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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.

目的

腹主动脉瘤（AAA）是一种心血管疾病，死亡率高，其发病机制与多种细胞死亡类型密切相关，如自噬、凋亡和焦亡。然而，AAA与铁死亡之间的关联尚不清楚。

方法

从基因表达综合数据库获取GSE57691和GSE98278数据集，并从FerrDb数据库下载铁死亡相关基因（FRG）集。对这些数据进行标准化处理，使用R语言中的limma软件包鉴定铁死亡相关差异表达基因（FDEG，AAA样本与正常样本比较）。通过基因集表达分析（GSEA）分析FRG表达，使用R语言中的clusterProfiler软件包和Cytoscape中的ClueGO对FDEG进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。使用Cytoscape构建蛋白质-蛋白质相互作用网络，并使用CytoHubba鉴定关键FDEG。使用iRegulon预测关键FDEG转录因子（TF）。在GSE98278数据集中验证FDEG，并鉴定AAA（与正常样本比较）和破裂性AAA（RAAA；与AAA样本比较）中的关键FDEG。通过CIBERSORT分析铁死亡相关免疫细胞浸润及其与关键基因的相关性。通过免疫荧光分析在载脂蛋白E和CD57B/6J小鼠的血管紧张素II诱导的AAA模型中再次验证关键FEDG。

结果

在AAA和正常样本中，鉴定出40个FDEG，GSEA显示抑制性FRG的表达显著下调。对于FDEG，GO术语为对氧化应激的反应和细胞对外部刺激的反应，KEGG通路为TNF和NOD样受体信号通路。IL6、ALB、CAV1、PTGS2、NOX4、PRDX6、GPX4、HSPA5、HSPB1和NCF2是关键基因簇中最富集的基因。CEBPG、NFAT5、SOX10、GTF2IRD1、STAT1和RELA是影响这些关键基因的潜在TF。参与AAA形成的铁死亡相关免疫细胞为CD8 + T细胞、初始CD4 + T细胞和调节性T细胞（Treg）；M0和M2巨噬细胞；以及嗜酸性粒细胞。Treg也参与RAAA。RAAA和AAA样本中GPX4、SLC2A1和PEBP1的表达均下调。GPX4和PEBP1在AAA中更重要，因为它们影响铁死亡相关免疫细胞浸润，而SLC2A1在RAAA中更重要。