FTO suppresses PRTN3 translation via m⁶A demethylation to block the CXCL9/CXCR3 axis and attenuate atherosclerosis.

Coronary artery disease (CAD) involves intricate immune-related pathways; however, the contribution of epigenetic mechanisms remains inadequately defined. The fat mass and obesity-associated protein (FTO), which functions as an N⁶-methyladenosine (m⁶A) demethylase, has been implicated in the progression of atherosclerotic conditions. This study sought to elucidate how FTO downregulates proteinase 3 (PRTN3) through m⁶A demethylation and inhibits neutrophil activation via the C-X-C motif chemokine ligand 9/C-X-C motif chemokine receptor 3 (CXCL9/CXCR3) signaling pathway in the context of CAD. RNA sequencing was carried out on peripheral blood mononuclear cells (PBMCs) obtained from CAD patients and healthy individuals to detect gene expression differences. Functional enrichment analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, were performed along with experimental validation in endothelial progenitor cells (EPCs). The regulatory relationship between FTO and PRTN3 was examined via luciferase reporter assays, RNA immunoprecipitation, and methylated RNA immunoprecipitation quantitative polymerase chain reaction. Neutrophil activation was evaluated by measuring CXCL9/CXCR3 expression, tracking cell migration, and assessing reactive oxygen species (ROS) generation in HL-60 cells. For in vivo validation, apolipoprotein E-deficient (ApoE⁻/⁻) mice were maintained on a high-fat diet and treated with the AAV9 vector carrying FTO via tail vein injection to evaluate effects on atherosclerotic development. Findings indicated that PRTN3 is significantly upregulated in CAD patients, which was corroborated in EPCs. FTO was shown to directly bind to PRTN3 and decrease its expression by reducing m⁶A methylation. Overexpression of FTO enhanced proliferation and migration of EPCs and reduced apoptosis, whereas FTO silencing produced opposing outcomes. Furthermore, PRTN3 was found to stimulate the CXCL9/CXCR3 axis, leading to increased neutrophil migration and ROS production. In vivo, FTO inhibits the activation of neutrophils by down-regulating the expression of PRTN3, reduces the inflammatory response, and protects the occurrence and development of atherosclerosis in mice. These results uncover a novel regulatory pathway involving FTO, m⁶A, PRTN3, and CXCL9/CXCR3 in CAD pathogenesis, highlighting FTO as a promising target for therapeutic intervention.