To further validate the relationship between SPP1+ macrophages and atherosclerosis progression, the gene signatures of SPP1+ macrophages were tested using bulk mRNA arrays from isolated macrophage-rich regions of stable and ruptured human atherosclerotic plaques by performing GSEA, which revealed that the gene signatures were significantly upregulated in ruptured plaques (Additional file 5: Figure S5e). This evidence concerns the gene SPP1 and atherosclerosis.