Table_3_Identification of key monocytes/macrophages related gene set of the early-stage abdominal aortic aneurysm by integrated bioinformatics analysis and experimental validation.XLSX

ObjectiveAbdominal aortic aneurysm (AAA) is a lethal peripheral vascular disease. Inflammatory immune cell infiltration is a central part of the pathogenesis of AAA. It’s critical to investigate the molecular mechanisms underlying immune infiltration in early-stage AAA and look for a viable AAA marker.
MethodsIn this study, we download several mRNA expression datasets and scRNA-seq datasets of the early-stage AAA models from the NCBI-GEO database. mMCP-counter and CIBERSORT were used to assess immune infiltration in early-stage experimental AAA. The scRNA-seq datasets were then utilized to analyze AAA-related gene modules of monocytes/macrophages infiltrated into the early-stage AAA by Weighted Correlation Network analysis (WGCNA). After that, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for the module genes was performed by ClusterProfiler. The STRING database was used to create the protein-protein interaction (PPI) network. The Differentially Expressed Genes (DEGs) of the monocytes/macrophages were explored by Limma-Voom and the key gene set were identified. Then We further examined the expression of key genes in the human AAA dataset and built a logistic diagnostic model for distinguishing AAA patients and healthy people. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immunosorbent Assay (ELISA) were performed to validate the gene expression and serum protein level between the AAA and healthy donor samples in our cohort.
ResultsMonocytes/macrophages were identified as the major immune cells infiltrating the early-stage experimental AAA. After pseudocell construction of monocytes/macrophages from scRNA-seq datasets and WGCNA analysis, four gene modules from two datasets were identified positively related to AAA, mainly enriched in Myeloid Leukocyte Migration, Collagen-Containing Extracellular matrix, and PI3K-Akt signaling pathway by functional enrichment analysis. Thbs1, Clec4e, and Il1b were identified as key genes among the hub genes in the modules, and the high expression of Clec4e, Il1b, and Thbs1 was confirmed in the other datasets. Then, in human AAA transcriptome datasets, the high expression of CLEC4E, IL1B was confirmed and a logistic regression model based on the two gene expressions was built, with an AUC of 0.9 in the train set and 0.79 in the validated set. Additionally, in our cohort, we confirmed the increased serum protein levels of IL-1β and CLEC4E in AAA patients as well as the increased expression of these two genes in AAA aorta samples.
ConclusionThis study identified monocytes/macrophages as the main immune cells infiltrated into the early-stage AAA and constructed a logistic regression model based on monocytes/macrophages related gene set. This study could aid in the early diagnostic of AAA.

研究目的：腹主动脉瘤（Abdominal aortic aneurysm, AAA）是一种致死性外周血管疾病。炎症性免疫细胞浸润是AAA发病机制的核心环节，探究早期AAA免疫浸润的分子机制并寻找可行的AAA标志物具有重要意义。
研究方法：本研究从NCBI-GEO数据库下载了早期AAA模型的多个mRNA表达数据集及单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据集。采用mMCP-counter与CIBERSORT评估早期实验性AAA的免疫浸润水平。随后，利用加权基因共表达网络分析（Weighted Correlation Network Analysis, WGCNA）分析scRNA-seq数据集，筛选浸润至早期AAA的单核细胞/巨噬细胞相关基因模块。通过ClusterProfiler对模块基因开展基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）功能富集分析。借助STRING数据库构建蛋白质相互作用（Protein-Protein Interaction, PPI）网络。采用Limma-Voom分析单核细胞/巨噬细胞的差异表达基因（Differentially Expressed Genes, DEGs），并筛选得到关键基因集。随后在人类AAA数据集验证关键基因的表达，构建用于区分AAA患者与健康人群的逻辑回归诊断模型。最后，通过实时定量聚合酶链反应（Real-time quantitative polymerase chain reaction, RT-qPCR）与酶联免疫吸附试验（Enzyme Linked Immunosorbent Assay, ELISA）验证本队列中AAA患者与健康供体样本的基因表达及血清蛋白水平差异。
研究结果：单核细胞/巨噬细胞被鉴定为浸润早期实验性AAA的主要免疫细胞。对scRNA-seq数据集的单核细胞/巨噬细胞进行伪细胞构建并开展WGCNA分析后，从两个数据集中共筛选出4个与AAA显著正相关的基因模块；功能富集分析显示，这些模块主要富集于髓系白细胞迁移、含胶原蛋白的细胞外基质及PI3K-Akt信号通路。在模块核心基因中鉴定出Thbs1、Clec4e与Il1b为关键基因，且在其他数据集中证实在Clec4e、Il1b及Thbs1的表达水平显著升高。进一步在人类AAA转录组数据集中验证发现CLEC4E、IL1B呈高表达，基于这两个基因的表达构建逻辑回归模型，训练集AUC为0.9，验证集AUC为0.79。此外，在本队列中证实AAA患者血清IL-1β与CLEC4E蛋白水平升高，且AAA主动脉样本中这两个基因的表达亦显著上调。
研究结论：本研究鉴定出单核细胞/巨噬细胞是浸润早期AAA的主要免疫细胞，并构建了基于单核细胞/巨噬细胞相关基因集的逻辑回归诊断模型，可为AAA的早期诊断提供参考依据。