IGFBP6 Is Downregulated in Unstable Carotid Atherosclerotic Plaques According to an Integrated Bioinformatics Analysis and Experimental Verification

Figures

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  Fig. 1.. Volcano map analysis of the upregulated and downregulated genes in the stable and unstable carotid plaques in the three datasets A. 1575 DEGs, including 680 upregulated and 875 downregulated DEGs, were identified in the unstable plaques compared with the stable plaques in the GSE41571 dataset. B. 760 DEGs were screened, with 471 upregulated and 289 downregulated in the GSE118481 dataset. C. 1186 DEGs were identified, with 619 upregulated and 567 downregulated in the E-MTAB-2055 dataset. D. 14 upregulated DEGs were identified after merging the three datasets. E. Two downregulated DEGs were identified after merging the three datasets. DEGs, differentially expressed genes

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  Fig. 2.. Bubble diagram (top) results of the functional enrichment analyses of upregulated and downregulated DEGs The size of an individual point represents the degree of enrichment. The color depth represents the P-value. A. GO-BP enrichment analysis of upregulated DEGs. B. GO-BP enrichment analysis of downregulated DEGs. C. KEGG pathway enrichment analysis for upregulated genes. D. KEGG pathway enrichment analysis for downregulated genes. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes

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  Fig. 3.. PPI network and subnet module analysis The violet circles represent the upregulated DEGs, and the green diamonds represent the downregulated DEGs. The size of the nodes represents the number of protein-protein interaction pairs of a certain protein. The lines between two nodes represent the interaction of two proteins. A. Construction of PPI networks. B, C, D. Subnet module analysis of PPI. B, mcode 1; C, mcode 2; D, mcode 3. PPI, protein–protein interactions; DEGs, differentially expressed genes.

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  Fig. 4.. miRNA/TF targeted DEGs regulatory networks The violet circles represent the upregulated DEGs, and the green diamonds represent the downregulated DEGs. The red triangle represents miRNA, and the yellow hexagon represents TF. The arrows indicate the regulatory direction.

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  Fig. 5.. Drug–gene interaction networks The violet rectangles represent the up-DEGs, and green rectangles represent the downregulated DEGs. The gray boxes indicate the drugs.

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  Fig. 6.. Representative histologic images of the stable and unstable plaques and the classification system A–E: Representative image and stability scoring of the stable plaques. F–J: representative image and stability scoring of the unstable plaques. A, F indicate hematoxylin–eosin staining (scale bar: 1,000 µm); B, G indicate Masson's trichrome staining (scale bar: 1,000 µm); C, H indicate CD68 immunostaining; D, I indicate CD31 immunostaining (scale bar: 1,000 µm); E: associated scoring chart for the stable plaques; J: associated scoring chart for the unstable plaques.

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  Fig. 7.. Experimental verification of the mRNA expression of six candidate genes identified from the co-regulated DEGs and PPI networks in the tissue samples A. qRT-PCR analysis of COL1A2, TYROBP, ADCY3, PLA2G7, CYP27A1, and IGFBP6 in 54 carotid atherosclerotic plaques (27 stable and 27 unstable plaques according to the clinical criteria; n = 54). B. qRT-PCR analysis of the 6 genes in the same patient cohort (28 stable and 26 unstable plaques according to the histologic criteria; n = 54). ***p < .001. Three independent experiments were performed.

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  Fig. 8.. Immunochemistry analysis of IGFBP6 in histologically stable and unstable plaques A. IGFBP6-positive area (brown) is significantly decreased in the unstable plaque compared with that in the stable plaque (×10) (****p < .0001) B. Colocalization of CD68+ macrophages in the border between the fibrous cap and necrotic core, in the area where IGFBP6 is expressed (×10). The black rectangle indicates the local enlarged view (×40). C. CD31+ endothelial cells are colocalized with IGFBP6 (× 10). The black rectangle indicates the local enlarged view (×40).

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  Supplemental Fig. 1.. A. The brown region indicates the α-SMA+ cells in the tunica media in carotid atherosclerotic plaques. B. IGFBP6 is not colocalized with α -SMA+ cells in the tunica media. C. The brown region indicates the α-SMA+ cells in the intima layer. D. IGFBP6 is not colocalised with the α-SMA+ cells in the intima layer.

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  Fig. 9.. A. Concentration of IGFBP6 protein in the plasma of patients with histologically stable versus unstable plaque. (****p < .0001) B. ROC curves of IGFBP6, models 1, 2, and 3 for the discrimination between patients with stable and unstable plaques. The discriminative power of IGFBP6: the AUC was 0.894 (95% CI: 0.810–0.977), the sensitivity was 1.000, and the specificity was 0.655. The discriminative power of model 1: AUC was 0.903 (95% CI: 0.823–0.982), the sensitivity was 0.739, and the specificity was 0.931. The discriminative power of model 2: AUC was 0.906 (95% CI: 0.821–0.990), the sensitivity was 0.870, and the specificity was 0.828. The discriminative power of model 3: AUC was 0.952 (95% CI: 0.900–1.000), sensitivity was 0.913, and specificity was 0.897. Model 1, IGFBP6 adjusted for age and gender; model 2, age, gender, smoke, hypertension, weight, diabetes, creatinine, triglyceride, and total high-density lipoprotein and low-density lipoprotein cholesterol levels; model 3, IGFBP6 adjusted for all the above cofounders. ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval.