MLK3 promotes atherosclerosis by regulating ferroptosis in macrophages

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.

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.

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.

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