miR487a-3p and miR6855-3p Facilitate Macrophage Proinflammatory Polarization and Lipid Accumulation in Atherosclerosis

BACKGROUND: The integrated regulation of microRNAs on macrophage plasticity plays a key role in atherosclerosis. We tested the hypothesis that miR487a-3p and miR6855-3p accelerate atherosclerosis by intensifying macrophage inflammatory response and metabolic dysregulation. METHODS: The microRNA sequencing and messenger RNA sequencing were conducted in peripheral monocytes from patients with coronary artery disease (CAD) and healthy controls. Macrophages in mouse aortas and human coronary arteries were characterized using flow cytometry and immunostaining. Atherosclerosis development was evaluated in male PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpression mice harboring myeloid cell-specific deficiency of CPE (carboxypeptidase E) or RRM2 (ribonucleotide reductase regulatory subunit M2) and challenged with a high-fat diet. RESULTS: miR487a-3p and miR6855-3p were the top microRNA candidates identified by microRNA sequencing in peripheral monocytes and validated by quantitative real-time polymerase chain reaction, with significant differences between patients with CAD and controls. Both microRNAs were lipid-inducible and secreted extracellularly. KLF (Krüppel-like factor 5) and IRF1 (interferon regulatory factor 1) bound to the promoter regions of miR487a-3p and miR6855-3p, respectively, to enhance their transcription. Accordingly, patients with CAD exhibited significantly elevated plasma miR487a-3p and miR6855-3p levels compared with controls, which positively correlated with blood lipid levels and Gensini score (reflecting CAD severity and prognosis). The area under the receiver operating characteristic curve (≈0.83 for each) supported their diagnostic accuracy. Of note, miR487a-3p and miR6855-3p were predominantly expressed in coronary arterial macrophages. The dramatic expansion of miR487a-3p + and miR6855-3p + macrophages and the elevated expression of both microRNAs in coronary arteries were positively associated with lesion area in patients with CAD. Mechanistically, transcriptomic analyses and functional assays revealed that elevated miR487a-3p or miR6855-3p promoted macrophage proinflammatory responses, lipid metabolic dysregulation, and foam cell formation. Conversely, inhibition of either microRNA alleviated ox-LDL (oxidized low-density lipoprotein)–induced macrophage inflammatory responses and lipid metabolic dysfunction. Moreover, conditioned medium from miR487a-3p- or miR6855-3p-overexpressing macrophages promoted endothelial cell apoptosis, whereas this effect was attenuated when endothelial cells were exposed to medium from ox-LDL-treated macrophages with microRNA inhibition. Furthermore, integration of downregulated genes from monocyte and macrophage messenger RNA sequencing with TargetScan-predicted targets identified CPE and RRM2 as targets of miR487a-3p and miR6855-3p, respectively. Direct binding was confirmed by dual-luciferase assays and microRNA pulldown. Overexpression of CPE or RRM2 partially reversed the detrimental effects of miR487a-3p and miR6855-3p, respectively, on macrophage phenotypic switching and metabolic dysregulation. Conversely, monocyte-/macrophage-specific depletion of CPE or RRM2 aggravated atherosclerosis progression in hypercholesterolemic mice by instigating macrophage inflammatory responses and lipid metabolic disturbance. CONCLUSIONS: miR487a-3p and miR6855-3p fulfill the criteria of promising biomarkers for CAD diagnosis and prognosis. Mechanistically, they intensify inflammatory responses and disrupt lipid metabolism in macrophages, identifying both microRNAs as potential therapeutic targets for CAD.