ABSTRACT
This study aimed to investigate the role and underlying mechanism of exosomes secreted by oxidized low-density lipoprotein (oxLDL)-stimulated macrophages in the progression of atherosclerosis (AS).
Exosomes from peripheral blood of AS patients or oxLDL-treated macrophages were co-cultured with human neutrophils. Neutrophil extracellular traps (NETs) were detected by immunofluorescence staining. The levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). The expression levels of miR-146a and superoxide dismutase 2 (SOD2) were determined by quantitative real-time PCR (qRT-PCR) and western blot. The generation of intracellular reactive oxygen species (ROS) was observed by using dichlorofluorescin diacetate (DCFH-DA). ApoE-deficient mice were fed with high-fat diet (HFD) to induce AS. Atherosclerotic plaques were evaluated by Oil red O (ORO) and hematoxylin-eosin (HE) staining.
Our results showed that miRNA-146a was enriched in serum-derived exosomes of AS patients and oxLDL-treated macrophage THP-1-derived exosomes. Importantly, exosomal miR-146a secreted by oxLDL-treated macrophages promoted ROS and NETs release via targeting SOD2. In addition, intravenous administration of oxLDL-treated THP-1 cells-derived exosomes into AS mice significantly deteriorated AS in vivo.
Our findings indicate that exosomal miR-146a derived from oxLDL-treated macrophages promotes NETs formation via inducing oxidative stress, which might provide a novel scientific basis for the understanding of AS progression.
KEYWORDS: Atherosclerosis, exosomes, macrophages, neutrophil extracellular traps, oxidized low-density lipoprotein
Introduction
Atherosclerosis (AS) is a chronic inflammatory disease, which is also the common pathophysiological basis of cardiovascular diseases [1]. Data from recent studies correlate atherosclerosis with autoimmune mechanisms [2,3]. Various immune cells are involved in the pathogenesis of AS, such as macrophages, lymphocytes, dendritic cells and neutrophils, which cause the dysfunction of endothelial cells via the activation of adhesion molecules and inflammatory cytokines, and ultimately lead to the formation of atherosclerotic plaque [4]. As the first line of defense against invading pathogens, neutrophils resist microbial invasion by classical phagocytosis and degranulation. A growing body of evidence suggests that neutrophils also respond to infection from pathogenic microorganisms via the release of neutrophil extracellular traps (NETs), web-like structures comprised of decondensed chromatin nuclear histone and granular antibacterial proteins [5]. The process of NETs formation is termed as NETosis, which is different from other forms of cell death such as necrosis and apoptosis. In recent years, abundant studies focus on the involvement of NETosis in AS progression [6].
Macrophages serve as the major sources of inflammatory cytokines and critical mediators of innate immune responses and play a key role in the occurrence and development of AS [7]. Convincing evidence suggests that cholesterol crystals trigger NETosis, which in turn prime macrophages for cytokine release, thereby amplifying immune cell recruitment in atherosclerotic plaques [8,9]. However, whether macrophages involve in the formation of NETs remains unclear. Recently, Arroyo et al [10] provided evidence that miR-146a regulated the generation of NETs in adverse cardiovascular diseases such as atrial fibrillation (AF). Furthermore, Nguyen et al [11] found that miR-146a was enriched in extracellular vesicles (EVs) derived from oxidized low-density lipoprotein (oxLDL)-loaded macrophages. Accordingly, we speculated that exosomes secreted by oxLDL-treated macrophages transferred miR-146a to promote the formation of NETs and accelerate the development of AS.
NETosis is recognized as nicotinamide adenine dinucleotide phosphate (NADPH)-dependent specific cell death program [12]. Oxidative stress mediated by reactive oxygen species (ROS) not only plays an important role in the pathogenesis of AS, but also is necessary for NETosis [13]. Superoxide dismutase 2 (SOD2) is a pivotal component of endogenous antioxidant defense barrier, which is essential for balancing the intracellular ROS via functioning as a radical scavenger [14]. Olsson et al [15] proved that the decreased expression of SOD2 in neutrophils induced by lipopolysaccharide (LPS) is one of the potential causes of neutrophil dysfunction. Cui et al [16] recently reported that miR-146a enhanced ROS generation and decreased the expression of SOD2 at mRNA and protein levels in epithelial ovarian cancer (EOC) cells. Overall, we hypothesized that oxLDL-treated macrophages downregulated SOD2 expression in neutrophils through transferring exosomal miR-146a and thereby resulted in the overproduction of ROS and the formation of NETs, leading to AS deterioration.
