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