MLK3 promotes atherosclerosis by regulating ferroptosis in macrophages

Background: Atherosclerosis (AS) is the leading cause of cardiovascular death worldwide. Macrophage Ferroptosis is implicated in AS progression. The precise mechanism by which Mixed Lineage Kinase 3 (MAP3K11, MLK3) regulates macrophage Ferroptosis in atherosclerosis is poorly understood.

Methods: From the Gene Expression Omnibus (GEO) database, bulk and single-cell RNA-sequencing (scRNA-seq) datasets from vascular tissues were obtained. MLK3 was discovered to be a crucial ferroptosis-related gene (FRG) implicated in AS by means of differential analysis, weighted gene co-expression network analysis (WGCNA), and a Ferroptosis gene set. Macrophages were shown to be the main cell type that expressed MLK3 when immune-infiltration profiling and single-cell RNA Sequencing were combined. In vitro, macrophages were treated with Oxidized low-density lipoprotein (ox-LDL) or erastin, with or without MLK3 knockdown. Animal models assessed AS progression, Ferroptosis, Collagen deposition, and JNK/p53 activation. Immunofluorescence and clinical plasma MLK3 assays were performed.

Results: MLK3 was predominantly expressed in plaque macrophages. Ox-LDL upregulated MLK3 and induced Ferroptosis, while MLK3 knockdown attenuated ox-LDL-induced Ferroptosis by suppressing JNK/p53 signaling, without affecting erastin-induced Ferroptosis. Animal models showed increased MLK3 expression, Ferroptosis, JNK/p53 activation, plaque area, and Collagen deposition during AS progression. Immunofluorescence confirmed MLK3 co-localization with ferroptosis-associated proteins in plaque macrophages. Plasma MLK3 levels were consistently elevated in AS patients.

Conclusion: MLK3 accelerates the development of atherosclerotic lesions by driving macrophage Ferroptosis through JNK/p53 signaling.