Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Abstract Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3ʹ untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.


| INTRODUCTION
Coronary heart disease is a leading cause of morbidity and mortality worldwide. 1 Treatment options include coronary artery bypass surgery and balloon angioplasty combined with stent implantation 2 ; however, the failure rate for treatment is high (8%-40%) because of restenosis (recurrence of blood vessel narrowing) predominantly caused by intimal hyperplasia. [3][4][5][6] Intimal hyperplasia is the thickening of a blood vessel wall in response to injury, potentially induced by a surgical procedure. Mesenchymal stem cells (MSCs) have been shown to exert an inhibitory effect on neointimal hyperplasia (the new layer of arterial intima formed particularly on a prosthesis) via rapid re-endothelialisation. 7 Another study reported the therapeutic effects of human MSCs by decreasing the inflammatory response to carotid artery ligation. 8 Exosomes secreted by MSCs have been shown to mediate intercellular communications between these cells and their target cells 9 ; however, the role of MSC-derived exosomes (MSC-Exo) in neointimal hyperplasia remains to be fully elucidated.
Exosomes, small membraned vesicles (30-100 nm) that are generated through inward budding of the endosomal membrane, modulate the function of recipient cells by transferring complex biological information, including mRNAs, microRNAs (miRNAs) and soluble proteins into these target cells in a functional form. [10][11][12] Liang et al showed that transfer of miR-125a to endothelial cells via MSC-Exo promoted angiogenesis in these target cells by miR-125a-mediated repression of DLL4. 9 The direct effect of exosomes on target cell function provides a wide range of potential therapeutic applications, including cellular renewal and tissue repair following myocardial ischaemia-reperfusion, 13 neurological stroke 14 and skin wounds, 15 likely mediated by mechanisms involving exosome-transferred miR-NAs. 16,17 However, whether exosome-mediated miRNA transfer plays a role in neointimal hyperplasia remains poorly understood.
In this study, to evaluate MSC-Exo, and more specifically MSC-Exo-derived miRNA transfer on neointimal hyperplasia, we assessed their effects on the proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro and in vivo by the determination of neointima formation following balloon-induced vascular injury. Previous studies in VSMCs have reported a number of key miRNAs (miR-21, miR-143, miR-145 and miR-221) that play important roles in proliferation, migration and neointimal thickening. [18][19][20][21] Another key miRNA, miR-125b, has been shown to inhibit the process of calcification of VSMCs, a pathology linked to the development of cardiovascular disease, 22 by inhibiting osteoblastic differentiation and proliferation. [23][24][25] In our study, the delivery of miR-125b into VSMCs via MSC-Exo was investigated along with the potential role of MSC-Exo-transferred miR-125b in neointimal hyperplasia.
Myosin-1E (Myo1e) is an actin-dependent molecular motor whose translocation is essential for lamellipodium extension and subsequent cellular motility and cell migration. 26 Heim et al 27

reported that
Myo1e binds to the FERM domain of focal adhesion kinase (FAK), activating FAK and inducing Y397 phosphorylation. Previous studies revealed that FAK is involved in the regulation of aortic smooth muscle cell motility and growth, and the regulation of smooth muscle cell recruitment during blood vessel morphogenesis. [28][29][30] The potential role of Myo1e and FAK activation in VSMCs in response to MSC-Exoderived signals was also investigated in this study. Our findings provide insight into the role of MSC-Exo-mediated miR-125b transfer on neointimal hyperplasia following arterial injury, offering potential therapeutic targets for vascular diseases.

| Cell culture
Bone marrow MSCs were isolated as previously described. 31  Primary rat VSMCs were isolated as previously described 32 with some modifications. Briefly, the aorta of an adult Sprague-Dawley rat was excised, cleaned of connective tissue, fat and endothelium and cut into 1-mm 2 pieces. The pieces were treated with 1 mL (1 mg/mL) of collagenase II (Sigma-Aldrich, St. Louis, MO, USA) and 0.125% trypsin for 1 hour at 37°C, followed by centrifugation at 300 g for 5 minutes to harvest the cells. Cells were cultured in DMEM supplemented with 10% FBS, and passages 3-7 were used for subsequent experiments. HEK293 cells were cultured in DMEM containing 10% FBS.

| Isolation and characterisation of exosomes
To isolate the exosomes of MSCs, the cells were cultured in DMEM/ F12 containing 10% exosome-free FBS for 48 hours and the supernatants were collected and centrifuged at 3000 g for 15 minutes to remove the cells and cell debris. The exosomes were isolated from the supernatants using ExoQuick-TC Kit (System Biosciences, Mountain View, CA, USA) according to the manufacturer's instructions. The pelleted exosomes were fixed in 2% paraformaldehyde in PBS, pH 7.4 and the morphology of the exosomes was observed using transmission electron microscopy (TEM) as previously described. 33 The exosomes were further characterized by Western blot analysis with three exosome-specific biomarkers: CD9, CD63 and CD81 (Abcam, Cambridge, UK).

