Abstract

Background Large-artery (LAA-stroke) is a leading cause of cardiovascular morbidity and mortality worldwide. Recent evidence indicates that lysosomal dysfunction plays a critical role in its pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Identifying characteristic lysosome-related genes (LRGs) offers new perspectives for deciphering disease mechanisms and developing novel therapeutic strategies. Methods This study integrated multi-omics data and experimental validation to identify key LRGs in LAA-stroke. We analyzed transcriptomic datasets ( GSE100927 and GSE28829 ) through differential expression analysis, protein-protein interaction(PPI) network construction, and machine learning algorithms to screen characteristic LRGs. Single-cell RNA sequencing data ( GSE159677 ) were utilized to resolve gene expression patterns across cellular subpopulations in atherosclerotic plaques. A series of experimental approaches, including in silico knockout, RNA sequencing, and in vitro assays, were employed to validate the role of ANPEP in macrophage foam cell formation. Results We identified seven characteristic genes from 63 differentially expressed LRGs. Among them, Aminopeptidase N(ANPEP) demonstrated outstanding diagnostic performance in both training and external validation cohorts (AUC: 0.803–1.000). Single-cell analysis revealed specific enrichment of ANPEP in macrophages, particularly in the TREM2+ lipid-associated macrophage subset. Pseudotime trajectory analysis indicated that ANPEP expression closely aligned with lipid transport genes such as SPP1, CD36, and ABCA1. Functional experiments confirmed that ANPEP knockdown significantly suppressed ox-LDL-induced foam cell formation and intracellular lipid deposition by modulating lipid metabolism pathways, including VDR and MAPK signaling. Conclusions This multi-level study suggests that ANPEP may serve as a key regulator of macrophage lipid metabolism in LAA-stroke. Our finding