Materials and methods
Patients and controls
A total of 22 patients with AS (ranges from 48–68 years with mean age 59.8 ± 7.2 years; 12 male; mean height 169.96 ± 10.49 cm) and 18 normal volunteers (ranges from 48–68 years with mean age 58.9 ± 9.2 years; 10 male; mean height 170.01 ± 11.28 cm) who underwent physical examinations during the same period were enrolled in this study as AS group and healthy controls (HC), respectively. There were no significant difference in age, distribution of gender, and height between AS and HC group. AS was diagnosed if brachial-ankle pulse wave velocity (baPWV) >1400 cm/s. The exclusion criteria were as follows: severe arrhythmia, valvular heart disease, malignant tumor, diabetes, and severe liver and kidney dysfunction. All experimental procedures were approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University and signed consent was given. Peripheral blood was collected from each subject for the following experiments.
Serum exosomes isolation and authentication
Blood samples (1 ml) were collected in 1.5 ml Eppendorf tubes and centrifuged at 500 g for 10 min at 4°C. The supernatant was transferred to new centrifugal tubes followed by centrifugation again at 2,000 × g for 10 min to discard dead cells. The supernatant was subject to additional centrifugation at 10,000 × g for 30 min to discard cell debris. Afterward, the supernatant was centrifuged again at 100,000 × g for 70 min. The remaining pellets were exosomes which were then re-suspended in PBS for identification and stored at −80°C. The presence of exosomes was morphologically confirmed by transmission electron microscopy (TEM; JEOL Ltd., Tokyo, Japan). Exosomal markers CD9 and Tsg101 were determined by western blot.
Cell line and cell culture
Human macrophage THP-1 cells (American Type Culture Collection; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) containing 10% exosome-depleted fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO2.
Cell transfection and treatment
THP-1 cells at the logarithmic phase were transfected with miR-146a mimic, mimic negative control (NC), miR-146a inhibitor or inhibitor NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 24 h post-transfection, THP-1 cells were stimulated with 50 µg/ml oxidized low-density lipoprotein (oxLDL; Guangzhou Yiyuan Biotech Co., Ltd., Guangzhou, China) for 48 h.
Isolation of THP-1 cells-derived exosomes
The culture medium was centrifuged for 15 min at 3,000 × g to eliminate dead cells and filtrated through a 0.22-μm filter (Millipore, Carrigtwohill, Ireland) to remove cell debris. After centrifugation at 1,000 × g for 30 min at 4°C, the medium was incubated overnight with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). The obtained exosomes were eventually re-suspended in 50 μl PBS.
Neutrophils isolation and treatment
Meanwhile, neutrophils were separated from peripheral blood mononuclear cells (PBMCs) of healthy subjects by density gradient centrifugation. After washed with PBS, PBMCs were layered over a Percoll gradient (78%/69%/52%) and centrifugated at 1, 200 × g for 30 min. Erythrocytes were removed by adding the erythrocyte lysis buffer (Qiagen, Hilden, Germany). The dense band at the 69%/78% interface was collected as the neutrophil fraction. Neutrophils were identified by expression of CD16 surface marker as assessed by flow cytometry. The isolated neutrophils were co-cultured with serum exosomes or exosomes (10 µg/ml) derived from oxLDL-treated THP-1 cells for 24 h following 72 h of treatment with ROS scavenger N-acetyl-L-cysteine (NAC, 10mM) (Sigma-Aldrich).
In vivo experiments
The male ApoE-deficient (ApoE−/-) mice obtained from the Experimental Animal Central Laboratory were fed with a high-fat diet (HFD) to establish a mouse AS model. The HFD prescription: 2% cholesterol, 8% egg yolk powder, 10% lard, 0.2% bile salt, and 80% basic forage. After 7–14 weeks feeding with HFD, ApoE−/- mice were intravenously injected with exosomes (5 µg exosome protein/mouse) derived from THP-1 cells which were treated with or without oxLDL or isopyknic PBS (n = 6 per group). After 1 week, blood, aorta and atherosclerotic tissues were collected. All the protocols in this study were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University.
