expression and BK-β1-mediated channel activation by MG132. A, protein expression of BK-β1 in STZ-induced diabetic aortas with or without 24-h treatment of MG132 (10 μm). Inhibition of proteasomal activity by MG132 increased BK-β1 protein expression in diabetic vessels. B, robust BK channel openings by DHS-1 (0.1 μm) were observed in freshly isolated diabetic coronary SMCs after 12-h incubation with MG132. Group data with statistical analysis are shown in the bar graphs. Dashed lines represent the channel closed state (c). W/O, washout.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of MuRF1 and BK-β1 expression by NF-κB. A, protein expression of NF-κB1/p105, NF-κB1/p50, and RelA/p-65 in the aortas of control and STZ-induced diabetic mice. The levels of NF-κB/p105, NF-κB1/p50, and p-65 protein expression were significantly increased in diabetic mice compared with controls (Ctrl). B, 24-h incubation with 0.5 μm TPCA-1 suppressed the levels of NF-κB1/p105, NF-κB1/p50, and MuRF1 expression but markedly increased that of IκB-β in both NG- and HG-cultured human coronary SMCs. TPCA-1 treatment did not alter BK-β1 expression in NG-cultured cells but significantly enhanced that in HG-cultured cells. Group data and statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Control, Incubation, Cell Culture

Journal: Nature Communications

Article Title: Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis

doi: 10.1038/s41467-022-35330-1

Figure Lengend Snippet: a Regions of 10 weeks, 15 weeks, and 20 weeks HFD-fed mice’s adjacent series sections plaque had neutrophils (LY6G) co-located with H3CIT, or macrophages (CD68) co-localized with citrullinated histone H3 (H3CIT). Scale bar = 200 μm, 100μm, 25μm respectively. The white arrow showed the H3CIT + cells of Macrophages or Neutrophils. b Quantification of the ratio of ETs + neutrophil area/ETs + total area within plaque lesions harvested from three-time points HFD fed Ldlr -/- mice (each time point, n = 5 mice). (10w vs 15w **** p < 0.0001; 10w vs 20w **** p < 0.0001) c Quantification of ETs + macrophage area/ETs + total area (each time point, n = 5 mice). (10w vs 15w **** p < 0.0001; 10w vs 20w **** p < 0.0001). d Gating strategy for CD68 + ETs + macrophages, LY6G + ETs + Neutrophils, and α-SMA + CD68 + ETs + VSMCs. e Quantification of CD68 + ETs + macrophages, LY6G + ETs + neutrophils, and α-SMA + CD68 + ETs + vascular smooth muscle cells (VSMCs) in the plaque harvested from n = 5, 24 weeks HFD fed mice. (CD68 + ETs + vs LY6G + ETs + **** p < 0.0001; α-SMA + CD68 + ETs + vs α-SMA - CD68 + ETs + **** p < 0.0001). f Representative images within plaque from HFD fed Ldlr -/- mice’s ascending aorta. Scale bar = 30 μm. The white arrow showed the α-SMA + H3CIT + CD68 + cells. g Immunofluorescence staining (IF) within plaque from human artery aspiration plaque. Scale bar = 10 μm. h , i IF staining of H3CIT and CD68 on the early or late stage of aortic arch plaque harvested from B6-G/R Myh11 Cre mice fed on HFD diet, Scale bar = 200 μm, 50 μm, respectively. White arrows are pointed at the H3CIT positive cells within the plaque. The side of the white star represents the lumen side. For all panels, error bars represent SD. p -value was determined by unpaired two-tailed Student’s t -test ( e ) or one-way ANOVA with Bonferroni post-test ( b , c ). Source data are provided as a Source Data file. Each experiment was repeated independently 3 times for ( f – i ).

Article Snippet: Thus, rat aortic vascular smooth muscle cells (RASMCs) from 25 male Sprague-Dawley rats (200–250 g) were used for vitro experiments.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis

doi: 10.1038/s41467-022-35330-1

Figure Lengend Snippet: a Individual cell area-under-the-curve (AUC) values overlay for selected differential canonical pathway activities. b Volcano plots of differentially expressed genes (DEGs) were screened by comparing Tdtomato + cells harvested from B6-G/R Myh11 Cre Pad4 flox/flox mice ( Pad4 Δ/Δ Tdtomato + cells) with that harvested from B6-G/R Myh11 Cre mice ( Pad4 +/+ Tdtomato + cells) in scRNAseq data. c Volcano plot of DEGs screened by comparing H3CIT + dsDNA challenged rat aortic vascular smooth muscle cells (RASMCs) with control RASMCs of RNA-seq data. d Heatmap showed different gene expression patterns between groups of RASMCs in c . e Western blot analysis showed RASMCs treated with H3CIT + dsDNA induced a marked decrease in the levels of phosphorylated STAT3, and STING-SOCS1 signaling pathway was activated compared with control RASMCs. f , g The Quantification of WB results in e ( n = 3 independent experiments). Ox-LDL 0 h p = 0.2067, STAT3 p = 0.4199, **** p < 0.0001. h Western blot analysis showed RASMCs treated with H3CIT + dsDNA induced a marked increase in the protein levels of TLR4 and MYD88. i , j The Quantification of WB results in h ( n = 3 independent experiments). **** p < 0.0001. k Violin plot of Gsdmd or Mmp9 between two groups of scRNA-seq results. l IF staining of SOCS1 in atherosclerosis plaque of BCA lesions of Pad4 flox/flox mice and Myh11 Cre Pad4 flox/flox mice, respectively. Scale bar = 50 μm. m The Quantification of the ratio of SOCS1 positive area in plaque lesion area between 2 groups (each group n = 3 mice). **** p < 0.0001. n IF staining of GSDMD in atherosclerosis plaque of BCAs of n = 3 Pad4 flox/flox mice and n = 3 Myh11 Cre Pad4 flox/flox mice, respectively. Scale bar = 50 μm. o The Quantification of the ratio of GSDMD positive area in plaque lesion area between 2 groups (each group n = 3 mice). p = 0.001.NS. means no significance. The side of the white star represents the lumen side. For all panels, error bars represent SD. Source data are provided as a Source Data file. p -value was determined by unpaired two-tailed Student’s t -test.

Article Snippet: Thus, rat aortic vascular smooth muscle cells (RASMCs) from 25 male Sprague-Dawley rats (200–250 g) were used for vitro experiments.

Techniques: Control, RNA Sequencing, Gene Expression, Western Blot, Staining, Two Tailed Test