Periodontitis‐compromised dental pulp stem cells secrete extracellular vesicles carrying miRNA‐378a promote local angiogenesis by targeting Sufu to activate the Hedgehog/Gli1 signalling

ObjectivesPreviously, our investigations demonstrated robust pro‐angiogenic potentials of extracellular vesicles secreted by periodontitis‐compromised dental pulp stem cells (P‐EVs) when compared to those from healthy DPSCs (H‐EVs), but the underlying mechanism remains unknown.Materials and methodsHere, circulating microRNAs (miRNAs) specifically found in P‐EVs (compared with H‐EVs) were identified by Agilent miRNA microarray analysis, and the roles of the candidate miRNA in P‐EV‐enhanced cell angiogenesis were confirmed by cell transfection and RNA interference methods. Next, the direct binding affinity between the candidate miRNA and its target gene was evaluated by luciferase reporter assay. CCK‐8, transwell/scratch wound healing and tube formation assays were established to investigate the proliferation, migration, and tube formation abilities of endothelial cells (ECs). Western blot was employed to measure the protein levels of Hedgehog/Gli1 signalling pathway components and angiogenesis‐related factors.ResultsThe angiogenesis‐related miRNA miR‐378a was found to be enriched in P‐EVs, and its role in P‐EV‐enhanced cell angiogenesis was confirmed, wherein Sufu was identified as a downstream target gene of miR‐378a. Functionally, silencing of Sufu stimulated EC proliferation, migration and tube formation by activating Hedgehog/Gli1 signalling. Further, we found that incubation with P‐EVs enabled the transmission of P‐EV‐contained miR‐378a to ECs. Subsequently, the expressions of Sufu, Gli1 and vascular endothelial growth factor in ECs were significantly influenced by P‐EV‐mediated miR‐378a transmission.ConclusionsThese data suggest that P‐EVs carrying miR‐378a promote EC angiogenesis by downregulating Sufu to activate the Hedgehog/Gli1 signalling pathway. Our findings reveal a crucial role for EV‐derived miR‐378a in cell angiogenesis and hence offer a new target for modifying stem cells and their secreted EVs to enhance vessel regenerative potential.     

microRNA‐19b‐3p‐containing extracellular vesicles derived from macrophages promote the development of atherosclerosis by targeting JAZF1

Atherosclerosis has been regarded as a major contributor to cardiovascular disease. The role of extracellular vesicles (EVs) in the treatment of atherosclerosis has been increasingly reported. In this study, we set out to investigate the effect of macrophages‐derived EVs (M‐EVs) containing miR‐19b‐3p in the progression of atherosclerosis, with the involvement of JAZF1. Following isolation of EVs from macrophages, the M‐EVs were induced with ox‐low density lipoprotein (LDL) (ox‐LDL‐M‐EVs), and co‐cultured with vascular smooth muscle cells (VSMCs). RT‐qPCR and western blot assay were performed to determine the expression of miR‐19b‐3p and JAZF1 in M‐EVs and in VSMCs. Lentiviral infection was used to overexpress or knock down miR‐19b‐3p. EdU staining and scratch test were conducted to examine VSMC proliferation and migration. Dual‐luciferase gene reporter assay was performed to examine the relationship between miR‐19b‐3p and JAZF1. In order to explore the role of ox‐LDL‐M‐EVs carrying miR‐19b‐3p in atherosclerotic lesions in vivo, a mouse model of atherosclerosis was established through high‐fat diet induction. M‐EVs were internalized by VSMCs. VSMC migration and proliferation were promoted by ox‐LDL‐M‐EVs. miR‐19b‐3p displayed upregulation in ox‐LDL‐M‐EVs. miR‐19b‐3p was transferred by M‐EVs into VSMCs, thereby promoting VSMC migration and proliferation. mir‐19b‐3p targeted JAZF1 to decrease its expression in VSMCs. Atherosclerosis lesions were aggravated by ox‐LDL‐M‐EVs carrying miR‐19b‐3p in ApoE^−/− mice. Collectively, this study demonstrates that M‐EVs containing miR‐19b‐3p accelerate migration and promotion of VSMCs through targeting JAZF1, which promotes the development of atherosclerosis.     

miR‐4286 functions in osteogenesis and angiogenesis via targeting histone deacetylase 3 and alleviates alcohol‐induced bone loss in mice

