Exploration and validation of the influence of angiogenesis-related factors in aortic valve calcification

Figures

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  FIGURE 1. Differentially expressed genes between calcified and normal aortic valve samples. Volcano plots for differential gene expression data from: (A,D) GSE83453, (B,E) GSE51472, (C,F) GSE153555. In the volcano plots and heatmap, the red sections show upregulated genes (log2FC ≥ 0.5 and adjusted p-value < 0.05), whereas the green/blue sections represent downregulated genes; black dots represent no significant difference.

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  FIGURE 2. (A) The Venn plot of DEGs from GSE83453, GSE51472, and GSE153555. (B) Venn plots showing the relationship between DEGs and angiogenesis-related genes across all three gene sets. Venn plots show the relationship between DEGs and angiogenesis-related genes in all three gene sets. Intersected DEGs and angiogenesis-related genes in at least two CAVD datasets were screened as strongly correlated genes, and one key gene was identified.

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  FIGURE 3. Boxplots represent the gene expression levels of calcified samples compared to normal samples in the three datasets. (A) GSE83453, (B) GSE51472, (C) GSE153555. Wilcoxon rank-sum test, normal, control; CAV, calcified aortic valve. *p < 0.05, **p < 0.01, ****p < 0.0001.

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  FIGURE 4. Enrichment analysis of disease genes. Gene Ontology (GO) (A) biological process, (B) cellular component, (C) molecular function, Kyoto Encyclopedia of Genes and Genomes (KEGG) (D) enrichment analysis.

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  FIGURE 5. The protein-miRNA interaction network diagram, in which the blue squares represent miRNAs, the orange origins represent the related genes we screened out, with the top five miRNAs with the highest comprehensive scores displayed.

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  FIGURE 6. (A) Upstream miRNAs and transcription factors of the key gene SCG2. (B) Interaction network diagram of the key transcription factor ZEB1.

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  FIGURE 7. The protein-chemical-molecular interaction network diagram of the key protein gene SCG2.

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  FIGURE 8. Validation of SCG2 and ZEB1 in the calcified aortic valve. (A,B) The mRNA expression levels of SCG2 and ZEB1 in valves from normal patients (n = 5 biologically independent samples) and calcification patients (n = 5 biologically independent samples) were obtained by RT-PCR and normalized with GAPDH. (C,D) The protein expression level of SCG2. The bar graphs represent the expression levels of SCG2 protein in normal and calcified tissues. Valves from normal patients and CAVD patients were validated by Western Blotting. N1-N4 represent different samples of normal individuals, C1-C4 represent four individuals with CAV. ImageJ and GraphPad Prism softwares were used to analyze the expression level of SCG2 protein in normal and calcified aortic valves. Mann–Whitney U, *p < 0.05, **p < 0.01, ****p < 0.001, normal, control; CAV, calcified aortic valve.

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  FIGURE 9. Immunohistochemical staining of SCG2 and ZEB1 in human aortic valve, Bar chart for quantitative assessment of immunostaining. normal: normal aortic valve (n = 5 biologically independent samples), CAV: CAVD patients (n = 5 biologically independent samples). *p < 0.05, **p < 0.01 (A,B) immunohistochemical staining of the panorama of SCG2 and ZEB1. (C,D) Comprehensive quantitative expression analysis based on immunohistochemically stained sections.