Mir-147 limits the contribution of inflammatory macrophages to atherosclerosis

Abstract Introduction Atherosclerosis is a chronic inflammatory disease of the arteries that occurs when lipoproteins become trapped in arterial walls, attracting monocytes that mature into inflammatory macrophages. These macrophages ingest lipoproteins and fill with lipid droplets (LD). Although inflammatory macrophages are central contributors to atherosclerotic lesion development, the underlying mechanism is incompletely understood. miR-147 has been linked to anti-inflammatory effects, but its role in macrophages within atherosclerotic plaques remains unknown. Methods A live plaque confocal imaging approach was established to investigate the role of miR-147 in macrophages in atherosclerotic plaques, using aortic arch or root tissue from the Apoe–/–mTmG/Mir147flox/floxLysMCre+ or Apoe–/–mTmG/Mir147WT/WTLysMCre+ mice after Western-type diet (WD) feeding. Myeloid cells in these mice expressed mEGFP. In the live plaque, apoptosis was indicated by Biotracker Caspase 3 dye and LD was labeled by LipidSpot dye. In situ PCR was used to detect the expression of miR-147. Cholesterol crystal formation was detected by polarized microscopy and confocal reflection microscopy. Moreover, we generated Apoe–/–LSL-tAgo2/Mir147WT/WTLysMCre+ and Apoe–/–LSL-tAgo2/Mir147flox/floxLysMCre+ mice that express tAgo2 in myeloid cells. After WD feeding, the aorta from these mice was used to perform GFP-tagged Ago2 immunoprecipitation combined with RNA prime-sequencing to identify miR-147 targets in plaque macrophages. Results We found that miR-147 is mainly expressed in macrophages with low LD levels (LDlow), which enhanced their mitochondrial activity and survival compared to macrophages with high LD levels (LDhigh). Knockout of miR-147 in myeloid cells exacerbated atherosclerosis, characterized by increased lipid and macrophage accumulation, and enhanced formation of cholesterol crystals and necrotic core. Moreover, miR-147 deficiency in myeloid cells enhanced LDlow macrophage-induced caspase-3 activation in endothelial cells and inhibited apoptotic DNA uptake by inflammatory LDlow macrophages. The increased endothelium caspase3 activation facilitated the projection by LDhigh macrophages of tubular membrane extensions across the damaged endothelium. Mechanistically, the effect of miR147 on LDlow macrophages was due to the targeting of galectin-3, since galectin 3 inhibitor GB1107 reversed the effect of miR-147 knockout. Conclusion We provide new insights into how macrophage subsets influence atherosclerosis. Inflammatory macrophages that lacking LDs exert cytotoxic effects on endothelial cells and remove extracellular chromatin within the plaque. miR-147 plays a key role in limiting atherosclerosis by counteracting the effects of inflammatory non-foamy macrophages through targeting galectin-3.