源自巨噬细胞的细胞外囊泡 miR-32 通过抑制 VSMC 自噬促进 2 型糖尿病小鼠的动脉钙化。
Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy.
机构信息
The First Affiliated Hospital, Institute of Clinical Medicine, Department of Endocrinology and Metabolism, Hengyang Medical School, University of South China, Hengyang, 421000, Hunan, China.
The First Affiliated Hospital, Department of Laboratory Medicine, Hengyang Medical School, University of South China, Hengyang, 421000, China.
出版信息
J Transl Med. 2022 Jul 6;20(1):307. doi: 10.1186/s12967-022-03502-8.
BACKGROUND
The development of diabetes vascular calcification (VC) is tightly associated with the inhibition of vascular smooth muscle cell (VSMC) autophagy. Previously, our team found that miR-32-5p (miR-32) promotes macrophage activation, and miR-32 is expressed at higher level in the plasma of patients with coronary calcification. However, whether miR-32 mediates the function of macrophages in type 2 diabetes (T2D) VC is still unclear.
METHODS
Wild-type (WT) and miR-32 mice were used in this study. qRT-PCR and western blotting were used to analyze gene expression. Flow cytometry was used to analyze the influence of glucose concentration on macrophage polarization. Nanoparticle tracking analysis (NTA), transmission electron microscopy, and confocal microscopy were used to identify macrophage extracellular vehicles (EVs). Immunofluorescence, in situ hybridization (ISH), immunohistochemistry, and alizarin red staining were used to analyze the influence of macrophage EVs on autophagy and calcification of the aorta of miR-32 mice. A luciferase assay was used to analyze the effect of miR-32 on myocyte enhancer factor 2D (Mef2d) expression. Co-IP combined with mass spectrometry (MS) and transcriptome sequencing was used to analyze the signalling pathway by which Mef2d acts in VSMC autophagy.
RESULTS
We found that high glucose conditions upregulate miR-32 expression in macrophages and their EVs. Importantly, macrophages and their EVs promote VSMC osteogenic differentiation and upregulate miR-32 expression in VSMCs. Moreover, miR-32 mimics transfection promoted osteogenic differentiation and inhibited autophagy in VSMCs. In vitro and in vivo experiments showed that Mef2d is the key target gene of miR-32 that inhibits VSMC autophagy. Furthermore, MS and transcriptome sequencing found that cGMP-PKG is an important signalling pathway by which Mef2d regulates VSMC autophagy. In addition, after T2D miR-32 mice were injected with macrophage EVs via the caudal vein, miR-32 was detected in aortic VSMCs of miR-32 mice. Moreover, autophagy was significantly inhibited, and calcification was significantly enhanced in aorta cells.
CONCLUSIONS
These results reveal that EVs are the key pathway by which macrophages promote T2D VC, and that EVs miR-32 is a key cause of autophagy inhibition in VSMCs.
背景
糖尿病血管钙化（VC）的发展与血管平滑肌细胞（VSMC）自噬的抑制密切相关。此前，我们的团队发现 miR-32-5p（miR-32）促进巨噬细胞活化，并且 miR-32 在冠状动脉钙化患者的血浆中表达水平更高。然而，miR-32 是否介导巨噬细胞在 2 型糖尿病（T2D）VC 中的功能尚不清楚。
方法
本研究使用野生型（WT）和 miR-32 小鼠。使用 qRT-PCR 和 Western blot 分析基因表达。使用流式细胞术分析葡萄糖浓度对巨噬细胞极化的影响。使用纳米颗粒跟踪分析（NTA）、透射电子显微镜和共聚焦显微镜鉴定巨噬细胞细胞外囊泡（EVs）。免疫荧光、原位杂交（ISH）、免疫组织化学和茜素红染色分析巨噬细胞 EVs 对 miR-32 小鼠主动脉自噬和钙化的影响。使用荧光素酶测定分析 miR-32 对肌细胞增强因子 2D（Mef2d）表达的影响。使用 Co-IP 结合质谱（MS）和转录组测序分析 Mef2d 在 VSMC 自噬中作用的信号通路。
结果
我们发现高葡萄糖条件上调巨噬细胞及其 EVs 中的 miR-32 表达。重要的是，巨噬细胞及其 EVs 促进 VSMC 成骨分化，并上调 VSMCs 中的 miR-32 表达。此外，miR-32 模拟物转染促进 VSMCs 成骨分化并抑制自噬。体内外实验表明，Mef2d 是抑制 VSMC 自噬的 miR-32 的关键靶基因。此外，MS 和转录组测序发现 cGMP-PKG 是 Mef2d 调节 VSMC 自噬的重要信号通路。此外，在 T2D miR-32 小鼠经尾静脉注射巨噬细胞 EV 后，miR-32 在 miR-32 小鼠的主动脉 VSMCs 中被检测到。此外，自噬明显受到抑制，主动脉细胞的钙化明显增强。
结论
这些结果表明 EVs 是巨噬细胞促进 T2D VC 的关键途径，而 EVs miR-32 是 VSMCs 自噬抑制的关键原因。
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