VSMCs
Purified exosomes were labelled with 5 mmol/L of the fluorescent dye DIO (Invitrogen) by incubation for 15 minutes at 37°C. Any remaining WANG ET AL. | 1529 free dye was removed by ultracentrifugation at 120 000 g for 90 minutes, followed by two washes in PBS with ultracentrifugation.
To analyse exosome uptake by VSMCs, cells were incubated with DIO-labelled exosomes for 3 hours and then stained with DAPI (Invitrogen). The internalisation of DIO-labelled exosomes by VSMCs was visualized using an Eclipse TE2000 fluorescence microscope (Nikon, Tokyo, Japan).
MSCs were transfected with Cy3-labelled miR-125b mimics and incubated for 48 hours in medium containing exosome-free FBS.
Then, exosomes were isolated and subsequently incubated with VSMCs for 3 hours. Finally, cells were visualized under a fluorescence microscope as discussed above.

| Luciferase reporter assay
A synthetic fragment of the rat Myo1e 3′-UTR containing either wild-type (WT) or mutant (MUT) miR-125b binding sites, was inserted into the pRL-TK-Report vector (Promega, Madison, WI, USA). HEK293 cells were cotransfected with 200 ng of WT or MUT construct or the vehicle control and the miR-125b mimic or inhibitor or negative control (50 nmol/L) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. After 48 hours, the firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo ™ Luciferase Assay system (Promega) and a GloMax microplate luminometer (Promega). The relative luciferase activity was normalized to the Renilla luciferase activity.

| Scratch wound healing assay
Vascular smooth muscle cells were seeded in 6-well plates at a concentration of 2 × 10 5 cells per well. After incubation with starvation medium for 24 hours, a sterile pipette tip was used to inflict a linear scratch wound in the centre of the cell monolayer. After washing with PBS to remove any cellular debris, cells were stimulated with or without exosomes (200 μg/mL) in the presence or absence of PDGF-BB (20 ng/mL) for 24 hours, and the wound was monitored under a phase-contrast microscope (Olympus IX51, Tokyo, Japan), and the percentage of cell closure was calculated by measurements of the scratch width using ImageJ software.

| In vivo effects of MSC-Exo in a rat model of balloon-induced vascular injury
The balloon-induced vascular injury model was performed as previously described. 34 Briefly, Sprague-Dawley rats weighing 250-300 g (SLAC Laboratory Animal Co., Ltd, Shanghai, China) were anesthetised with 75 mg/kg pentobarbital and heparinised with 100 U/kg heparin sodium. To induce balloon injury, a 2F Fogarty arterial embolectomy balloon catheter (Edwards Lifesciences, Irvine, CA, USA) was introduced through the left external carotid artery and the artery was distended by the passage of saline three times. After balloon injury, 20 μg of exosomes was injected intravenously every other 2 days for 14 days. The left common carotid artery was harvested for analysis. Immunofluorescence was used to detect the localisation of exosomes in the carotid artery. DIO-labelled exosomes were injected intravenously into rats following vascular injury. The left common carotid artery was harvested for cryostat sectioning.
Carotid artery tissue was embedded in OCT compound and snap frozen for cryostat sectioning (7 μm). Artery tissue sections were then examined under a fluorescence microscope.
All procedures involving animals were approved by the Animal Research Committee of Zhongshan Hospital Fudan University (Reference number: SCXK2009-0019).

| MSC-Exo reduce VSMC proliferation and migration
The establishment of an MSC culture was confirmed visually by the presence of elongated and linear cells and by flow cytometry with antibodies directed against CD90, CD29, CD44 and CD34 ( Figure 1A,B). To evaluate the effects of MSC-Exo on neointimal hyperplasia, MSC-Exo were isolated and the morphology of the exosomes was observed using TEM. The exosomes purified from the MSC culture supernatants were round membrane-bound vesicles that were~50-100 nm in diameter ( Figure 1C). Then, Western blot analysis was performed on the proteins from MSC lysates and purified exosomes to confirm the presence of exosome/extracellular vesicle-specific markers: CD63, CD81 and CD9. As expected, all three markers were strongly expressed in the purified exosomes ( Figure 1D). Next, VSMCs were incubated with DIO-labelled exosomes and confocal microscopy was performed to demonstrate the internalisation of DIO-labelled exosomes (green) by VSMCs, whose nuclei were counterstained with DAPI (blue), and uptake of exosomes by VSMCs was observed ( Figure 1C

