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

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Abstract
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.

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