Abstract Body: Background: During atherosclerotic plaque formation, arterial smooth muscle cells (SMCs) migrate from the medial layer into the intima and undergo complex phenotypic modulation (PM), characterized by loss of contractile marker expression and heterogeneous ectopic expression of markers of macrophages, fibroblasts, osteogenic cells, and stem cells. Our previous work has established that cholesterol-induced endoplasmic reticulum stress, particularly PERK signaling, is a key driver of SMC migration and PM in atherosclerosis. However, the molecular cues that direct SMC migration into the plaque, especially against the significant arterial transmural pressure gradient, remain incompletely defined.
Methods: We analyzed single cell RNA sequencing datasets from both murine and human atherosclerotic plaques to identify macrophage-derived ligands predicted to signal to SMCs. SMC migration, PERK signaling and PM were assessed in vitro using recombinant proteins, antibody-mediated neutralization and pharmacological intervention.
Results: Single-cell analyses revealed enrichment of secreted phosphoprotein 1 (SPP1) or osteopontin in plaque macrophages and modulated SMCs, while contractile SMCs preferentially expressed SPP1 receptors including integrin α8β1 and CD44, with the latter’s expression increasing with SMC modulation. Recombinant SPP1 induced robust SMC migration and PM comparable to cholesterol loading, accompanied by PERK activation. Conditioned media from cholesterol-loaded macrophages similarly induced SMC migration, PERK signaling and PM, effects that were abolished by antibody-mediated SPP1 neutralization. Mechanistically, pharmacologic inhibition of focal adhesion kinase (FAK) blocked SPP1-induced migration, PERK activation, and expression of PM markers, identifying a SPP1–FAK–PERK signaling axis linking macrophage-derived cues to SMC stress responses.
Conclusion: These results identify SPP1 as a macrophage-derived regulator of SMC migration, PERK activation and PM. Since circulating SPP1 levels correlate with plaque burden and adverse cardiovascular outcomes in humans, these findings provide mechanistic context for SPP1 as an active regulator of SMC migration and PM in atherosclerosis, instead of being just a correlative biomarker. Together, this work provides a mechanistic foundation for future studies dissecting the cell type–specific roles of SPP1 during atherosclerotic plaque progression.