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

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 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.

Pravastatin promotes type 2 diabetes vascular calcification through activating intestinal Bacteroides fragilis to induce macrophage M1 polarization

“…T2D mice were generated as previously reported. 19 , 20 Mice were group housed (2–5 mice per cage) at a controlled temperature (22 ± 2°C) under a 12‐h light/dark cycle with free access to food and water. The mice adapted to their new environment for 7 days before the behavioral tests.…”

“… 17 We previously confirmed that peripheral macrophages in T2D patients are prone to polarize into the M1 phenotype and secrete inflammatory factors and extracellular vesicles to promote VC. 18 , 19 , 20 Are macrophages also the key target cells of BF to promote T2D VC?…”

Emerging role of exosomes in vascular diseases

“…Ca 2+ uptake by cells via exosomes or Ca 2+ channels further contributed to an increase in Nox5 activity (90). Under hyperglycaemic conditions, EV miR-32 in macrophages was upregulated and delivered to VSMCs, promoting osteogenic differentiation and inhibiting autophagy in VSMCs via the miR-32/Mef2d/cGMP-PKG signaling pathway (91). Lipoprotein(a) stimulation led to an increase in the annexin level in the EV cargo of primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs), and these EVs readily aggregated in the collagen matrix and induced its mineralization, leading to the formation of microcalcifications that coalesced into macrocalcifications (92).…”

Role of advanced glycation end products on vascular smooth muscle cells under diabetic atherosclerosis

“…Severe vascular calcification in diabetic has also been confirmed to be related to VSMCs ( 117 , 118 ). In addition, AGEs ( 119 , 120 ), apoptosis ( 121 , 122 ), oxidative stress ( 123 , 124 ) and a variety of noncoding RNAs ( 125 , 126 ) regulate AS under diabetic state. Recent studies about the effect of high glucose on VSMCs was listed in Table 3 .…”