Recent Advances in Single-Cell Analysis of Atherosclerotic Plaque Biology

Atherosclerosis, the leading cause of coronary artery disease, stroke, and peripheral arterial disease, is now recognized as a lipid-driven disease complicated by an immune response that plays a major role in its pathogenesis. The response-to-injury hypothesis proposed by Ross et al. laid the foundation for understanding atherosclerosis as a chronic inflammatory process, in which endothelial injury and lipid insudation trigger immune activation, smooth muscle cell proliferation, and plaque formation. Traditional approaches, such as immunohistochemistry, flow cytometry, and bulk RNA sequencing, have identified macrophages and T cells as the key immune players in plaques. However, these methods lack the resolution to differentiate among diverse immune cell states or to detect rare but functionally significant populations. Recent advances in single-cell and spatial transcriptomic technologies have revolutionized our understanding of atherosclerotic plaques. These methods have generated detailed cellular atlases in murine models and human atherosclerotic tissues, revealing previously unrecognized immune cell subsets and novel pathogenic pathways. Single-cell analyses have identified a heterogeneous spectrum of macrophages, including resident-like, inflammatory, and TREM2^high foamy subsets, in addition to a CD163⁺ macrophage subset, including the hemoglobin-stimulated macrophage [M(Hb)] phenotype. In parallel, functionally diverse T-cell subsets with specialized pro- and anti-inflammatory roles have also been characterized. Spatial transcriptomics has provided further insights into the anatomical organization of these immune populations within plaques, highlighting region-specific inflammatory niches and fibrous-cap dynamics. Furthermore, single-cell T-cell receptor sequencing has identified antigen-specific T-cell expansions, supporting the hypothesis that atherosclerosis exhibits autoimmune-like characteristics. These findings have major therapeutic implications. The selective targeting of specific types of pro-inflammatory macrophages and tailored immunomodulation of T-cell subsets may provide new strategies to stabilize plaques and other novel and targeted immunomodulatory approaches to prevent cardiovascular events. As single-cell and spatial technologies continue to evolve, they will further refine our ability to design precision immunotherapies for atherosclerosis by integrating classical inflammatory models with high-resolution molecular insights.