Figure 2

Extracellular NLRP3-YFP inflammasome particles regulate gene expression in HCASMC. (A) Heatmap of genes upregulated in HCASMC treated with extracellular NLRP3-YFP inflammasome particles (N = 2) for 24 h. (B) List of the ten most upregulated genes. (C) Validation of four listed genes (NLRP3, ICAM1, CADM1, SPON1) using qPCR after 4 and 24 h treatment with extracellular NLRP3-YFP particles. Data were normalized on the mean of two housekeeping genes (RPLP0 and TBP) and were log2-transformed for equal distribution. Untreated cells (control) were set at 1. (D) Gene enrichment analysis of the 200 most upregulated genes (mean log2 fold > 3.5 vs. control) was performed using EnrichR and NCATS BioPlanet 2019 comprehensive integrated pathway analysis. Pathways with p < 0.05 are shown. mRNA expression of (E) adhesion molecule ICAM1 and (F) NLRP3 in HCASMC (N = 9) treated with extracellular NLRP3-YFP inflammasomes (3:1 particles/ cell) for 24 h with or without pre-incubation of caspase-1 inhibitor (Ac-YVAD-cmk, 25 µg/ml), NLRP3 inhibitor (MCC950, 1 µM) and NFκB inhibitor (IKK-16, 2 µM). Data are expressed as mean ± SEM and were normalized on the mean of two housekeeping genes (RPLP0 and TBP) and referenced to untreated control which was set at 1. Differences between the groups were calculated using One-way ANOVA and uncorrected Fisher´s LSD post-hoc test (*p < 0.05). (G) Selected genes (NLRP3, ICAM1, CADM1, NUP210) were re-analysed using a pre-existing microarray dataset and the gene expression was compared between atheroma plaque and adjacent macroscopically intact tissue from the same patient (N = 34). Data are expressed as mean ± SEM and differences between the groups were calculated using unpaired, two-tailed Student´s T-Test (***p < 0.001, ****p < 0.0001).