MGST3 defines a mitochondrial-health-associated M2 macrophage state in atherosclerosis: integrative multi-omics, single-cell mediation, and functional validation.

Abstract Background: Mitochondrial dysfunction is increasingly recognized as a driver of maladaptive macrophage polarization in atherosclerosis (AS), but the mitochondrial genes that shape disease-relevant macrophage states remain poorly defined. Methods: We integrated weighted gene co-expression network analysis (WGCNA), machine learning, two-sample Mendelian randomization (two-sample MR), bulk transcriptomics, single-cell analysis, disease-level validation, single-cell mediation, and Results: Eight candidate genes were consistently prioritized by WGCNA and machine learning; among them, microsomal glutathione S-transferase 3 (MGST3) showed the strongest protective genetic signal. Two-sample MR supported a protective association between genetically predicted MGST3 expression and coronary artery disease/coronary heart disease risk (training set OR = 0.989, 95% CI 0.978-0.999, P = 0.042; test set OR = 0.977, 95% CI 0.960-0.995, P = 0.013). Human carotid plaque validation confirmed reduced MGST3 at both mRNA and protein levels in AS lesions. Notably, despite this whole-plaque downregulation, single-cell and disease-level analyses revealed an expanded MGST3-high M2-like macrophage state in AS, suggesting a compensatory protective macrophage program within diseased plaques. MGST3-high M2-like cells displayed an OXPHOS-oriented, antioxidant, and less inflammatory transcriptional state, acted as an active signaling population, and showed higher composite MitoHealth scores. Single-cell mediation analysis further indicated that MitoHealth statistically links higher MGST3 expression to lower inflammatory activity, lower M1-like polarization, and lower atherogenic macrophage programming. In M2-polarized macrophages, MGST3 knockdown reduced M2-associated and mitochondrial/antioxidant transcripts while increasing pro-inflammatory transcripts, providing transcript-level functional support. PheWAS analysis detected no significant phenotype associations at the predefined threshold. Conclusions: These findings define MGST3 as a marker of a mitochondrial-health-associated M2-like macrophage state that appears expanded within AS plaques despite reduced whole-plaque MGST3 expression. The MGST3-MitoHealth axis may represent a compensatory macrophage program linked to mitochondrial preservation and lower pro-atherogenic activation, warranting further diagnostic and therapeutic investigation in AS.