Fig 2: Diagnostic performance of S100A8/S100A9 expression levels for calcific aortic valve disease (CAVD) patients in the datasets. (A–C) The expression levels of S100A8/S100A9 in CAVD were based on the GSE databases (GSE153555, GSE51472, and GSE83453). (D–F) Diagnostic value of S100A8 and S100A9 in GSE databases (GSE153555, GSE51472, and GSE83453). Receiver operating characteristic (ROC) curve of normal and CAVD valve tissues. ** P < 0.01, *** P < 0.001 when two groups were compared as indicated or compared with the corresponding control.

Fig 3: Weighted co-expression network analysis (WGCNA) of GSE12644 dataset and Venn diagram to obtain the key genes S100A8 and S100A9. (A) Sample clustering of the GSE12644 dataset. The sample was clustered into two significantly different clusters. (B) Selection of optimal thresholds. The threshold was 16. (C) By aggregating genes with strong correlations in the same module, different modules were obtained and are displayed in different colors. (D) A network heatmap plot was created through WGCNA showing overall module-related gene branches in hierarchical clustering dendrograms. (E) Correlation analysis between modules and calcific aortic valve disease (CAVD). (F) The brown module was significantly positively correlated with CAVD (COR = 0.95, P < 0.001). The genes in the brown module are labeled as the WGCNA-hub genes. (G) Intersection of GeneCards, WGCNA, and differential expression data, displayed in the Venn diagram. The two most significant genes, S100A8 and S100A9, were obtained.

Fig 4: Expression of immune checkpoint-related mRNAs in calcific aortic valve disease (CAVD) patients. (A, B) Comparison of expression levels of 38 immune checkpoint-related RNAs. Immune checkpoint-related RNA expression between S100A8/S100A9 high- and low-expression groups of patients. Blue represents high expression, whereas red represents low expression. (C, D) Visualization of differentially expressed immune checkpoint-related genes in CAVD. S100A8/S100A9 high expression in CAVD patients is marked with blue, and S100A8/S100A9 low expression is marked with red. (E) Spearman correlation analysis of 38 immune checkpoint-related RNAs in CAVD. A higher number in the circle indicates a stronger correlation. The change in color on the right represents a positive or negative correlation. * P < 0.05, ** P < 0.01, *** P < 0.001 when two groups were compared as indicated or compared with the corresponding control.

Fig 5: Validation of S100A8 and S100A9 expression in ApoE-/- mice. (A) Echocardiography of hearts from APOE-/- mice in each group. Quantification of flow-velocity maximum of the aortic valve, mm/s. Blue represents a normal diet, and red represents a western diet administered to APOE-/- mice. (B) Von Kossa staining of the mouse valves. The areas of valve calcification are stained black. (C) Immunofluorescence showed increased S100A8 and S100A9 expression levels in aortic valve tissues from ApoE-/- mice fed a western diet for 4 months. ***P< 0.001 when two groups were compared as indicated or compared to the corresponding control.