Abstract Body: Introduction: Carotid artery disease involves atherosclerotic plaque formation within the arterial wall, which can lead to stroke. In advanced atherosclerosis, macrophage (Mφ) efferocytosis, a process of apoptotic cell (AC) clearance, is impaired, leading to AC accumulation and plaque progression. Plaque progression involves coordinated communication among multiple plaque cell types, mediated by extracellular vesicles (EVs). The role of plaque EV-mediated communication in modulating Mφ efferocytosis remains unknown. We hypothesize that plaque-EVs regulate Mφ efferocytosis.
Methods: EVs were isolated from human carotid atherosclerotic plaques using size-exclusion chromatography and characterized according to MISEV2023 guidelines. Proteomic analysis of plaque-EVs was performed using a published dataset generated by our lab. Human Mφ were treated with plaque-EVs (10¹⁰EVs; 24h). RNA sequencing was performed, followed by differential gene expression analysis (Fold change>1.5; P_adj<0.05) and pathway enrichment (overrepresentation analysis), with validation of efferocytosis-related transcripts by RT–qPCR. Mφ efferocytic capacity was assessed using an in vitro efferocytosis assay, in which treated Mφ were incubated with ACs at a 1:3 ratio for 2h and quantified by confocal microscopy, with plaque eluate-treated Mφ as a control.
Results: Carotid plaque-EVs were isolated at a yield of ~8.88×10⁸ particles/mg of plaque, with a mean diameter of ~175 nm (n=10 plaques), and intact EVs were visualized by cryo-EM. Plaque-EV proteome enriched for phagocytosis, AC clearance, and Mφ activation pathways. Transcriptomic profiling of Mφs treated with plaque-EVs revealed upregulation of 925 genes and downregulation of 1182 genes compared to naïve Mφs (n=4). Pathway analysis of downregulated genes enriched for efferocytosis, phagosome, phagocytosis and AC clearance pathways, while upregulated genes enriched for lipids and atherosclerosis, and chemotaxis pathways (P_adj<0.05). Plaque-EV treated Mφs showed a downregulation in pro-efferocytic genes MERTK, AXL, and GAS6 compared to naïve Mφs (n=6, P<0.05). Plaque-EV treated Mφs had ~15% reduced efferocytic capacity (45% vs 60%) compared to naïve and plaque eluate-treated Mφs (n=7-10, P<0.05).
Conclusion: Our study showed that carotid plaque-EVs downregulate Mφ efferocytosis. These findings position plaque-EVs as both therapeutic targets and potential delivery vehicles, where modulation of EV cargo may be leveraged to restore efferocytosis.