ObjectivesAlcohol consumption is one of the leading factors contributing to premature osteopenia. MicroRNA (miRNA) coordinates a cascade of anabolic and catabolic processes in bone homeostasis and dynamic vascularization. The aim was to investigate the protective role of miR‐4286 in alcohol‐induced bone loss and its mechanism.Materials and MethodsThe effect of miR‐4286 and alcohol on bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) was explored via multiple in vitro assays, including cell proliferation, QPCR, Western blot, osteogenesis, angiogenesis etc miR‐4286 directly regulated HDAC3 was investigated by luciferase reporter assay, and the function of HDAC3 was also explored in vitro. Moreover, alcohol‐induced bone loss in mice was established to reveal the preventive effect of miR‐4286 by radiographical and histopathological assays.ResultsIn vitro, ethanol dramatically inhibited the proliferation and osteogenesis of BMSCs, and substantially impaired the proliferation and vasculogenesis of HUVECs. However, a forced overexpression of miR‐4286 within BMSCs and HUVECs could largely abolish inhibitory effects by alcohol. Furthermore, alcohol‐induced inhibition on osteogenic and vasculogenic functions was mediated by histone deacetylase 3 (HDAC3), and dual‐luciferase reporter assay showed that HDAC3 was the direct binding target of miR‐4286. In vivo, micro‐CT scanning and histology assessment revealed that miR‐4286 could prevent alcohol‐induced bone loss.ConclusionsWe firstly demonstrated that miR‐4286 might function via intimate osteogenesis‐angiogenesis pathway to alleviate alcohol‐induced osteopenia via targeting HDAC3.     

CD33‐targeting extracellular vesicles deliver antisense oligonucleotides against FLT3‐ITD and miR‐125b for specific treatment of acute myeloid leukaemia

IntroductionAcute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%–65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs).MethodsIn this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33‐binding monoclonal antibody onto the EV surface.ResultsThe conjugation of RBCEVs with the CD33‐binding antibody significantly increases the uptake of RBCEVs by CD33‐positive AML cells, but not by CD33‐negative cells. We also load CD33‐targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML.ConclusionTargeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

In this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them with surface functionalization and exogenous drug loading to develop an efficient drug delivery system for AML. Anti‐CD33 antibody was conjugated to RBCEVs using an enzymatic method combined with the streptavidin‐biotin system. We load the antibody conjugated RBCEVs with ASOs targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the treatment with engineered EVs improve leukaemia suppression both in vitro and in vivo.     

METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.     

PTH‐induced EndMT via miR‐29a‐5p/GSAP/Notch1 pathway contributed to valvular calcification in rats with CKD

BackgroundEndothelial‐to‐mesenchymal transition (EndMT) is a common pathophysiology in valvular calcification (VC) among non‐chronic kidney disease (CKD) patients. However, few studies were investigated in CKD‐induced VC. Parathyroid hormone (PTH) was considered to be an important component of EndMT in CKD‐induced cardiovascular diseases. Therefore, determining whether PTH could induce valvular EndMT and elucidating corresponding mechanism involved further study.MethodsPerforming a 5/6 nephrectomy with a high phosphorus diet was done to construct VC models in rats with CKD. miRNA sequencing was used to ascertain changes in microRNA in human umbilical vein endothelial cells (HUVECs) intervened by PTH. VC was observed by Von Kossa staining and scanning electron microscope.ResultsPTH induced valvular EndMT in VC. Global microRNA expression profiling of HUVECs was examined in PTH versus the control in vitro, in which miR‐29a‐5p was most notably decreased and was resumed by PTHrP(7‐34) (PTH‐receptor1 inhibitor). Overexpression of miR‐29a‐5p could inhibit PTH‐induced EndMT in vitro and valvular EndMT in vivo. The dual‐luciferase assay verified that γ‐secretase‐activating protein (GASP) served as the target of miR‐29a‐5p. miR‐29a‐5p‐mimics, si‐GSAP and DAPT (γ‐secretase inhibitor) inhibited PTH‐induced γ‐secretase activation, thus blocking Notch1 pathway activation to inhibit EndMT in vitro. Moreover, Notch1 pathway activation was observed in VC. Blocking Notch1 pathway activation via AAV‐miR‐29a and DAPT inhibited valvular EndMT. In addition, blocking Notch1 pathway activation was also shown to alleviate VC.ConclusionPTH activates valvular EndMT via miR‐29a‐5p/GSAP/Notch1 pathway, which can contribute to VC in CKD rats.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

HIF‐1α‐induced up‐regulation of microRNA‐126 contributes to the effectiveness of exercise training on myocardial angiogenesis in myocardial infarction rats