| MSC-Exo deliver miR-125b into VSMCs
To investigate the MSC-Exo-mediated transfer of the miRNA, miR-125b, into VSMCs, the relative expression levels of miR-125b in MSCs and MSC-Exo were first determined by quantitative reversetranscription PCR (RT-qPCR). Expression levels of miR-125b were significantly higher in MSC-Exo than in MSCs (P < 0.05; Figure 2A). Next, the expression of miR-125b in VSMCs after incubation with MSC-Exo was determined by RT-qPCR and significantly higher miR-125b expression was detected in VSMCs incubated with MSC-Exo compared with control VSMCs (P < 0.05; Figure 2B). To demonstrate the transfer of miRNA from MSCs into VSMCs via exosomes, exosomes were isolated from MSCs transfected with Cy3-miR-125b for 48 hours and were then incubated with VSMCs for 3 hours. Localisation of Cy3-miR-125b (red) in the exosome-treated VSMCs (counterstained with DAPI, blue) was observed using a confocal microscope ( Figure 2C). This provided evidence for the exosome-mediated transfer of miR-125b from MSCs into VSMCs. Next, exosomes derived from these MSC strains were incubated with VSMCs to investigate the effects of exosomal-transferred miR-125b on VSMC proliferation and migration. VSMC proliferation was decreased following treatment with exosomes from MSCs transfected with miR-125b mimic compared with exosomes from NC-transfected MSCs (P < 0.05; Figure 3C). Conversely, VSMC proliferation was increased following treatment with exosomes from MSCs transfected with miR-125b inhibitor compared with exosomes from IC-transfected MSCs (P < 0.05; Figure 3D).
Next, the effects of MSC-derived exosomally transferred miR-125b on VSMC migration were assessed using the same MSC transfected strains and a scratch-wound assay. VSMC migration was decreased following treatment with exosomes from MSCs transfected with miR-125b mimic compared with exosomes from NC-transfected MSCs (P < 0.05; Figure 3E). Conversely, VSMC migration was increased following treatment with exosomes from MSCs transfected with miR-125b inhibitor compared with exosomes from IC-transfected MSCs (P < 0.05; Figure 3F). Taken together, these findings indicate that miR-125b delivered by MSC-Exo inhibits VSMC proliferation and migration.

| Exosomal miR-125b suppresses VSMC proliferation and migration by targeting Myo1e
Next, the mechanism responsible for exosomal miR-125b-mediated suppression of VSMC proliferation and migration was investigated. A miR-125b binding site was identified in the 3ʹUTR of rat Myo1e ( Figure 4A). Myo1e is an actin-dependent molecular motor previously reported to play a role in lamellipodium extension and subsequent cell migration. 26 Figure 4E). VSMCs overexpressing Myo1e were then subjected to functional assays including a cell proliferation assay and a scratch-wound assay to measure cell migration.
Myo1e overexpression restored the inhibition of VSMC proliferation ( Figure 4F) and migration ( Figure 4G) in response to treatment with exosomes isolated from MSCs transfected with miR-125b mimic.

F I G U R E 3 miR-125b delivered by exosomes modulates vascular smooth muscle cells (VSMC) proliferation and migration. (A, B)
Mesenchymal stem cells (MSCs) were transfected with 50 nmol/L miR-125b mimic or mimic control (NC) (A), or 100 nmol/L inhibitor or inhibitor control (IC) (B) as indicated. miR-125b expression was analysed in MSCs or in MSC-derived exosomes by RT-qPCR. **P < 0.01 vs NC or IC. (C, D) Analysis of VSMC proliferation by MTT assays. VSMCs were treated with exosomes isolated from MSCs transfected with miR-125b mimic (C) or inhibitor (D) in the presence or absence of PDGF-BB (20 ng/mL) for 48 h and cell proliferation was determined. *P < 0.05, **P < 0.01. (E, F) Cell migration was measured after treatment with exosomes isolated from MSCs transfected with miR-125b mimic (E) or inhibitor (F) in the presence or absence of PDGF-BB (20 ng/mL) by scratch-wound assays and is presented as the percentage of cell closure. Experiments were repeated at least three times (n = 3) and data are the average of repeat experiments. *P < 0.05, **P < 0.01 Focal adhesion kinase has been reported to play a role in the proliferation and migration of aortic smooth muscle cells, and the regulation of smooth muscle cell recruitment during blood vessel morphogenesis. [28][29][30] Myo1e has previously been reported to induce FAK phosphorylation and FAK kinase activity. 27 Here, the phosphorylation of FAK was examined by Western blot analysis in the VSMCs after the indicated treatment ( Figure 4H). FAK phosphorylation was promoted by the overexpression of Myo1e but decreased by treatment with exosomes isolated from MSCs transfected with miR-125b mimic. Transfection of VSMCs with lentiviral particles of Myo1e shRNA reduced the expression of Myo1e mRNA and the levels of protein ( Figure 4I). Moreover, cell proliferation and migration were both reduced in VSMCs with silenced Myo1e expression ( Figure 4J,K). Stimulation by PDGF-BB led to increased levels of cell proliferation and migration in the control VSMCs but these levels were significantly lowered in VSMCs with Myo1e silenced (Figure 4J,K). Taken together, these findings indicate that exosomal miR-125b suppresses VSMC proliferation and migration by targeting Myo1e and the subsequent FAK activation.

