CXCL16 promotes macrophage-driven inflammation and vascular smooth muscle cell phenotypic switching during carotid plaque destabilization

Carotid plaque instability is a major determinant of ischemic stroke and is characterized by heightened inflammation and structural remodeling of the vessel wall. Although macrophages and vascular smooth muscle cells (VSMCs) are central to plaque vulnerability, the mechanisms coordinating immune activation with vascular remodeling remain incompletely understood. Bulk transcriptomic data from multiple Gene Expression Omnibus (GEO) datasets were integrated to compare unstable and stable carotid plaques. Differential gene expression analysis, weighted gene co-expression network analysis, immune-gene curation, and machine learning methods (least absolute shrinkage and selection operator [LASSO] and random forest) were used to identify key genes and construct a nomogram. The findings were validated using independent datasets, single-cell RNA sequencing, and human carotid plaque specimens. The mechanistic roles of CXCL16 were examined using macrophage functional assays, NF-κB inhibition, VSMC co-culture with macrophage-conditioned media, and the establishment of an ApoE −/− carotid atherosclerosis model with local CXCL16 suppression. Cell-cell communication and pseudotime analyses were performed to explore macrophage-VSMC interactions. CXCL16 , CCL2 , and MMP9 were consistently upregulated in unstable plaques and showed robust diagnostic performance across datasets. A three-gene nomogram generated from this study suggested potential clinical utility. Single-cell analyses indicated that CXCL16 was enriched in plaque-associated M1 macrophages and was associated with inflammatory activation states. Human plaque staining confirmed higher CXCL16/CCL2/MMP9 expression in unstable plaques with increased macrophage and leukocyte infiltration. In vitro, CXCL16 knockdown attenuated NF-κB activation and reduced downstream inflammatory mediators (including CCL2), accompanied by decreased macrophage migration; NF-κB inhibition phenocopied these effects. In vivo, CXCL16 suppression reduced carotid plaque formation and inflammatory cell infiltration. Cell-cell communication analysis revealed enhanced SPP1/osteopontin signaling from M1 macrophages toward VSMCs, with higher SPP1 expression in CXCL16-high M1 macrophages. Co-culture experiments showed that macrophage-derived CXCL16 promoted VSMC migration and phenotypic switching, which was reversed by CXCL16 knockdown. CXCL16 acts as a central inflammatory mediator in carotid plaque destabilization by promoting NF-κB–dependent macrophage activation and migration. It also enhances SPP1/osteopontin-associated macrophage-VSMC crosstalk that drives phenotypic remodeling in VSMCs. Our results collectively suggest CXCL16 as a diagnostic biomarker and a potential therapeutic target for carotid atherosclerosis. • CXCL16 drives carotid plaque destabilization by promoting macrophage inflammation and VSMC switching. • Transcriptomic and single-cell analyses highlight CXCL16 as a key inflammatory mediator in plaque instability. • CXCL16 suppression reduces macrophage activation and VSMC migration, suggesting a therapeutic target.