Exercise training (ET) is a non‐drug natural rehabilitation approach for myocardial infarction (MI). Among the numerous beneficial effects of ET, myocardial angiogenesis is indispensable. In the present study, we investigated the role and mechanism of HIF‐1α and miR‐126 in ET‐induced MI myocardial angiogenesis which may provide new insights for MI treatment. Rat model of post‐MI and human umbilical vein endothelial cells (HUVECs) were employed for our research. Histomorphology, immunohistochemistry, quantitative real‐time PCR, Western blotting and small‐interfering RNA (siRNA) transfection were applied to evaluate the morphological, functional and molecular mechanisms. In vivo results showed that 4‐week ET could significantly increase the expression of HIF‐1α and miR‐126 and reduce the expression of PIK3R2 and SPRED1, while 2ME2 (HIF‐1α inhibitor) partially attenuated the effect of ET treatment. In vitro results showed that HIF‐1α could trigger expression of miR‐126 in HUVECs in both normoxia and hypoxia, and miR‐126 may be involved in the tube formation of HUVECs under hypoxia through the PI3K/AKT/eNOS and MAPK signalling pathway. In conclusion, we revealed that HIF‐1α, whose expression experiences up‐regulation during ET, could function as an upstream regulator to miR‐126, resulting in angiogenesis promotion through the PI3K/AKT/eNOS and MAPK signalling pathway and subsequent improvement of the MI heart function.     

Inhibition of exosomal miR‐24‐3p in diabetes restores angiogenesis and facilitates wound repair via targeting PIK3R3

Diabetic foot ulcer (DFU) is one of the common ailments of elderly people suffering from diabetes. Exosomes containing various active regulators have been found to play a significant role in apoptosis, cell proliferation and other biological processes. However, the effect and the underlying mechanism of action of diabetes patients derived from circulating exosomes (Dia‐Exos) on DFU remain unclear. Herein, we aim to explore the potential regulatory role of Dia‐Exos. First, we attempted to demonstrate the harmful effect of Dia‐Exos both in vivo and in vitro. miRNA‐24‐3p (miR‐24‐3p) was found enriched with Dia‐Exos. Hence, inhibition of this miRNA could partially reverse the negative effect of Dia‐Exos, thus, in ture, accelerates wound repair. Luciferase assay further verified the binding of miR‐24‐3p to the 3′‐UTR of phosphatidylinositol 3‐kinase regulatory subunit gamma (PIK3R3) mRNA and the PIK3R3 expression enhanced human umbilical vein endothelial cells functionality in vitro. Hence, the findings of this study reveal the regulatory role of Dia‐Exos in the process of wound healing and provide experimental evidence for the therapeutic effects of knocking down miR‐24‐3p in DFU treatment.     

MiR‐223‐3p inhibits rTp17‐induced inflammasome activation and pyroptosis by targeting NLRP3

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.     

MiR‐103‐3p targets the m6A methyltransferase METTL14 to inhibit osteoblastic bone formation

Impaired osteoblast function is involved in osteoporosis, and microRNA (miRNA) dysregulation may cause abnormal osteoblast osteogenic activity. However, the influence of miRNA on osteoblast activity and the underlying mechanisms remain elusive. In this study, miR‐103‐3p was found to be negatively correlated with bone formation in bone specimens from elderly women with fractures and ovariectomized (OVX) mice. Additionally, miR‐103‐3p directly targeted Mettl14 to inhibit osteoblast activity, and METTL14‐dependent N⁶‐methyladenosine (m⁶A) methylation inhibited miR‐103‐3p processing by the microprocessor protein DGCR8 and promoted osteoblast activity. Moreover, miR‐103‐3p inhibited bone formation in vivo, and therapeutic inhibition of miR‐103‐3p counteracted the decreased bone formation in OVX mice. Further, METTL14 was negatively correlated with miR‐103‐3p but positively correlated with bone formation in bone specimens from elderly women with fractures and OVX mice. Collectively, our results highlight the critical roles of the miR‐103‐3p/METTL14/m⁶A signaling axis in osteoblast activity, identifying this axis as a potential target for ameliorating osteoporosis.     

A circular intronic RNA ciPVT1 delays endothelial cell senescence by regulating the miR‐24‐3p/CDK4/pRb axis

Circular RNAs (circRNAs) have been established to be involved in numerous processes in the human genome, but their function in vascular aging remains largely unknown. In this study, we aimed to characterize and analyze the function of a circular intronic RNA, ciPVT1, in endothelial cell senescence. We observed significant downregulation of ciPVT1 in senescent endothelial cells. In proliferating endothelial cells, ciPVT1 knockdown induced a premature senescence‐like phenotype, inhibited proliferation, and led to an impairment in angiogenesis. An in vivo angiogenic plug assay revealed that ciPVT1 silencing significantly inhibited endothelial tube formation and decreased hemoglobin content. Conversely, overexpression of ciPVT1 in old endothelial cells delayed senescence, promoted proliferation, and increased angiogenic activity. Mechanistic studies revealed that ciPVT1 can sponge miR‐24‐3p to upregulate the expression of CDK4, resulting in enhanced Rb phosphorylation. Moreover, enforced expression of ciPVT1 reversed the senescence induction effect of miR‐24‐3p in endothelial cells. In summary, the present study reveals a pivotal role for ciPVT1 in regulating endothelial cell senescence and may have important implications in the search of strategies to counteract the development of age‐associated vascular pathologies.     