| miR-125b delivered by MSC-Exo reduces neointima formation in rat carotid arteries after angioplasty
To analyse the effects of exosome-derived miR-125b on neointima formation in rat carotid arteries after angioplasty, a rat model of balloon-induced vascular injury was employed. Following balloon injury,

| DISCUSSION
All types of surgical coronary intervention result in some degree of arterial injury, the response to which determines the success of the procedure and the long-term prognosis of the patient. Stent implantation, for example, promotes neointimal hyperplasia, a major cause of in-stent restenosis. [3][4][5][6] Current strategies to prevent restenosis are focused on the inhibition of neointimal hyperplasia through drugeluting stents and vascular brachytherapy. It is, therefore, crucial to understand the molecular mechanisms involved in neointima formation.
Intimal hyperplasia is the thickening of a blood vessel wall in response to injury and involves the proliferation and migration of VSMCs and the deposition of extracellular matrix. When this process of rapid re-endothelialisation occurs on a prosthesis, the new layer of arterial intima is referred to as the neointima. VSMC proliferation and subsequent migration into the neointima layer following injury have been shown to be promoted by PDGF-BB, 35 but the molecular mechanism involved remained to be fully elucidated. It has previously been established that MSCs affect VSMCs via exosome-mediated intercellular communication between these cells. 9 Furthermore, it was reported that the exosome-mediated transfer of biological material and signalling molecules (such as mRNA and miRNA) between these cell types could induce altered behaviour in the recipient cells, such as triggering angiogenesis. 13,36,37 Similarly, the exosome-mediated transfer of miRNAs from MSCs to endothelial cells has been reported to promote angiogenesis. 38 Extracellular vesicles, of which exosomes are the smallest in size   50 demonstrated that miR-143-3p exosomally transferred from pulmonary artery smooth muscle cells exerted a paracrine pro-migratory and pro-angiogenic effect on pulmonary arterial endothelial cells. In another study, miR-125a-5p, which was downregulated following vascular injury, was shown to modulate phenotypic switching of VSMCs, a key event in the development of restenosis following coronary intervention. miR-125a-5p had a direct effect on VSMC proliferation and migration.
Several studies have recognized miR-125b as an important mediator for the modulation of the SMC phenotype. 23,25,51 Deregulation of VSMC phenotype switching is associated with vascular proliferative and migration disorders. 52 Through literature searches, we found that miR-125b was enriched in MSC-Exo, 53,54 therefore, we investigated the role of MSC-Exo-mediated miR-125b transfer on neointimal hyperplasia following arterial injury. The findings from our study indicated that MSC-Exo are enriched in miR-125b and can transfer this miRNA to VSMCs. In previous studies, miR-125b was reported to play a role in osteoblast differentiation by regulating cell proliferation, 23 and osteogenic transdifferentiation of VSMCs and the proliferation of human coronary artery smooth muscle cells. 25 In our study, we demonstrated that miR-125b targets the 3ʹUTR of Myo1e in VSMCs repressing its expression and affecting subsequent FAK activation.
The interaction between Myo1e and FAK and the resulting phosphorylation-activation of FAK has been shown to be crucial for cell motility, 27,28 and therefore the transcriptional repression of Myo1e would be expected to inhibit the migratory behaviour of VSMCs.
Myosin 1E is an actin-dependent molecular motor that is expressed in a range of tissues and has the ability to bind to both cell membranes and actin filaments. Myo1e also binds phospholipids with high affinity 55 and regulates podocyte function and glomerular filtration. 56 Furthermore, the ERK-mediated translocation of Myo1e from the cytosol to the tips of lamellipodia is essential for cell motility and migration 57 and Myo1e regulates TLR4-triggered macrophage spreading and chemokine release as part of the innate immune response. 58 Myo1e has therefore been linked to a number of diseases such as atherosclerosis, 59 coronary artery disease and kidney disease. 60 In our study, using a rat model of balloon-induced vascular injury, we revealed that exosome-derived miR-125b repressed Myo1e expression, suppressed VSMC proliferation and migration and inhibited neointima formation in rat carotid arteries following angioplasty.
Our findings suggest that miR-125b may be a potential therapeutic candidate for the treatment of vascular diseases and arterial injury. It is conceivable that this miRNA could be delivered to the site of injury by incorporation into a drug-eluting stent.

ACKNOWLEDG EMENTS
This work was supported by the National Natural Science Foundation of China (81470581).

CONFLI CTS OF INTEREST
The authors confirm that there are no conflicts of interest.