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

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.