Fig 2: IL-1R1 knockdown prevents OSS-induced EndMT in vitro. (A) Module of Endothelial phenotypic switches to EndMT in atherosclerosis. (B) immunofluorescent images of mock vs. IL-1R1 siRNA HAECs subjected to oscillatory shear (OSS; 1 dynes/cm2 for 24 h), from n = 3. The cells were stained for Phalloidin (F-actin, purple), N-cadherin (red; mesenchymal marker), and CD31 (green; endothelial marker). (C,D) Immunofluorescent images of Snail1 (red) and DAPI (white: shows nuclei) in mock vs. IL- 1R1 siRNA HAOECs subjected to oscillatory shear (OSS; 24 h) and assessed for Snail1 nuclear translocation. Snail1 nuclear translocation was compared between IL-1R1 siRNA cells and mock controls in static and OSS conditions. Data are mean ± SEM, multiple unpaired t test, ***p < 0.001, n = 6 independent experiments. Scale bars = 400 - 100 µm.

Fig 3: EndMT and IL-1R1 complex are highly expressed in human atheromatous lesions compared to intact tissue. (A) GSE43292 dataset was analyzed and (B) the heat map to SNAI1, IRAK1, NF-kB, IRAK4, IL-1R1 and SNAI2 was shown. (B–E) expression levels of the IL-1R, IRAK1, NFKB1 and SNAI1 genes from n = 32 individuals per group, the value (intensity) of each probe represents the RNA expression level. Data was analyzed as mean ± SEM, and statistical analysis was conducted by unpaired t test, *p < 0.05, **p < 0.01.

Fig 4: IL-1R1 expression is associated with EndMT under disturbed blood flow areas. (A) Representative image of Oil O Red-stained aortic arch section of ApoE KO. Scale bars = 1 mm. Dash boxes represent the athero-resistant and atheroprone areas. (B,C) Immunofluorescent staining of IL-1R1 (red) and CD31 (green). Scale bars = 50–100 µm. (D) Shows immunofluorescence staining of CD31 (purple) Snail (red) and IL-1R1 (green) in atheroprone and athero-resistant areas in the EC-specific lineage tracing mice, YFP positive expression in ECs as stained by GFP (grey) (n = 3). Scale bars = 500 - 100 µm. (E,F) Human aortic ECs (HAOECs) exposed to laminar shear stress (LSS 10 dynes/cm2; mimicking in vivo athero-protective flow and oscillatory shear stress (OSS; 1 dynes/cm2; mimicking in vitro atheroprone flow) for 24 h. and lysed and mRNA was quantified using qRT-PCR for IL-1R1 (E) and SNAI1 (F). Data are represented as mean ± SEM, and 1-way ANOVA and Tukey's post-test test, *p < 0.05, n = 4 independent experiments.

Fig 5: EndMT in human unstable atheroma is associated with IL-1R1 expression. (A) Sections of human coronary atherosclerosis graded into very stable, moderately stable, and unstable lesions according to AHA recommendations and stained with H&E, scale bars = 1,000 µm, black boxes indicate the areas of interest that were stained for immunofluorescence and captured in B. (B,C) Human lesions were stained for IL-1R1 (white), vWF (green) and Snail1 (red) and showing increase expressions between these proteins within unstable lesions. Scale bars = 100 µm, white box indicates the higher magnification area demonstrated in (C), scale bar in C indicates 50 µm. (D,E) Semi-quantification to the expression levels of IL-1R1 and Snail1 (Mean Intensity) per high power field (HPF) and F is an analytic correlation between Snail1 and IL-1R1 expressions in unstable lesions, red dots indicate the 95% Confidence Intervals, Pearson r = 0.9808, p (two tailed) = 0.0032. Data are from n = 5 different human samples, and are mean ± SEM, analyzed by 1-way ANOVA and Tukey's post-test, **p < 0.01, ns, non-significant.