Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy

(2022) 20:307 Cao et al. Journal of Translational Medicine https://doi.org/10.1186/s12967-022-03502-8 Journal of Translational Medicine Open Access RESEARCH Extracellular vesicle miR‑32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy Jingsong Cao1† , Cong Chen2†, Qian Chen1†, Yan Gao1, Zhibo Zhao1, Qing Yuan1, Anqi Li1, Shiqi Yang1, Yuqi He2, Xuyu Zu3* and Jianghua Liu1* Abstract 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 † Jingsong Cao, Cong Chen, and Qian Chen are contributed equally to this work. *Correspondence: ; 1 The First Affiliated Hospital, Institute of Clinical Medicine, Department of Endocrinology and Metabolism, Hengyang Medical School, University of South China, Hengyang 421000, Hunan, China 3 The First Affiliated Hospital, Institute of Clinical Medicine, Department of Tumor Research, Hengyang Medical School, University of South China, Hengyang 421000, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cao et al. Journal of Translational Medicine (2022) 20:307 Page 2 of 15 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. Keywords: Macrophage, EVs, miR-32, Diabetes, Vascular calcification Introduction Diabetes easily induces microvascular and macrovascular complications that lead to peripheral vascular disease [1]. Vascular calcification (VC) is an important clinicopathological feature of diabetes and is considered as a major independent risk factor for cardiovascular diseases [2]. In the process of VC, at least 4 kinds of cells may lead to VC. These cell types include pericytes in microvessels, pericyte-like calcifying vascular cells in the aortic intima, smooth muscle cells (SMCs) in the media, and myofibroblasts in the adventitia [3]. Among the 4 cell types, local smooth muscle cells are an important source of calcifying vascular cells [3–5]. The key to VSMC calcification is the transformation from a contractile phenotype to an osteoblast-like phenotype [6]. However, the mechanism of VSMC calcification is still unclear. Our previous research determined that the expression of miR-32 is increased in VSMCs during the progression of vascular calcification, and miR-32 was expressed at high level in the plasma of VC patients compared with non-VC patients [7]. Interestingly, we further found that miR-32 is also involved in the activation of microglia, which are resident macrophages in the central nervous system [8]. Therefore, exploring the relationship and mechanisms among macrophages, miR-32 and VSMC calcification is meaningful. Macrophages are the major participators of innate immunity and exist in all human tissues, including the aortic wall [9]. In response to infection, peripheral monocytes, derived from the bone marrow, are the major source of macrophages in the aortic wall [10]. In the plaques of human and mouse models, macrophages are abundant immune cells, and they are the primary cell type among total plaque cells [11, 12]. Infiltrated macrophages polarize to the proinflammatory M1 phenotype and secrete inflammatory factors and exosomes, leading to increased plaque stability [13, 14]. Macrophage polarization is a response to environmental stimulation and involves coordinated metabolic and transcriptional rewiring [15]. In diabetes, high glucose induces M1 macrophage polarization and the secretion of exosomes to regulate the development of cardiovascular diseases [16, 17]. Recent research found that the exosomes recovered by centrifugation include oncosomes, ectosomes, microvesicles and membrane vesicles, so the production is better called EVs [18]. However, the roles of macrophag (...truncated)