Abstract Body (Do not enter title and authors here): Background: The hallmark of high-risk atheroma is presence of a large lipid/necrotic core (LNC) containing macrophages (MF), foam cells and necrotic tissue. Enlargement of the core contributes to plaque rupture. Cell senescence is an irreversible cell proliferation arrest associated with vascular aging. Senescent MF are reported to be present in atherosclerotic plaques; however, their functional role remains to be elucidated.
Hypothesis: Senescent MF promote expansion of the LNC contributing to plaque destabilization.
Methods: Coronary sections were obtained from atherosclerotic hypercholesteremic pigs. LNCs were identified by staining of cryosections with Oil Red. MF cell marker (scavenger receptor A, SRA), and senescence markers (β-galactosidase (βGal) and γ-H2AX histone) were quantified by immunohistochemistry. βGal activity was assessed with X-Gal assay. Spatial transcriptomics (ST) and single cell RNA seq (scRNA seq) analysis was used to identify differentially expressed genes (DEGs) and quantify senescence module scores in plaque compartments.
Results: Increased βGal activity (2.5-fold increase) and upregulated γ-H2AX histone levels (1.8-fold increase) (both are P<0.05) were detected in cells inside LNCs and in a 50um-thick plaque area surrounding LNCs. The number of senescent MF (SRA+/βGal+ cells) was increased (P<0.05) in the area surrounding LNCs compared to other plaque compartments. Senescent marker positivity was higher in cells in areas surrounding LNCs in plaques with large cores (>40% of core area normalized per plaque area) compared to plaques with smaller cores (<15%). These results suggest that development of senescence correlated with LNC size. Top DEGs in cells inside LNCs were molecules associated with induction or development of cell senescence (cathepsin D, cystatin 3 and ferritin light chain) and MF/foam cell markers (SRA, CD68 and lipoprotein lipase). Senescence module score was higher in LNCs vs. other compartments. scRNA seq and ST data were used to identify and locate MF subtype contributing to senescence in LNCs. M2 polarized MF have higher senescence module scores compared to M1 MF. M2 MF were highly enriched in the area between the LNC and plaque fibrous cap.
Conclusions: Our data suggest that upregulated levels of senescent MF in LNCs and in the surrounding area promote expansion of the LNC and contribute to plaque destabilization. These results identify senescent MF as a potential novel therapeutic target.