Gene expression profiling of peripheral blood mononuclear cells in the setting of peripheral arterial disease

Peripheral arterial disease (PAD) is a relatively common manifestation of systemic atherosclerosis that leads to progressive narrowing of the lumen of leg arteries. Circulating monocytes are in contact with the arterial wall and can serve as reporters of vascular pathology in the setting of PAD. We performed gene expression analysis of peripheral blood mononuclear cells (PBMC) in patients with PAD and controls without PAD to identify differentially regulated genes. We identified 87 genes differentially expressed in the setting of PAD; 40 genes were upregulated and 47 genes were downregulated. We employed an integrated bioinformatics pipeline coupled with literature curation to characterize the functional coherence of differentially regulated genes. Notably, upregulated genes mediate immune response, inflammation, apoptosis, stress response, phosphorylation, hemostasis, platelet activation and platelet aggregation. Downregulated genes included several genes from the zinc finger family that are involved in transcriptional regulation. These results provide insights into molecular mechanisms relevant to the pathophysiology of PAD. PAD was defined as an ankle brachial index (ABI) <= 0.9 (n = 19) while age and gender matched controls had an ABI > 1.0 (n = 18). Microarray analysis was performed using Affymetrix HG-U133 plus 2.0 gene chips and analyzed using GeneSpring GX 11.0. Gene expression data was normalized using Robust Multichip Analysis (RMA) normalization method, differential expression was defined as a fold change [greater than or equal to]1.5, followed by unpaired Mann-Whitney test (P < 0.05) and correction for multiple testing by Benjamini and Hochberg False Discovery Rate. Meta-analysis of differentially expressed genes was performed using an integrated bioinformatics pipeline with tools for enrichment analysis using Gene Ontology (GO) terms, pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular event enrichment using Reactome annotations and network analysis using Ingenuity Pathway Analysis suite. Extensive biocuration was also performed to understand the functional context of genes.

外周动脉疾病（Peripheral arterial disease, PAD）是一类较为常见的系统性动脉粥样硬化表现，可导致下肢动脉管腔进行性狭窄。循环单核细胞可与动脉壁直接接触，可作为PAD发生时血管病理状态的报告指标。本研究对PAD患者与非PAD对照人群的外周血单个核细胞（Peripheral blood mononuclear cells, PBMC）开展基因表达分析，以筛选差异调控基因。最终共鉴定出87个在PAD状态下存在差异表达的基因，其中40个基因上调，47个基因下调。本研究采用整合生物信息学流程结合文献人工注释，对差异调控基因的功能一致性进行系统解析。值得注意的是，上调基因主要参与免疫应答、炎症反应、细胞凋亡、应激反应、磷酸化过程、止血功能、血小板活化及血小板聚集等生物学过程；下调基因则包含多个参与转录调控的锌指家族基因。本研究结果为解析PAD病理生理学相关的分子机制提供了重要见解。本研究中PAD的诊断标准为踝肱指数（ankle brachial index, ABI）≤0.9（病例组样本量n=19），年龄与性别匹配的对照组人群踝肱指数＞1.0（对照组样本量n=18）。基因表达微阵列分析采用Affymetrix HG-U133 Plus 2.0基因芯片完成，数据分析使用GeneSpring GX 11.0软件。基因表达数据采用稳健多芯片平均法（Robust Multichip Analysis, RMA）进行标准化处理；差异表达基因的筛选标准为倍数变化≥1.5，随后采用独立样本曼-惠特尼U检验（P＜0.05），并通过Benjamini与Hochberg错误发现率进行多重检验校正。针对差异表达基因的荟萃分析采用整合生物信息学流程完成，涵盖基于基因本体（Gene Ontology, GO）术语的富集分析、基于京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）的通路分析、基于Reactome注释的分子事件富集分析，以及采用Ingenuity Pathway Analysis套件进行的网络分析。本研究同时开展了大规模生物编注工作，以深入解析基因的功能背景。