miR‐126‐5p enhances radiosensitivity of lung adenocarcinoma cells by inhibiting EZH2 via the KLF2/BIRC axis

Radiotherapy is a common method for the treatment of lung adenocarcinoma, but it often fails due to the relative non‐susceptibility of lung adenocarcinoma cells to radiation. We aimed to discuss the related mechanisms by which miR‐126‐5p might mediate radiosensitivity of lung adenocarcinoma cells. The binding affinity between miR‐126‐5p and EZH2 and between KLF2 and BIRC5 was identified using multiple assays. A549 and H1650 cells treated with X‐ray were transfected with miR‐126‐5p mimic/inhibitor, oe‐EZH2, or si‐KLF2 to detect cell biological functions and radiosensitivity. Finally, lung adenocarcinoma nude mouse models were established. miR‐126‐5p and KLF2 were poorly expressed, while EZH2 and BIRC5 were upregulated in lung adenocarcinoma tissues and cells. miR‐126‐5p targeted EZH2 to promote the KLF2 expression so as to inhibit BIRC5 activation. Both in vitro and in vivo experiments verified that elevated miR‐126‐5p inhibited cell migration and promoted apoptosis to enhance the sensitivity of lung adenocarcinoma cells to radiotherapy via the EZH2/KLF2/BIRC5 axis. Collectively, miR‐126‐5p downregulated EZH2 to facilitate the sensitivity of lung adenocarcinoma cells to radiotherapy via KLF2/BIRC5.     

Bone marrow macrophage‐derived exosomal miR‐143‐5p contributes to insulin resistance in hepatocytes by repressing MKP5

ObjectiveIn this study, we aim to explore the role of bone marrow macrophage‐derived exosomes in hepatic insulin resistance, investigate the substance in exosomes that regulates hepatic insulin signalling pathways, reveal the specific molecular mechanisms involved in hepatic insulin resistance and further explore the role of exosomes in type 2 diabetes.Materials and methodsHigh‐fat diet (HFD)‐fed mice were used as obesity‐induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD‐fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR‐143‐5p mimics. Luciferase assay and western blot were used to assess the target of miR‐143‐5p.ResultsBMMs from HFD‐fed mice were polarized towards M1, and miR‐143‐5p was significantly upregulated in exosomes of BMMs from HFD‐fed mice. Overexpression of miR‐143‐5p in Hep1‐6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual‐luciferase reporter assay and western blot demonstrated that mitogen‐activated protein kinase phosphatase‐5 (Mkp5, also known as Dusp10) was the target gene of miR‐143‐5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR‐143‐5p mimics in Hep1‐6.ConclusionBone marrow macrophage‐derived exosomal miR‐143‐5p induces insulin resistance in hepatocytes through repressing MKP5.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.     

Macrophage‐derived exosomal miR‐4532 promotes endothelial cells injury by targeting SP1 and NF‐κB P65 signalling activation

Atherosclerosis is a complex pathological process involving macrophages, endothelial cells and vascular smooth muscle cells that can lead to ischemic heart disease; however, the mechanisms underlying cell‐to‐cell communication in atherosclerosis are poorly understood. In this study, we focused on the role of exosomal miRNAs in crosstalk between macrophages and endothelial cells and explored the rarely studied molecular mechanisms involved. Our in vitro result showed that macrophage‐derived exosomal miR‐4532 significantly disrupted human umbilical vein endothelial cells (HUVECs) function by targeting SP1 and downstream NF‐κB P65 activation. In turn, increased endothelin‐1 (ET‐1), intercellular cell adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) and decreased endothelial nitric oxide synthase (eNOS) expression in HUVECs increased attraction of macrophages, exacerbating foam cell formation and transfer of exosomal miR‐4532 to HUVECs. MiR‐4532 overexpression significantly promoted endothelial injury and pretreatment with an inhibitor of miR‐4532 or GW4869 (exosome inhibitor) could reverse this injury. In conclusion, our data reveal that exosomes have a critical role in crosstalk between HUVECs and macrophages. Further, exosomal miR‐4532 transferred from macrophages to HUVECs and targeting specificity protein 1 (SP1) may be a novel therapeutic target in patients with atherosclerosis.