Abstract
BACKGROUND: Dysregulated macrophages are important causes of Atherosclerosis (AS) formation and increased plaque instability, but the heterogeneity of these plaques and the role of macrophage subtypes in plaque instability have yet to be clarified. METHODS: This study integrates single-cell and bulk-seq data to analyze atherosclerotic plaques. Unsupervised clustering was used to reveal distinct plaque subtypes, while survival analysis and gene set variation analysis (GSVA) methods helped in understanding their clinical outcomes. Enrichment of differential expression of macrophage genes (DEMGs) score and pseudo-trajectory analysis were utilized to explore the biological functions and differentiation stages of macrophage subtypes in AS progression. Additionally, CellChat and the BayesPrism deconvolution method were used to elucidate macrophage subtype interaction and their prognostic significance at single-cell resolution. Finally, the expression of biomarkers was validated in mouse experiments. RESULTS: Three distinct AS plaque subtypes were identified, with cluster 3 plaque subtype being particularly associated with higher immune infiltration and poorer prognosis. The DEMGs score exhibited a significant elevation in three macrophage subtypes (SPP1(+)/VCAN(+) macrophages, IL1B(+) macrophages, and FLT3LG(+) macrophages), associated with cluster 3 plaque subtype and highlighted the prognostic significance of these subtypes. Activation trajectory of the macrophage subtypes is divided into three states (Pre-branch, Cell fate 1, and Cell fate 2), and Cell fate 2 (SPP1(+)/VCAN(+) macrophages, IL1B(+) macrophages, and FLT3LG(+) macrophages dominant) exhibiting the highest DEMGs score, distinct interactions with other cell components, and relating to poorer prognosis of ischemic events. This study also uncovered a unique SPP1(+)/VCAN(+) macrophage subtype, rare in quantity but significant in influencing AS progression. Machine learning algorithms identified 10 biomarkers crucial for AS diagnosis. The validation of these biomarkers was performed using Mendelian Randomization analysis and in vitro methods, supporting their relevance in AS pathology. CONCLUSION: Our study provides a comprehensive view of AS plaque heterogeneity and the prognostic significance of macrophage subtypes in plaque instability.