Western blot
Total protein were extracted from serum or THP-1 cells-derived exosomes and human neutrophils using Radio-Immunoprecipitation Assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocked with 5% skim milk, the membranes were incubated with rabbit monoclonal antibodies against CD9 (ab92726), Tsg101 (ab125011), SOD2 (ab13533) and β-actin (ab179467) purchased from Abcam (Cambridge, UK) at a dilution of 1:5000 overnight at 4°C, followed by incubation with secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000 dilution; Abcam) for 2 h. The enhanced chemiluminescence reagent (Beckman Colter, Brea, CA, USA) was used to detect the protein bands.
RNA extraction and qRT-PCR analysis
Total RNA was extracted from serum or THP-1 cells-derived exosomes and human neutrophils using Trizol reagent (Invitrogen), and reverse transcribed into cDNAs using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq TM II Kit (Takara, Shiga, Japan) on an ABI 7500 Real-Time PCR system (Applied Biosystems). The relative expression levels of miR-146a and SOD2 were normalized to U6 and GAPDH, respectively.
Immunofluorescence
NETs formation was examined by immunofluorescence staining. In brief, human neutrophils at a density of 5 × 105 cells/ml were seeded into 24-well plates and stimulated with exosomes followed by fixed with 4% paraformaldehyde and permeabilized with Triton X-100. Meanwhile, formalin-fixed, paraffin-embedded atherosclerotic tissues were pretreated with a 0.25% trypsin solution. Trypsinized tissue sections and fixed neutrophils were stained with rabbit polyclonal anti-citrullinated histone H3 (H3Cit) rabbit antibody (1:50 dilution; ab5103; Abcam). The slides were incubated with goat anti-rabbit IgG labeled-secondary antibodies for 2 h and the DNA was stained with 4′6-diamidino-2-phenylindole (DAPI) for 5 min. Images were captured with a confocal microscope (Zeiss, Jena, Germany).
Measurement of intracellular ROS
Human neutrophils were incubated with dichlorofluorescin-diacetate (DCFH-DA; 10 µM; Sigma-Aldrich) solution and washed with serum-free DMEM. Intracellular ROS accumulation was measured using spectrofluorophotometer (Shimadzu Corporation, Tokyo, Japan) at the wavelength 488/525 nm.
Enzyme-linked immunosorbent assay (ELISA)
Quantitative analyses of interleukin (IL)-8, tumor necrosis factor (TNF)-α, IL-6, and IL-1β in cell culture supernatants or mouse sera were performed following the ELISA procedures using a quantitative ELISA kit (R&D Systems, Minneapolis, MN, USA).
Determination of serum TC, TG and LDL
Total cholesterol (TC), triglycerides (TG) and low-density lipoprotein (LDL) were detected by automatic biochemical analyzer (Hitachi, Tokyo, Japan) using a commercial kit (Beijing North Institute of biological technology, Beijing, China).
Histopathological analysis
The formalin-fixed, paraffin-embedded aorta sections were incubated with Oil Red O solution (Sigma-Aldrich) for 10 min, washed with 60% isopropanol, and counterstained with hematoxylin and eosin. The atherosclerotic lesions were quantified using microscope (Carl Zeiss, Jena, Germany).
Statistical analysis
All results were represented as mean ± standard deviation (SD) and analyzed using Graphpad Prism version 4.0 software (GraphPad, San Diego, CA, USA). Differences between groups were assessed using analysis of variance (ANOVA) and P-value <0.05 indicated as significantly different.
