Mir147 Limits the Contribution of Non-Foamy Macrophages to Atherosclerosis

BACKGROUND: Hypercholesterolemia and a high-fat diet promote 2 macrophage subtypes involved in atherosclerosis by inducing lipid droplet accumulation in foamy macrophages (FMs) and inflammatory activation in non-foamy macrophages (NFMs). MicroRNAs are key regulators of macrophage function; for instance, miR-10a-5p reduces atherosclerosis and improves mitochondrial health in FMs, whereas miR-155-5p accelerates atherosclerosis by impairing efferocytosis. miR-147-3p is upregulated by inflammatory stimuli in macrophages and in atherosclerotic lesions, suggesting a role in NFMs. METHODS: The role of miR-147-3p in myeloid cells, with or without enhanced green fluorescent protein expression, on atherosclerosis was examined in Apoe-/-Mir147flox/floxLysMCre+ mice. Using live-plaque 4D confocal imaging, we assessed lipid droplets, caspase-3 activation, apoptotic DNA, cholesterol crystal (CC) formation, and mitochondrial function. We also imaged macrophage migration, phagocytosis of apoptotic DNA, and the formation of tubular membrane extensions. We tested mitochondrial function in live-plaque tissue by Seahorse assay. GFP-tagged Argonaute 2 immunoprecipitation combined with prime RNA sequencing was performed using atherosclerotic aortas from Apoe-/-LSL-tAgo2/Mir147flox/floxLysMCre+ and control mice. The effect of the galectin-3 inhibitor GB1107 was studied using 4D live-plaque imaging. RESULTS: Unlike FMs, NFMs are primarily located in the plaque core and show higher miR-147-3p levels in both mouse and human atherosclerosis. Knocking out Mir147 in myeloid cells increases atherosclerosis, with enhanced CC formation and apoptotic DNA accumulation in necrotic cores. Removing Mir147 reduces mitochondrial activity and elevates caspase-3 activity in NFMs, but not in FMs, and lowers the spare respiratory capacity of plaque macrophages. Moreover, deleting Mir147 impairs NFM uptake of apoptotic DNA, increases extracellular apoptotic DNA, and promotes CC formation. Additionally, Mir147 deficiency in NFMs induces caspase-3 activation in endothelial cells, facilitating the transendothelial extension of FM projections. The Lgals3 transcript, encoding galectin-3, was reduced in the tagged Argonaute 2 immunoprecipitate after Mir147 knockout. A miR-147-3p binding site in the Lgals3 3'-UTR was functionally confirmed. GB1107 treatment reversed the Mir147 knockout effect in macrophages. CONCLUSIONS: miR-147-3p reduces atherosclerosis by suppressing the harmful effects of NFMs on endothelial cells and by enhancing their clearance of apoptotic DNA through targeting galectin-3. Increasing miR-147-3p levels might thus slow the expansion of the necrotic core and reduce atherothrombosis caused by NFM-induced endothelial damage.