Adipose tissue-derived fibroblasts engage in immune-stromal crosstalk during obesity-aggravated atherosclerosis in mice

Background Atherosclerosis is a common disorder where changes in vascular wall and in the surrounding perivascular adipose tissue are evident. However, how multiple cell types contribute to the disease at the cellular level remains poorly understood. Moreover, obesity can exacerbate atherogenesis, but it remains to be revealed which cell types drive the aggravation. Our objectives were to define the key cell populations across tissues in a highly atherogenic mouse model of atherosclerosis under obese and normal-weight conditions, and to explore tissue-specific cellular changes and interactions associated with obesity. Methods We employed 5-prime single-cell RNA sequencing combined with antibody staining in Ldlr-/- Apob100/100 male mice, comparing two groups: animals fed a high-fat diet to induce obesity and those maintained on regular chow as non-obese controls. We harvested aorta, epididymal and perivascular adipose tissues, and spleen from each animal. Aorta samples were CD45+ enriched, and a panel of 138 antibodies was used to generate CITE-seq libraries. Spatial transcriptomics of human carotid arteries and aorta were used to confirm the expression of top targets from the mouse model. Immunohistochemistry and multiplexed immunofluorescence were used to validate the findings. Results We generated data from almost 46000 cells enabling analyses of cell phenotypes, gene set enrichment, regulon activity, and cell interactions. We found fibroblast subpopulations derived from the adipose tissues to be involved in local immune responses and potential contributors to atherosclerosis in obesity. Pi16+ progenitor fibroblasts were significantly reduced and PVAT morphology was remarkably changed in obesity. We also observed clear spatial patterning of the top differentially expressed genes from our mouse model in human aorta and carotid artery. Conclusions We present a comprehensive multi-tissue single-cell analysis of the immune-stromal landscape in a murine model of atherosclerosis in obesity and normal state. Fibroblasts show distinct immune activation especially in perivascular adipose tissue.