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

Journal: Stem Cell Research & Therapy

Article Title: Spatio-temporal model of Meox1 expression control involvement of Sca-1-positive stem cells in neointima formation through the synergistic effect of Rho/CDC42 and SDF-1α/CXCR4

doi: 10.1186/s13287-021-02466-8

Figure Lengend Snippet: CDC42 was involved in SDF-1α expression in VSMC and CXCR4 expression in Sca-1+ stem cells mediated by Meox1. The aim of the experiment was to explore possible molecular mechanisms of Meox1-induced SDF-1α expression in VSMC and CXCR4 expression in HUCSCs, using RhoA inhibitor CCG1423, CDC42 blocker ZCL278, or Rac1 inhibitor Azathioprine. A , B Overexpressing Meox1 (Ad-Meox1) increased SDF-1α expressions while knockdown of Meox1 by shRNA (Ad-shMeox1) in VSMCs transfected with treatment of Ad-Meox1 or Ad-shMeox1 for 3 days as determined by western blot ( A ) and semi-quantitative analysis ( B ). Three independent experiments were performed, n = 3, * P < 0.05 vs. VSMC transfected with Ad-Null; # P < 0.05 vs. VSMCs transfected with Ad-Meox1. C , D The SDF-1α expressions in VSMCs transfected with Ad-Meox1, following the addition of RhoA inhibitor CCG1423, CDC42 blocker ZCL278, or Rac1 inhibitor Azathioprine, as determined by western blot ( C ) and semi-quantitative analysis ( D ). Three independent experiments were performed, n = 3, * P < 0.05 vs. VSMCs transfected with Ad-Null; # P < 0.05 vs. VSMCs transfected with Ad-Meox1. E , F CXCR4 expressions in HUCSCs transfected with Ad-Meox1 for 24 h, following the addition of CCG1423, or Azathioprine for 48 h, as determined by western blot ( E ) and semi-quantitative analysis ( F ). Three independent experiments were performed, n = 3, * P < 0.05 vs. HUCSCs treated with Ad-Null; # P < 0.05 vs. HUCSCs treated with Ad-Meox1; & P < 0.05 vs. Ad-Meox1-transfected HUCSCs treated with CDC42 blocker ZCL278

Article Snippet: Primary vascular smooth muscle cells (VSMC) were isolated from aortic artery of Sprague Dawley rats (280–300 g) and cultured as previously described [ ].

Techniques: Expressing, Knockdown, shRNA, Transfection, Western Blot

Journal: Cell reports

Article Title: Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and Rho

doi: 10.1016/j.celrep.2021.109019

Figure Lengend Snippet: VSMCs were isolated from SMA-GFP reporter mice, transfected with siRNAs targeting YAP (si159 and si160) or TAZ (si843 and si924) mRNAs, or a control (Cntrl) siRNA. The cells were cultured on a stiff collagen-coated hydrogel for 24 h in the presence of 20% FBS. (A) Representative images of SMA promoter activity and traction force in response to YAP and TAZ siRNA. See for results for all siRNAs used. Cell scale bars, 50 μm; the traction force scale bar, = 0–1,200 nN. (B) Quantification of traction forces from (A). The dashed line shows the traction force threshold identified in that binned the VSMCs into high and low contractile states; n ≈ 45–80 cells analyzed per siRNA. (C) VSMCs isolated from WT mice were transfected with siRNAs and cultured on coverslips with 20% FBS and EdU for 24 h as in (A) and (B). The fraction of EdU-positive nuclei was determined relative to Hoechst-stained nuclei and normalized to the control siRNA for each experiment. Results show mean + SD; n = 3. (D) Model of VSMC phenotypic plasticity emerging from a null state as regulated by ECM stiffness, the balance between Rac and Rho activities, and the effects of YAP and TAZ.

Article Snippet: Primary mouse vascular smooth muscle cells (VSMCs) were isolated from explants of the descending thoracic aorta of 10-12 week-old male C57BL/6 (Jackson Laboratories) or SMA-GFP reporter mice (C57BL/6 background) as described ( ; ).

Techniques: Isolation, Transfection, Control, Cell Culture, Activity Assay, Staining