Results
Serum exosomes derived from AS patients stimulated formation of NETs in human blood-derived neutrophils
Serum-derived exosomes were extracted from AS patients and HCs and confirmed by TEM and western blot. Consistent with the morphology of exosomes, serum exosomes displayed spherical structures with a diameter of approximately 100 nm (Figure 1(a). As shown in Figure 1(b), the proteins of CD9 and Tsg101 were positive expressed in serum exosomes derived from AS patients and HCs. The relative abundance of serum exosomal miR-146a was further detected by qRT-PCR. As illustrated in Figure 1(c), miR-146a expression was significantly higher in the AS group than that in the HC group. To evaluate whether serum exosomes derived from AS patients could stimulate NETs formation, we co-cultured serum exosomes together with HC-derived neutrophils identified by CD16, a membrane marker for neutrophils (Figure 1(d)). The results demonstrated that serum exosomes derived from AS patients induced NETs formation evidenced by immunofluorescent staining for H3Cit (Figure 1(e)) compared with those derived from HCs. Moreover, ROS production was augmented (Figure 1(f)), while SOD2 mRNA and protein levels (Figure 1(g,h)) were decreased in human neutrophils when co-cultured with AS patients-derived exosomes compared with the HC group. As expected, the levels of pro-inflammatory cytokines IL-8, TNF-α, IL-6, and IL-1β (Figure 1(i–l)) were significantly induced in a co-culture system containing AS patients-derived exosomes and HC-derived neutrophils.
Figure 1.
Serum exosomes derived from AS patients stimulated formation of NETs in human blood-derived neutrophils. Electron microscopic images (a), detection of exosomal markers (b), and expression of miR-146a (c) in serum exosomes collected from AS patients (n = 22) and HC (n = 18) using TEM (scale bar: 500 nm), western blot, and qRT-PCR, respectively; Identification of human neutrophils derived from HCs using CD16 surface marker as assessed by flow cytometry (d); Quantification of NETs using immunofluorescence staining (e) for Cit-histone H3 (H3Cit), intracellular ROS levels using DCFH-DA staining (f), SOD2 mRNA (g) and protein levels (h) and the secretion of IL-8 (i), TNF-α (j), IL-6 (k), and IL-1β (l) determined by ELISA in the neutrophils after stimulation with exosomes of AS patients and HC. **P < 0.01 vs. HC group.
Macrophages-derived exosomes induced NETs formation in response to oxLDL
Exosomes were isolated from oxLDL-treated or untreated THP-1 cells. The morphology of isolated exosomes was confirmed under TEM (Figure 2(a)). Furthermore, the results of western blot analysis demonstrated that exosomes were enriched with exosomal markers CD9 and Tsg101 (Figure 2(b)), thus confirming the effective isolation of exosomes. Following this, miR-146a expression was examined in isolated exosomes by qRT-PCR, and the results revealed that exosomal miR-146a expression was significantly higher in oxLDL-treated exosomes when compared with exosomes excreted by untreated controls (Figure 2(c)). We then investigated the role of oxLDL-treated THP-1 cells-derived exosomes in regulating NETs release, oxidative stress and inflammatory response. To this end, oxLDL-stimulated or untreated THP-1 cells-derived exosomes were co-cultured with normal neutrophils. As shown in Figure 2(d,e), exosomes secreted by oxLDL-treated THP-1 cells induced the excessive release of NETs and ROS from human neutrophils. Furthermore, the mRNA and protein levels of SOD2 (Figure 2(f,g)) were downregulated, whereas, the levels of IL-8, TNF-α, IL-6, and IL-1β (Figure 2(h–k)) were markedly upregulated in the oxLDL group when compared with the control group.
Figure 2.
Macrophages-derived exosomes induced NETs formation in response to oxLDL. Electron microscopic images using TEM (a; scale bar: 500 nm), detection of exosomal markers (b), and the mRNA levels of miR-146a (c) in oxLDL-treated or untreated THP-1 cells-derived exosomes; Immunofluorescence staining (d) for H3Cit, intracellular ROS levels (e), SOD2 mRNA (f) and protein levels (g) and the secretion of IL-8 (h), TNF-α (i), IL-6 (j), and IL-1β (k) in the neutrophils after stimulation with oxLDL-treated or untreated THP-1 cells-derived exosomes. **P < 0.01 vs. Control group.
Exosomes secreted by oxLDL-treated macrophages transferred miR-146a to enhance ROS generation and NETs release through downregulating SOD2
To further verify whether exosomal miR-146a participated in the above-mentioned biological roles, miR-146a expression was overexpressed or knocked down in THP-1 cells and successful transfection efficiency was confirmed by qRT-PCR (Figure 3(a,b)). Furthermore, exosomes derived from miR-146a-overexpressing-THP-1 cells further promoted NETs formation (Figure 3(c)) and ROS generation (Figure 3(d)), decreased expression of SOD2 (Figure 3(e,f)), and increased levels of IL-8, TNF-α, IL-6, and IL-1β production (Figure 3(g–j)) under oxLDL exposure. In contrast, exosomes derived from miR-146a-silencing-THP-1 cells yielded an opposite effect.
