Supplementary Material for: Dynamic Gene Expression Signatures Across Stages of Carotid Plaque Calcification

Objective: To elucidate gene expression changes associated with different stages of calcification in carotid artery plaques and clarify the dynamic biological processes underlying plaque maturation. Methods: Carotid plaque specimens were classified into four groups—mildly, moderately, severely, and very severely calcified—based on calcium scores derived from computed tomography. RNA-Seq (SMART-Seq) analysis and qRT-PCR validation were performed to assess expression profiles of genes related to vascular smooth muscle cell (VSMC) phenotype, inflammation, extracellular matrix (ECM), and calcification. Results: Mildly calcified plaques exhibited elevated inflammatory (CD68, TNF) and osteogenic (RUNX2, SPP1/OPN) gene expression. Moderately calcified plaques showed transient suppression of these genes with upregulation of ELN, suggesting structural stabilization. In severely calcified plaques, osteogenic and inflammatory gene expression persisted, while contractile markers (ACTA2, MYH11) showed limited reactivation. Very severely calcified plaques demonstrated downregulation of VSMC-related genes and selective ECM remodeling, indicating a quiescent, structurally stabilized stage. Conclusion: Carotid plaque calcification represents a dynamic, multistage process characterized by reversible and irreversible transitions in VSMC phenotype and ECM composition. These findings emphasize that calcification is not merely passive deposition but reflects coordinated cellular and molecular remodeling, which may inform future stage-specific therapeutic strategies.