Exosomes from nicotine-stimulated macrophages accelerate atherosclerosis through miR-21-3p/PTEN-mediated VSMC migration and proliferation

Abstract

Rationale: During the development of atherosclerosis, macrophages secrete exosomes that regulate vascular smooth muscle cells (VSMCs); however, whether nicotine, a major constituent of cigarettes, can modulate this communication in the context of atherogenesis remains to be further studied. In this study, we hypothesized that nicotine induces macrophages to secrete atherogenic exosomes containing microRNAs (miRNAs) to mediate cell-to-cell crosstalk and encourage proatherogenic phenotypes of VSMCs.Methods: In an in vivo study, nicotine was administered subcutaneously to 8-week-old male ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks. Oil red O and hematoxylin and eosin (HE) were used to stain atherosclerotic lesions. Lesion macrophages, VSMCs and exosomes were stained for CD68, α-smooth muscle actin (α-SMA) and CD9, and plaque exosomes were observed by transmission electron microscopy (TEM). Exosomes derived from control macrophages (M-Exos) and from nicotine-treated macrophages (NM-Exos) were isolated by ultracentrifugation, purified by sucrose density gradient centrifugation and characterized based on specific morphology and surface markers. The IVIS® Spectrum in vivo imaging system showed the biodistribution of NM-Exos and M-Exos in circulation. Chitosan hydrogel-incorporated exosomes were applied to simulate exosome secretion in situ. Scratch wound assay, transwell assay and EdU staining were conducted to assess the effects of NM-Exos on the migration and proliferation of mouse VSMCs. RNA-seq was performed to determine the miRNA profiles of M-Exos and NM-Exos. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of miRNAs and mRNAs. The roles of the candidate miRNA and its target gene were assessed using specific RNA inhibitors, siRNAs and miRNA mimics. Western blotting was used to detect candidate protein expression levels. A dual-luciferase reporting system was utilized to confirm the binding of a specific miRNA to its target gene.Results: Nicotine induced atherosclerotic lesion progression and resulted in plaque exosome retention in vivo. The biodistribution of NM-Exos showed that plaque-resident exosomes might be secreted in situ. VSMCs cocultured in vitro with nicotine-stimulated macrophages presented an increased capacity for migration and proliferation, which was exosome-dependent. In addition, isolated NM-Exos helped promote VSMC migration and proliferation. miRNA profiling showed that miR-21-3p was enriched in NM-Exos, and this miRNA was shown to play a key role in regulating NM-Exos-induced effects by directly targeting phosphatase and tension homologue (PTEN).Conclusion: Exosomal miR-21-3p from nicotine-treated macrophages may accelerate the development of atherosclerosis by increasing VSMC migration and proliferation through its target PTEN.

RETRACTED: PRDM16 Upregulation Induced by MicroRNA-448 Inhibition Alleviates Atherosclerosis via the TGF-β Signaling Pathway Inactivation

“…CD31, also known as platelet/endothelial cell adhesion molecule-1, has been elucidated to be expressed at a high level at endothelial cell-cell junctions to maintain the integrity and accelerate the recovery of the vascular permeability barrier in response to inflammatory or thrombotic challenge (Liu and Shi, 2012;Lertkiatmongkol et al, 2016). Additionally, a functional study has deciphered that α-SMA-positive VSMCs appreciably elevated in the progression of AS (Zhu et al, 2019). Also, Vimentin has been recognized as an indicator of cell migration, and loss of vimentin triggers enhanced oxidative stress and promoted vascular inflammation in macrophages, alleviating AS in a mouse model (Battaglia et al, 2018;Haversen et al, 2018).…”

Macrophage-derived extracellular vesicles: diverse mediators of pathology and therapeutics in multiple diseases

“…Recently, EVs from M1-like macrophages have been proven to activate extracellular regulated protein kinase (ERK) and protein kinase B (Akt) pathway by transferring integrins, which in turn stimulate extracellular matrix (ECM) production and cell migration and adhesion in VSMCs, aggravating atherosclerosis 73 . Additionally, EVs from macrophages exposed to nicotine also induce VSMC proliferation and migration by delivering miR-21-3p to activate phosphatase and tension homolog (PTEN) in these cells 74 .…”

M1 Macrophage Derived Exosomes Aggravate Experimental Autoimmune Neuritis via Modulating Th1 Response

“…In tumor microenvironment, exosomes derived from tumor associated macrophages (TAMs) have been proven to promote migration and invasion of colorectal cancer cells and to regulate aerobic glycolysis of breast cancer cells, via transferring certain miRNAs or lncRNAs, respectively (19,20). Exosomes also contribute to crosstalk between nicotine-treated macrophages and vascular smooth muscle cells, exacerbating the development of atherosclerosis (21). Furthermore, systematic regulation of insulin sensitivity by miRNA-containing exosomes from adipose tissue macrophages (ATMs) has also been well-documented (22).…”