Key ferroptosis-related genes in abdominal aortic aneurysm formation and rupture as determined by combining bioinformatics techniques

Abstract

Objectives: Abdominal aortic aneurysm (AAA) is a cardiovascular disease with high mortality and pathogenesis closely related to various cell death types, e.g., autophagy, apoptosis and pyroptosis. However, the association between AAA and ferroptosis is unknown.

Methods: GSE57691 and GSE98278 dataset were obtained from the Gene Expression Omnibus database, and a ferroptosis-related gene (FRG) set was downloaded from the FerrDb database. These data were normalized, and ferroptosis-related differentially expressed genes (FDEGs, AAA vs. normal samples) were identified using the limma package in R. FRGs expression was analyzed by Gene Set Expression Analysis (GSEA), and FDEGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) pathway enrichment analyses using the clusterProfiler package in R and ClueGO in Cytoscape. Protein-protein interaction networks were assembled using Cytoscape, and crucial FDEGs were identified using CytoHubba. Critical FDEG transcription factors (TFs) were predicted with iRegulon. FDEGs were verified in GSE98278 set, and key FDEGs in AAA (compared with normal samples) and ruptured AAA (RAAA; compared with AAA samples) were identified. Ferroptosis-related immune cell infiltration and correlations with key genes were analyzed by CIBERSORT. Key FEDGs were reverified in Ang II-induced AAA models of ApoE^-/- and CD57B/6J mice by immunofluorescence assay.

Results: In AAA and normal samples, 40 FDEGs were identified, and the expression of suppressive FRGs was significantly downregulated with GSEA. For FDEGs, the GO terms were response to oxidative stress and cellular response to external stimulus, and the KEGG pathways were the TNF and NOD-like receptor signaling pathways. IL6, ALB, CAV1, PTGS2, NOX4, PRDX6, GPX4, HSPA5, HSPB1, and NCF2 were the most enriched genes in the crucial gene cluster. CEBPG, NFAT5, SOX10, GTF2IRD1, STAT1, and RELA were potential TFs affecting these crucial genes. Ferroptosis-related immune cells involved in AAA formation were CD8+ T, naive CD4+ T, and regulatory T cells (Tregs); M0 and M2 macrophages; and eosinophils. Tregs were also involved in RAAA. GPX4, SLC2A1, and PEBP1 expression was downregulated in both the RAAA and AAA samples. GPX4 and PEBP1 were more important in AAA because they influenced ferroptosis-related immune cell infiltration, and SLC2A1 was more important in RAAA.

Conclusions: This is the first study to show that ferroptosis is crucial to AAA/RAAA formation. The TNF and NOD-like signaling pathways and ferroptosis-related immune cell infiltration play key roles in AAA/RAAA. GPX4 is a key ferroptosis-related gene in AAA. Ferroptosis and related genes might be promising targets in the treatment of AAA/RAAA.

Keywords: AAA (abdominal aortic aneurysm); GPX4; RAAA; ferroptosis; infiltration of immune cells.

FIGURE 1

The workflow of this study.

FIGURE 2

FDEGs in AAAs aortic vessels samples and normal samples of GSE57691. The Principal Component Analysis of AAAs aortic vessels samples and normal samples in GSE57691 set (A). The volcano plots of FDEGs in FRGs subsets of GSE57691 (B). The heatmap of FDEGs (C).

FIGURE 3

The GO and KEGG analysis of FDEGs in AAAs aortic vessels samples and normal samples of GSE57691. The GO-BP, CC, and MF analysis of FDEGs (A,B). the KEGG analysis of FDEGs (C). The top 5 enriched GO (D) and KEGG (E) pathway of FDEGs.

FIGURE 4

The correlation analysis, the PPI networks and TFs prediction of FDEGs in AAAs aortic vessels samples and normal samples of GSE57691. The Pearson’s correlation analysis in FDEGs (A). The relationships of pathways and FDEGs using the Clue GO analysis (B). The function clusters of FDEGs using the MCL of Cytoscape (C). The top 10 hub genes of FDEGs using the Cytohubba of Cytoscape (D). The TFs of the hub FDEGs predicted by the iRegulon of Cytoscape (E).

FIGURE 5

Ferroptosis-related infiltration of immune cells in the FEGs subsets of GSE57691 and GSE98278. The infiltration of immune cells in FEGs subsets of GSE57691 (A). The correlation of immune cells in FEGs subsets of GSE57691 (B). The difference infiltration of immune cells in FEGs subsets of GSE57691 (C). The infiltration of immune cells in FEGs subsets of GSE98278 (D). The correlation of immune cells in FEGs subsets of GSE98278 (E). The difference infiltration of immune cells in FEGs subsets of GSE98278 (F).

FIGURE 6

The correlation of key FDEGs and infiltration of immune cells in the FRGs subsets of GSE57691 and GSE98278. The correlation of GPX4 and infiltration of immune cells in AAA and normal samples of GSE57691 (A). The correlation of GPX4 and infiltration of immune cells in RAAA and AAA samples of GSE98278 (B). The correlation of SLC2A1 and infiltration of immune cells in AAA and normal samples of GSE57691 (C). The correlation of SLC2A1 and infiltration of immune cells in RAAA and AAA samples of GSE98278 (D). The correlation of PEBP1 and infiltration of immune cells in AAA and normal samples of GSE57691 (E). The correlation of PEBP1 and infiltration of immune cells in RAAA and AAA samples of GSE98278 (F).

FIGURE 7

The reverified of key FDEGs with Ang II induced-AAA model in ApoE^–/– and CD57B/6J mice. The drams of abdominal aorta in groups (A). The HE staining of abdominal aorta in each group (B). The abdominal aorta diameters increased rate of AAA compared with control samples [(C), n = 5]. The IF of SLC2A1 on abdominal aorta in groups (D) and mean area of SLC2A1 in groups (E). The IF of GPX4 on abdominal aorta in groups (F) and mean area of GPX4 in groups (G). The IF of PEBP1 on abdominal aorta in groups (H) and mean area of PEBP1 in groups (I) (n: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).