miR487a-3p and miR6855-3p Facilitate Macrophage Pro-inflammatory Polarization and Lipid Accumulation in Atherosclerosis
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BACKGROUND
The integrated regulation of microRNAs (miRNAs) on macrophage plasticity plays a key role in atherosclerosis (AS). We tested the hypothesis that miR487a-3p and miR6855-3p accelerate AS by intensifying macrophage inflammatory response and metabolic dysregulation.
METHODS
MiRNA sequencing (miR-seq) and mRNA sequencing (mRNA-seq) were conducted in peripheral monocytes from CAD patients and healthy controls. Macrophages in mouse aortas and human coronary arteries were characterized using flow cytometry and immunostaining. AS development was evaluated in male PCSK9-overexpression mice harboring myeloid cell-specific deficiency of carboxypeptidase E (CPE) or ribonucleotide reductase subunit M2 (RRM2) and challenged with a high-fat diet.
RESULTS
miR487a-3p and miR6855-3p were the top miRNA candidates identified by miR-seq in peripheral monocytes and validated by qPCR, with significant differences between coronary artery disease (CAD) patients and controls. Both miRNAs were lipid-inducible and secreted extracellularly. Krüppel-like factor 5 and interferon regulatory factor 1 bound to the promoter regions of miR-487a-3p and miR-6855-3p, respectively, to enhance their transcription. Accordingly, CAD patients 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 miR-487a-3p⁺ and miR-6855-3p⁺ macrophages and the elevated expression of both miRNAs in coronary arteries were positively associated with lesion area in CAD patients. Mechanistically, transcriptomic analyses and functional assays revealed that elevated miR-487a-3p or miR-6855-3p promoted macrophage pro-inflammatory responses, lipid metabolic dysregulation, and foam cell formation. Conversely, inhibition of either miRNA alleviated ox-LDL-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 miRNA inhibition. Furthermore, integration of downregulated genes from monocyte and macrophage mRNA-seq with TargetScan-predicted targets identified CPE and RRM2 as targets of miR-487a-3p and miR-6855-3p, respectively. Direct binding was confirmed by dual-luciferase assays and miRNA 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 AS progression in hypercholesterolemic mice by instigating macrophage inflammatory responses and lipid metabolic disturbance.
CONCLUSIONS
miR487a-3p and miR6855-3p fulfil the criteria of promising biomarkers for CAD diagnosis and prognosis. Mechanistically, they intensify inflammatory responses and disrupt lipid metabolism in macrophages, identifying both miRNAs as potential therapeutic targets for CAD.
