Molecular and spatiotemporal characterization of cells in murine atherosclerotic plaques

Background: Single-cell technologies have revolutionized our understanding of the phenotypic and transcriptional diversity of aortic leukocytes in atherosclerotic humans and mice. However, many studies using enzymatic digestion methods lack the spatial context of plaque cells. Methods: We utilized imaging mass cytometry (IMC) in combination with single-cell RNA sequencing (scRNA-seq) to elucidate the spatial distribution dynamics, phenotypic transitions, metabolic and functional changes, and the intercellular interaction networks of plaque cells during atherosclerotic progression. Additionally, we characterized the dynamic immune landscape of circulating leukocytes using cytometry of time of flight (CyTOF). Results: A highly multiplexed IMC panel comprising 33 metal-conjugated antibodies was designed to generate 11 highly multiplexed histology images of aortic root tissues from high-fat diet feeding ApoE-/- mice at different stage of atherosclerosis. Using histoCAT, we identified 8 principal cell subtypes with distinct phenotypic profiles and dynamic geolocations. Moreover, these IMC-defined cell subsets exhibited partial correspondence with aortic cell subtypes identified through scRNA-seq analysis, including 4 macrophage subsets, neutrophils, and smooth muscle cells (SMCs), SMCs-derived SEMs. In the context of atherosclerosis, we observed the activation of inflammatory pathways, enhanced OXPHOS, and osteoclast differentiation in macrophage populations, SMCs and SEMs. Remarkably, cell neighborhood analysis by IMC unveiled cell-cell interactions within the plaque microenvironment, particularly highlighting interactions between neutrophils, smooth muscle cells, and macrophages. These interactions were further confirmed through ligand-receptor interactions based on scRNA-seq data. Additionally, using CyTOF, we characterized the immune landscape of peripheral blood, revealing elevated myeloid cell populations and significantly reduced lymphocytes, accompanying with upregulation of lipid metabolism and glycolysis-associated regulators and activated inflammatory signaling in specific neutrophil and monocyte subpopulations. Conclusions: These data comprehensively depicted the dynamic spatiotemporal landscapes of atherosclerotic lesions and peripheral blood, contributing to the advancement of our knowledge and identification of potential therapeutic targets for cardiovascular disease.