Single‐Cell Data and Weighted Correlation Network Analysis Revealed the Regulatory Mechanisms of Macrophages in Carotid Plaques

<b>Background:</b> Macrophages play a critical role in carotid plaque. Understanding the mechanisms of carotid plaque formation based on macrophage heterogeneity could provide valuable insights for clinical intervention. <b>Methods:</b> Single-cell transcriptome and bulk RNA-seq data of carotid plaque were obtained from public databases. Weighted gene correlation network analysis (WGCNA) identified gene modules linked to unstable plaques. Macrophage marker genes were intersected with module genes of WGCNA, followed by using randomForest and LASSO regression to pinpoint key genes. Quantitative real-time PCR (qRT-PCR) and Western blot were used to verify the regulation of key genes at the cellular level. The correlation between the key genes and inflammatory phenotypes was examined by single-sample gene set enrichment analysis (ssGSEA). <b>Results:</b> Single-cell clustering revealed major cellular subpopulations, with elevated macrophage infiltration in carotid plaque. Six key macrophage-associated genes (<i>ADPGK, ATP6V1F, CX3CR1, MYO9B, RNF135</i>, and <i>SLC7A8</i>) were discovered. The qRT-PCR results demonstrated upregulation of <i>ADPGK, ATP6V1F</i>, and <i>RNF135</i> genes in vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (ox-LDL), except for <i>CX3CR1</i>, which was downregulated. Protein expression results showed that expressions of <i>ADPGK, ATP6V1F, RNF135</i>, and <i>SLC7A8</i> were significantly elevated in the ox-LDL-VSMC group. In addition, most of the immune cells showed significant differences between the unstable arterial plaque group and the control group. <b>Conclusion:</b> This study discovered potential biomarkers that affected carotid plaque progression and macrophage regulation at the single-cell level, and examined their regulatory roles in immune regulation, programed cell death (PCD), and inflammatory factor modulation.