A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle.
A MicroRNA93-Interferon Regulatory Factor-9-Immunoresponsive Gene-1-Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle.
复制标题
DOI：
10.1161/circulationaha.116.025490
复制
发表时间：
2017-06-13
期刊：
影响因子：
37.8
通讯作者：
Annex BH
中科院分区：
文献类型：
--
作者：
Ganta VC;Choi MH;Kutateladze A;Fox TE;Farber CR;Annex BH
目前还没有治疗和改善严重外周动脉疾病（PAD）患者结局的疗法。MicroRNA 93（miR 93）已显示出在遗传性PAD模型中有利地调节血管生成并减少组织损失。然而，miR 93介导的缺血性肌肉新生血管形成中涉及的细胞特异性功能、下游机制或信号转导尚不清楚。巨噬细胞是调节PAD中动脉生成反应的最佳已知因子，巨噬细胞诱导的动脉生成反应的程度取决于M2至M1激活/极化状态的增强。在目前的研究中，我们确定了一种新的机制，miR 93调节巨噬细胞极化，以促进血管生成和动脉生成，使实验性PAD中的缺血肌肉再血管化。体外（正常和缺氧血清饥饿（HSS）条件下的巨噬细胞、内皮细胞、骨骼肌细胞）和临床前PAD模型中的体内实验（单侧股动脉结扎和切除））来检测miR 93-干扰素调节因子-9（IRF 9）-免疫应答基因-1（IRG 1）-衣康酸途径在巨噬细胞极化，血管生成，动脉生成和灌注恢复。在体内，与野生型（WT）对照相比，miR 106 b-93-25簇缺陷小鼠（miR 106 b-93-25−/−）显示出与实验性PAD后M1样巨噬细胞增加相关的血管生成和动脉生成减少。在miR 106 b-93-25−/− PAD小鼠中肌肉内递送miR 93增加了血管生成、动脉生成、灌注程度，这与近端和远端后肢肌肉中更多的M2样巨噬细胞相关。在体外，miR 93甚至在M1样极化条件（HSS）下促进和维持M2样极化。将骨髓来源的巨噬细胞从miR 106 b-93-25−/−递送至WT缺血肌肉减少了血管生成、动脉生成和灌注，而将野生型巨噬细胞转移至miR 106 b-93-25−/−具有相反的效果。对miR 106 b-93-25−/−和WT缺血肌肉之间RNA测序的最高差异上调基因的系统分析表明，miR 93调节IRG 1功能以调节衣康酸产生和巨噬细胞极化。通过3′UTR内切酶分析确定IRG 1是否是miR 93的直接靶点，结果显示IRG 1不是miR 93的靶点，但可调控IRG 1表达的IRF 9是miR 93的靶点。在体外，IRF 9，IRG 1和衣康酸处理的表达增加显着降低内皮血管生成的潜力。我们得出结论，miR 93抑制IRF 9以减少IRG 1-衣康酸的产生，从而诱导缺血肌肉中的M2样极化，从而增强实验性PAD中的血管生成、动脉生成和灌注恢复。
Currently no therapies exist for treating, and improving outcomes in patients with severe peripheral arterial disease (PAD). MicroRNA93 (miR93) has been shown to favorably modulate angiogenesis and reduce tissue loss in genetic PAD models. However, the cell specific function, downstream mechanisms or signaling involved in miR93 mediated ischemic muscle neovascularization is not clear. Macrophages were best known to modulate arteriogenic response in PAD and the extent of arteriogenic response induced by macrophages is dependent on greater M2 to M1-activation/polarization state. In the current study, we identified a novel mechanism by which miR93 regulates macrophage-polarization to promote angiogenesis and arteriogenesis to revascularize ischemic muscle in experimental-PAD. In vitro (macrophages, endothelial cells, skeletal muscle cells under normal and hypoxia serum starvation (HSS) conditions) and in vivo experiments in preclinical-PAD models (unilateral femoral artery ligation and resection)) were conducted to examine the role of miR93-interferon regulatory factor-9 (IRF9)-immune responsive gene-1 (IRG1)-itaconic acid pathway in macrophage-polarization, angiogenesis, arteriogenesis and perfusion recovery. In vivo, compared to wild type (WT) controls, miR106b-93-25 cluster deficient mice (miR106b-93-25−/−) showed decreased angiogenesis and arteriogenesis correlating with increased M1-like-macrophages following experimental-PAD. Intra-muscular delivery of miR93 in miR106b-93-25−/− PAD mice increased angiogenesis, arteriogenesis, the extent of perfusion which correlated with more M2-like-macrophages in the proximal and distal hind-limb muscles. In vitro, miR93 promotes and sustains M2-like-polarization even under M1-like-polarizing conditions (HSS). Delivery of bone marrow derived macrophages from miR106b-93-25−/− to WT ischemic-muscle decreased angiogenesis, arteriogenesis and perfusion, while transfer of wild-type macrophages to miR106b-93-25−/− had the opposite effect. Systematic analysis of top-differentially upregulated genes from RNA-sequencing between miR106b-93-25−/− and WT ischemic-muscle showed that miR93 regulates IRG1 function to modulate itaconic acid production and macrophage-polarization. 3′UTR luciferase-assays performed to determine whether IRG1 is a direct target of miR93 revealed that IRG1 is not a miR93 target but IRF9 that can regulate IRG1-expression is a miR93 target. In vitro, increased expression of IRF9, IRG1 and itaconic acid treatment significantly decreased endothelial angiogenic potential. We conclude that miR93 inhibits IRF9 to decrease IRG1-itaconic acid production to induce M2-like-polarization in ischemic muscle to enhance angiogenesis, arteriogenesis and perfusion recovery in experimental-PAD.