Identification of Diagnostic Genes of Aortic Stenosis That Progresses from Aortic Valve Sclerosis

Abstract

Background: Aortic valve sclerosis (AVS) is a pathological state that can progress to aortic stenosis (AS), which is a high-mortality valvular disease. However, effective medical therapies are not available to prevent this progression. This study aimed to explore potential biomarkers of AVS-AS advancement.

Methods: A microarray dataset and an RNA-sequencing dataset were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened from AS and AVS samples. Functional enrichment analysis, protein-protein interaction (PPI) network construction, and machine learning model construction were conducted to identify diagnostic genes. A receiver operating characteristic (ROC) curve was generated to evaluate diagnostic value. Immune cell infiltration was then used to analyze differences in immune cell proportion between tissues. Finally, immunohistochemistry was applied to further verify protein concentration of diagnostic factors.

Results: A total of 330 DEGs were identified, including 92 downregulated and 238 upregulated genes. The top 5% of DEGs (n = 17) were screened following construction of a PPI network. IL-7 and VCAM-1 were identified as the most significant candidate genes via least absolute shrinkage and selection operator (LASSO) regression. The diagnostic value of the model and each gene were above 0.75. Proportion of anti-inflammatory M2 macrophages was lower, but the fraction of pro-inflammatory gamma-delta T cells was elevated in AS samples. Finally, levels of IL-7 and VCAM-1 were validated to be higher in AS tissue than in AVS tissue using immunohistochemistry.

Conclusion: IL-7 and VCAM-1 were identified as biomarkers during the disease progression. This is the first study to analyze gene expression differences between AVS and AS and could open novel sights for future studies on alleviating or preventing the disease progression.

Keywords: aortic stenosis; aortic valve sclerosis; diagnostic genes; immune infiltration; immunohistochemistry; machine learning.

Figure 1

The possible mechanisms IL7 and VCAM1 involved in for the disease progression. The two diagnostic genes, IL7 and VCAM1 interact with immune cells to promote the progression from AVS to AS together. By Figdraw.

Figure 2

Study flowchart.

Figure 3

Identification of DEGs between sclerotic and stenotic tissues and enrichment analysis of DEGs. (A) PCA for sclerotic and stenotic tissues. (B) Volcanic map of identified DEGs (P < 0.05, |logFC| > 1) between sclerotic and stenotic tissues. Red dots represent upregulated genes and blue dots represent downregulated genes. (C) Heatmap of DEGs. Upregulated genes are shown in red and downregulated genes are shown in blue. (D) The top five GO analysis results of DEGs. (E) KEGG pathway enrichment analysis results of DEGs (top 10 according to adjusted P value).

Figure 4

Identification of node genes between sclerotic and stenotic tissues and enrichment analysis of node genes. (A) The top 17 node genes were identified from the PPI network. (B) Fifteen genes were identified from the interaction of node genes and immune-related genes were downloaded from ImmPort and placed in the Venn diagram. (C) The top five GO analysis results of node genes. (D) KEGG pathway enrichment analysis results of node genes (top 10 according to adjusted P value).

Figure 5

Construction of the diagnostic model. (A and B) LASSO regression was used to screen biomarkers. The dotted vertical lines show the optimal value through lambda.min and lambda.1 se. Diagnostic genes (n = 2) and their coefficients were selected according to lambda.1 se (C) Expression validations of the diagnostic genes IL-7 and VCAM-1 in the GSE138531 dataset. (D) ROC analysis of the diagnostic model; IL-7 and VCAM-1 in the GSE51472 dataset and GSE138531 dataset.

Figure 6

Immune cell infiltration analysis between AS and AVS. (A) The proportion of 22 immune cells in sclerotic and stenotic valves visualized by a bar plot. (B) Differences in immune infiltration between the AVS and AS groups. *P < 0.05. ns: P > 0.05. ?: The calculated proportion = 0 in both sclerosis and stenosis group.

Figure 7

Correlation analysis between IL-7 and VCAM-1 expression and immune cell infiltration. (A) Correlation analysis visualized using a heatmap. (B) Correlation analysis. (C) Bubble chart of correlations between IL-7 expressions and immune cell proportions. (D) Bubble chart of correlations between VCAM-1 expression and immune cell proportions. *P < 0.05. **P < 0.01.

Figure 8

Levels of IL-7 and VCAM-1 in normal, sclerotic, and stenotic valves. (A) hematoxylin-eosin (HE), Masson, and alizarin red staining revealed the highest levels of IL-7 and VCAM-1 in stenotic aortic valves and relatively higher expressions of IL-7 and VCAM-1 in sclerotic aortic valves than in normal aortic valves (bar = 50 μm). (B) Quantification of IL-7 and VCAM-1 expression among the three groups. ****P < 0.0001.