Purpose: Atherosclerosis is a chronic inflammatory disease exacerbated by obesity. Using an atherosclerosis murine disease model, our objectives were to map the presence of immune and structural cells, to explore their potential interactions, and to investigate how obesity impacts them in different tissues.

Methods: We used ten Ldlr-/- Apob100/100 mice: five non-obese mice with a standard chow diet and five obese mice with a high-fat diet. We collected perivascular adipose tissue (PVAT), aorta, spleen, and epididymal white adipose tissue (eWAT) from each mouse, creating eight samples pooled by the tissue types. Following the tissue dissociation and enrichment of CD45+ cells from aorta samples, single-cell RNA-sequencing was performed using a 5’ gene expression immune profiling protocol, combining it with a panel of 138 antibodies, resulting in a multimodal data of ~ 46k cells. For spatial validation we used immunohistochemistry and immunofluorescence in an independent cohort of similar mice.

Results: In addition to identifying various immune cell populations and their subtypes, we observed marked diversity in structural cells, with particularly notable gene expression changes in fibroblasts between the non-obese and obese states. The most substantial changes were evident in the aorta, PVAT and eWAT. PVAT- and eWAT-derived fibroblast populations were especially interesting as a gene set enrichment analysis, based on the genes that were differentially expressed between the obesity states, revealed multiple immune-related functions. We validated the location of one PVAT-derived fibroblast populations with immunohistochemistry/immunofluorescence and observed a difference in their abundance between the obesity states: these fibroblasts were more abundant in average in the non-obese mice in comparison with the obese mice. This could be due to morphological changes in the PVAT towards a white adipose tissue phenotype during obesity evident in a morphometric analysis.

Conclusion: Our findings provide novel insights into obesity-related alterations in adipose tissue-derived fibroblasts during atherosclerosis and their potential immune-related responses during the disease. Our results emphasize the significance of PVAT in atherosclerosis during obesity and how obesity might abolish the functionality of specific fibroblasts that could otherwise support normal immune responses during the disease.