背景:心脑血管事件的风险增加与动脉粥样硬化斑块的不稳定性增加有关。然而,目前缺乏有效的诊断性生物标志物阻碍了对斑块不稳定性的评估.本研究旨在通过整合各种生物信息学工具来调查和鉴定与不稳定斑块相关的hub基因。提供对这种情况的检测和治疗的新见解。
方法:结合两种机器学习方法的加权基因共表达网络分析(WGCNA)用于鉴定与斑块不稳定性强相关的枢纽基因。通过估计RNA转录本的相对子集(CIBERSORT)方法进行细胞类型鉴定,以评估动脉粥样硬化患者的免疫细胞浸润模式。此外,进行基因集变异分析(GSVA)以研究潜在的生物学功能,通路,以及与不稳定斑块相关的hub基因的机制。为了进一步验证hub基因的诊断效率和表达,免疫组织化学(IHC),定量实时聚合酶链反应(RT-qPCR),对采集的人颈动脉斑块和血液样本进行酶联免疫吸附试验(ELISA)。免疫荧光共染色也用于确认hub基因和免疫细胞之间的关联,以及它们与线粒体的共定位。
结果:TheCIBERSORT分析显示,动脉粥样硬化患者CD8T细胞浸润显着减少,M0巨噬细胞浸润明显增加。随后,我们发现了两个与动脉粥样硬化斑块不稳定性密切相关的高度相关模块(蓝色和绿色).通过与线粒体相关基因的交叉,确定了50个关键基因。采用最小绝对收缩和选择算子(LASSO)逻辑回归和支持向量机递归特征消除(SVM-RFE)算法的进一步分析显示,六个集线器基因与斑块不稳定性显着相关。其中,NT5DC3,ACADL,SLC25A4,ALDH1B1和MAOB与CD8T细胞呈正相关,与M0巨噬细胞呈负相关,犬尿氨酸3-单氧合(KMO)与M0巨噬细胞呈正相关,与CD8T细胞呈负相关。人颈动脉斑块样本的IHC和RT-qPCR分析,以及血液样本的ELISA分析,显示KMO和MAOB表达显著上调,随着ALDH1B1表达的减少,与对照样品相比,在稳定和不稳定样品中。然而,在上述三个关键基因中,与稳定斑块样品相比,仅KMO在不稳定斑块样品中显示表达显著增加。此外,使用免疫荧光共染色技术评估了KMO在人颈动脉不稳定斑块组织和培养的小鼠巨噬细胞系中的表达模式.最后,慢病毒介导的KMO沉默被成功地转导到高脂喂养的ApoE-/-小鼠的主动脉中,结果表明,KMO沉默可以减轻ApoE-/-小鼠的斑块形成并促进斑块稳定性。
结论:结果表明,KMO,与巨噬细胞相关的线粒体靶向基因,有望作为评估动脉粥样硬化斑块不稳定性的有价值的诊断生物标志物。
BACKGROUND: The heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition.
METHODS: Weighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria.
RESULTS: The CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice.
CONCLUSIONS: The results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.