Regulation of succinate signaling and immunometabolism in diabetes-associated atherosclerosis by miR-369-3p

Atherosclerosis is accelerated under diabetic conditions in part due to altered macrophage metabolism and function. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages and regulate inflammation, yet their therapeutic potential in diabetes-associated atherosclerosis remains poorly understood. miRNA profiling of aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose containing (HFSC) diet for 12 weeks revealed low expression of miR-369-3p. miR-369-3p was also reduced in peripheral blood mononuclear cells (PBMCs) from diabetic patients with coronary artery disease. Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. Treatment with oxLDL reduced miR-369-3p expression in-stimulation mouse bone marrow-derived macrophages (BMDMs) downregulated miR-369-3p. Overexpression of miR-369-3p restored the oxLDL-mediated decrease of OXPHOS in BMDMs. Mechanistically, we found that miR-369-3p targeted the succinate receptor (GPR91) to alleviate the oxLDL-induced activation of inflammasome signaling pathways. Therapeutic administration of miR-369-3p in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and the diabetes-mediated accelerated atherosclerosis, evident by a decrease in plaque size and in the accumulation of pro-inflammatory Ly6Chi macrophages. RNA-seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p treated mice, consistent with an increase in macrophage efferocytosis in lesions. These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism. Overall design: To investigate the effects of miR-369-3p mimic in atherosclerotic plaques, Ldlr-/- mice were placed on high fat high sucrose containing diet (HFSC) for 8 weeks to induce atherosclerosis after which the mice were randomized into two treatment groups : Non-specific mimic (NS)control and miR-369-3p mimic (369-m). Mice received 2 intravenous injections of 2 nmole NS or 369-m every week for a total of four weeks with the continuation of HFSC diet. At the time of sacrificing, aortas were isolated and aortic plaque macrophages were FACs-sorted. We then performed low input RNA-seq for gene expression analyses in FACs-sorted macrophages from plaques of NS and 369-m injected mice.