Figure 3.
Exosomes secreted by oxLDL-treated macrophages transferred miR-146a to enhance ROS generation and NETs release through downregulating SOD2. The expression of miR-146a in THP-1 cells transfected with mimic NC or miR-146a mimic (a) and in THP-1 cells transfected with inhibitor NC or miR-146a inhibitor (b). Immunofluorescence staining (c) for H3Cit, intracellular ROS levels (d), SOD2 mRNA (e) and protein levels (f) and the secretion of IL-8 (g), TNF-α (h), IL-6 (i), and IL-1β (j) in the neutrophils after stimulation with exosomes secreted by THP-1 cells transfected with miR-146a mimic, miR-146a inhibitor or their negative controls following treatment with or without oxLDL. *P < 0.05, **P < 0.01 vs. mimic NC group; #P < 0.05, ##P < 0.01 vs. inhibitor NC group; $P < 0.05, $$P < 0.01 vs. Control group.
OxLDL-stimulated macrophages via delivering exosomal miR-146a promoted ROS-dependent formation of NETs
We next determined whether oxLDL-treated macrophages-derived exosomes promoted NETs formation via miR-146a/SOD2/ROS pathway. Our data demonstrated that treatment with ROS scavenger NAC in THP-1 cells-derived exosomes impaired miR-146a overexpression-mediated induction of ROS overgeneration (Figure 4(a)), downregulation of SOD2 expression (Figure 4(b,c)), induction of NETs formation (Figure 4(d)) as well as upregulation of IL-8, TNF-α, IL-1β, and IL-6 levels (Figure 4(e–h)) in response to oxLDL stimulation.
Figure 4.
OxLDL-stimulated macrophages via delivering exosomal miR-146a promoted ROS-dependent formation of NETs. Intracellular ROS levels (a), SOD2 mRNA (b) and protein levels (c), immunofluorescence staining (d) for H3Cit, and the secretion of IL-8 (e), TNF-α (f), IL-1β (g), and IL-6 (h) in the neutrophils after stimulation with exosomes secreted by oxLDL-treated or untreated THP-1 cells which were transfected with miR-146a mimic or miR-146a mimic following treatment with NAC. **P < 0.01 vs. oxLDL group; #P < 0.05, ##P < 0.01 vs. mimic NC group; $P < 0.05, $$P < 0.01 vs. miR-146a group.
Exosomal miR-146a derived from oxLDL-treated macrophages improved atherosclerotic plaque progression via facilitating NETs formation
To verify the role and mechanism of oxLDL-treated THP-1 cells-derived exosomes in AS progression, HFD-induced AS mice were intravenously injected with exosomes derived from oxLDL-treated THP-1 cells or PBS. As shown in Figure 5(a–c), the serum levels of blood lipids including TC, TG, and LDL were remarkably increased by exosomes injection in the AS group. The atherosclerotic plaque size and plaque morphology were subsequently measured using ORO staining and H&E staining. The results showed that oxLDL-treated THP-1 cells-derived exosomes significantly enhanced the atherosclerotic plaque area and led to aortic injury in HFD-induced ApoE−/− AS model mice compared with that in the vehicle group (Figure 5(d,e)). We further evaluated the effects of THP-1 cells-derived exosomes on the formation of NETs. Immunofluorescence confocal microscopy confirmed NETs formation after stimulation of ox-LDL-treated THP-1 cells-derived exosomes (Figure 5(f)), which further triggered pro-inflammatory cytokine secretion as demonstrated by ELISA results (Figure 5(g–j)). In summary, all these data indicated that exosomal miR-146a derived from ox-LDL-induced THP-1 macrophages promoted NETosis, leading to AS deterioration.
Figure 5.
