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Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy
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 p...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9258116/ https://www.ncbi.nlm.nih.gov/pubmed/35794619 http://dx.doi.org/10.1186/s12967-022-03502-8 |
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author | Cao, Jingsong Chen, Cong Chen, Qian Gao, Yan Zhao, Zhibo Yuan, Qing Li, Anqi Yang, Shiqi He, Yuqi Zu, Xuyu Liu, Jianghua |
author_facet | Cao, Jingsong Chen, Cong Chen, Qian Gao, Yan Zhao, Zhibo Yuan, Qing Li, Anqi Yang, Shiqi He, Yuqi Zu, Xuyu Liu, Jianghua |
author_sort | Cao, Jingsong |
collection | PubMed |
description | 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-022-03502-8. |
format | Online Article Text |
id | pubmed-9258116 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-92581162022-07-07 Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy Cao, Jingsong Chen, Cong Chen, Qian Gao, Yan Zhao, Zhibo Yuan, Qing Li, Anqi Yang, Shiqi He, Yuqi Zu, Xuyu Liu, Jianghua J Transl Med Research 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-022-03502-8. BioMed Central 2022-07-06 /pmc/articles/PMC9258116/ /pubmed/35794619 http://dx.doi.org/10.1186/s12967-022-03502-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Cao, Jingsong Chen, Cong Chen, Qian Gao, Yan Zhao, Zhibo Yuan, Qing Li, Anqi Yang, Shiqi He, Yuqi Zu, Xuyu Liu, Jianghua Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title | Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title_full | Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title_fullStr | Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title_full_unstemmed | Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title_short | Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy |
title_sort | extracellular vesicle mir-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting vsmc autophagy |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9258116/ https://www.ncbi.nlm.nih.gov/pubmed/35794619 http://dx.doi.org/10.1186/s12967-022-03502-8 |
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