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. 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.

动脉粥样硬化（Atherosclerosis）在糖尿病状态下的进展加速，部分原因是巨噬细胞代谢与功能发生紊乱。探寻能够修复代谢紊乱、促进炎症消退的关键靶点，仍是尚未实现的研究目标。微小RNA（MicroRNAs，miRs）可调控巨噬细胞内的多种信号事件与炎症过程，但目前对其在糖尿病相关动脉粥样硬化中的治疗潜力仍知之甚少。
对喂食高脂高蔗糖（high-fat sucrose containing, HFSC）饲料12周的Ldlr⁻/⁻小鼠的主动脉内膜斑块进行miRNA表达谱分析，发现miR-369-3p的表达水平显著降低。合并冠状动脉疾病的糖尿病患者外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中，miR-369-3p的表达同样下调。细胞类型特异性表达谱分析显示，miR-369-3p在主动脉巨噬细胞中富集。氧化低密度脂蛋白（oxidized low-density lipoprotein, oxLDL）处理可下调小鼠骨髓源性巨噬细胞（bone marrow-derived macrophages, BMDMs）中miR-369-3p的表达。过表达miR-369-3p可逆转oxLDL诱导的BMDMs中氧化磷酸化（oxidative phosphorylation, OXPHOS）水平下调。
机制研究表明，miR-369-3p可靶向结合琥珀酸受体（succinate receptor, GPR91），从而缓解oxLDL诱导的炎症小体信号通路激活。对喂食HFSC饲料的Ldlr⁻/⁻小鼠进行miR-369-3p治疗性给药，可降低斑块巨噬细胞中GPR91的表达，并改善糖尿病介导的动脉粥样硬化加速进展：表现为斑块体积减小，促炎性Ly6C^hi巨噬细胞的浸润减少。RNA测序（RNA-seq）分析显示，经miR-369-3p处理的小鼠的斑块巨噬细胞中，更多炎症消退相关通路被激活，这与病灶内巨噬细胞胞葬作用（efferocytosis）增强的结果一致。上述研究证实，miR-369-3p可通过调控GPR91与巨噬细胞琥珀酸代谢，发挥阻断糖尿病相关动脉粥样硬化进展的治疗作用。
为探究miR-369-3p模拟物对动脉粥样硬化斑块的作用，我们将Ldlr⁻/⁻小鼠喂食高脂高蔗糖（HFSC）饲料8周以诱导动脉粥样硬化，随后将小鼠随机分为两组：非特异性模拟物（non-specific mimic, NS）对照组与miR-369-3p模拟物（miR-369-3p mimic, 369-m）实验组。每周经尾静脉注射2 nmol的NS或369-m，持续4周，期间继续喂食HFSC饲料。处死小鼠时，分离主动脉并通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）富集主动脉斑块巨噬细胞。随后对经FACS分选得到的两组小鼠（注射NS与369-m）的斑块巨噬细胞进行低起始量RNA-seq，以分析基因表达谱。