Exosomal miR-146a derived from oxLDL-treated macrophages aggravated atherosclerotic plaque progression via facilitating NETs formation. The serum levels of blood lipids including TC (a), TG (b), and LDL (c); quantitative analysis (d) and representative photographs (e) of atherosclerotic lesion areas using ORO staining and H&E staining; immunofluorescence staining (f) for H3Cit and serum levels of TNF-α (G), IL-6 (h), IL-8 (i) and IL-1β (j) in normal feeding mice and AS model mice intravenously injected with PBS or ox-LDL-treated or untreated THP-1 cells-derived exosomes (n = 6 per group). **P < 0.01 vs. Normal group; ##P < 0.01 vs. NC group.
Discussion
In this study, we revealed that oxLDL-treated macrophages secreted miR-146a-containing exosomes, and that these exosomes induced NETs formation by increasing ROS production in neutrophils via inhibiting SOD2 expression, which further deteriorated AS progression. Our data indicate that miR-146a emerges as a communication molecule between macrophages and neutrophils to function as a pro-atherosclerotic factor.
MiRNAs are small single-stranded non-coding RNAs containing approximately 22 nucleotides that play crucial roles in pathophysiological processes[17]. Exosomes commonly act as important players in different cell types via transferring miRNAs as a form of intercellular communication. For instance, Zheng et al[18] displayed that exosomes derived from vascular smooth muscle cells (VSMCs) impaired the proliferation of microvascular endothelial cells (HMVECs) via exosomal miR-155, which in turn led to an increased endothelial permeability and enhanced AS progression. Li et al[19] uncovered that mesenchymal stem cells (MSCs)-derived exosomes promoted M2 macrophage polarization and ameliorated atherosclerosis through delivering miR-let7. To the best of our knowledge, the pathogenesis of AS involves a large number of exosomes derived from immune cells. For example, Gao et al[20] suggested that intravenous administration of dendritic cells (DCs)-derived exosomes increased atherosclerotic lesion formation in ApoE−/− mice. It is well documented that AS is an inflammatory process due to oxLDL accumulation in macrophages[21]. Chen et al[22] demonstrated that oxLDL stimulated macrophages-derived exosomal lncRNA GAS5 promoted the apoptosis of endothelial cells. A recent study has shown that EVs from atherogenic macrophages were taken up by naïve macrophages and transferred miR-146a to inhibit macrophage migration and accelerate the development of AS[11]. Our data in the present study showed that the expression of miR-146a was significantly upregulated in exosomes derived from serum of AS patients and oxLDL-treated THP-1 macrophages. A miRNA expression profiling previously identified that miR-146a was highly expressed in atherosclerotic plaques[23]. Further results in the current study showed that after co-cultured with normal human neutrophils, these exosomes could induce NETs formation, ROS overproduction and SOD2 downregulation in vitro.
Neutrophils form an essential part of the inflammatory response to combat invading pathogens through phagocytosis, degranulation and ROS generation[24]. NETosis is an alternative form of programmed cell death and an emerging mechanism underlying atherogenic inflammation[25]. Oxidative stress is defined as an excessive production of ROS that has been indicated to be an inducer of inflammatory disease. The imbalance between oxidants and antioxidants response to the increase of chemical reactions or impaired antioxidant defense system (ADS) might lead to the extreme rise of ROS, resulting in the large accumulation of active oxidative molecules and defects in DNA, RNA and proteins[26]. It has been previously demonstrated that ROS production by the NADPH oxidase is required for the formation of NETs[27]. Our in vitro experiments confirmed that miR-146a-overexpressing THP-1 macrophages-derived exosomes under oxLDL stimulation enhanced ROS production via downregulating SOD2 expression and led to inflammation and NETosis. Further investigation implied that the potential effect of miR-146a on ROS production, SOD2 expression and NETs formation was all reversed by ROS scavenger NAC. Moreover, intravenous injection of oxLDL-treated THP-1 macrophages-derived exosomes in ApoE−/− mice displayed higher levels of TC, TG and LDL and pro-inflammatory cytokines in sera, larger atherosclerotic lesions, and enhanced formation of NETs.
In conclusion, our findings indicated that atherogenic macrophages delivered endogenous miR-146a via exosomes to deteriorate AS development via SOD2/ROS-mediated NETosis. These data will provide a novel scientific basis for the understanding of AS progression.
Acknowledgments
Not applicable.
Disclosure statement
No potential conflict of interest was reported by the authors.
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
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