Molecular and spatiotemporal characterization of cells in murine atherosclerotic plaques

Objective

Single-cell technologies have revolutionized our understanding of the phenotypic and transcriptional diversity of aortic leukocytes in atherosclerotic humans and mice. However, enzymatically dissociated tissues lose the spatial context of plaque cells in situ . Here we utilized imaging mass cytometry (IMC) combining with single-cell RNA sequencing (scRNA-seq) to characterize the spatial distribution dynamics, phenotypic transitions, metabolic and functional phenotypes, and the intercellular interaction networks of plaque cells during atherosclerotic progression. Additionally, the dynamic immune landscape of circulating leukocytes associated with atherosclerosis was characterized using cytometry of time of flight (CyTOF).

Approach and Results

A highly multiplexed IMC panel with 33 metal-conjugated antibodies was designed to generate 11 highly multiplexed histology images of aortic root tissues from ApoE ^-/- mice on high-fat diet at different stage of atherosclerosis. Using histoCAT, we identified 8 principal cell subtypes with distinct phenotypic and geographic dynamics. Furthermore, IMC-defined cell subsets partially corresponded to scRNA-seq-annotated aortic cell subtypes, including 4 macrophage subsets, neutrophils, smooth muscle cells (SMCs) and SMC-derived SEMs (Stem cell, endothelial cell and macrophage-like cell). Activation of inflammatory pathways, increased oxidative phosphorylation and augmented osteoclast differentiation were observed in macrophage populations, SMCs and SEMs from an early stage to advanced stage of atherosclerosis. Notably, cell neighborhood analysis by IMC uncovered multifaceted cell-cell interactions within the plaque, in particular in neutrophil-mediated interactions with smooth muscle cells and macrophages, which were confirmed by ligand-receptor interactions based on scRNA-seq data.

Additionally, characterization of the peripheral immune cells by CyTOF revealed an increased ratio of myeloid cells to lymphocytes, and certain neutrophil and monocyte subpopulations also exhibited enhanced lipid metabolism and glycolysis as well as activated inflammatory signaling.

Conclusion

This study provides a dynamic spatiotemporal landscape of atherosclerotic lesions and peripheral leukocytes. The new information based on IMC may help understand atherosclerotic pathology and develop novel therapeutic strategies.