Stage-resolved Spatial Multi-omics Reveals Myeloid Niches in Human Atherosclerotic Plaques

Background: Atherosclerosis, a leading cause of heart attack and stroke, involves intricate immune cell dynamics within arterial plaques, yet their spatial organization and functional roles remain elusive. Methods: We combined Visium HD spatial transcriptomics, imaging mass cytometry, and single-cell RNA-seq across fatty-streak, advanced, and restenotic plaques to map myeloid architectures and relate them to lesion geography and extracellular matrix features. Slingshot trajectory analysis resolved macrophages differentiation path. In vitro, we tested microenvironmental and lipid cues separately: fibronectin (FN) exposure during PMA-driven THP-1 differentiation and oxidized LDL (oxLDL)-induced foam cell formation in murine bone-marrow derived macrophages (BMDMs). Results: Our analysis identified seven macrophage subsets and two neutrophil populations with distinct spatial distribution and functional roles. In early lesions, neutrophils expressing MMP9, MPO, p47phox, TGF-β1 and arachidonate 5-lipoxygenase (ALOX5), aligned with proteolysis, inflammatory processes, and endothelial-mesenchymal transition features. In advanced plaques, macrophage subsets exhibit specialized functions: Ki67+ proliferative macrophages localized near necrotic cores, sustaining local population; SPP1+ macrophages, enriched in lipid handling and tissue remodeling, are prone to apoptosis/ferroptosis, potentially promoting necrotic core expansion; and C3aR+ macrophages form antigen-presenting niches with elevated HLA-DR and CD74, engaging T cells possibly through CXCL12?CXCR4 signaling. Slingshot trajectories indicated progression from C3aR+ toward SPP1+ remodeling states concentrated at fibronectin-rich rims. In vitro, FN increased MMP9 and TIMP1 in THP-1-derived macrophages, consistent with FN imprinting remodeling features characteristic of SPP1? macrophages in situ. Concurrently, oxLDL-treated BMDMs showed enhanced lipid-handling and remodeling modules consistent with the SPP1 program. Conclusions: These findings define conserved myeloid niches and support a microenvironment-imprinting model that links ECM composition and lipid loading to macrophage state transitions, providing a framework for microenvironment-targeted therapies to stabilize plaques and mitigate cardiovascular risk.