Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126

Journal Pre-proof

Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126 Wei Liu , Linwei Li , Yuluo Rong , Dingfei Qian , Jian Chen , Zheng Zhou , Yongjun Luo , Dongdong Jiang , Lin Cheng , Shujie Zhao , Fanqi Kong , Jiaxing Wang , Zhimin Zhou , Tao Xu , Fangyi Gong , Yifan Huang , Changjiang Gu , Xuan Zhao , Jianling Bai , Feng Wang , Wene Zhao , Le Zhang , Xiaoyan Li , Guoyong Yin , Jin Fan , Weihua Cai PII: DOI: Reference:

S1742-7061(19)30849-9 https://doi.org/10.1016/j.actbio.2019.12.020 ACTBIO 6503

To appear in:

Acta Biomaterialia

Received date: Revised date: Accepted date:

6 September 2019 5 December 2019 13 December 2019

Please cite this article as: Wei Liu , Linwei Li , Yuluo Rong , Dingfei Qian , Jian Chen , Zheng Zhou , Yongjun Luo , Dongdong Jiang , Lin Cheng , Shujie Zhao , Fanqi Kong , Jiaxing Wang , Zhimin Zhou , Tao Xu , Fangyi Gong , Yifan Huang , Changjiang Gu , Xuan Zhao , Jianling Bai , Feng Wang , Wene Zhao , Le Zhang , Xiaoyan Li , Guoyong Yin , Jin Fan , Weihua Cai , Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Title: Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126

Authors: Wei Liu1,#, Linwei Li1,#, Yuluo Rong1,#, Dingfei Qian1, Jian Chen1, Zheng Zhou1, Yongjun Luo1, Dongdong Jiang1, Lin Cheng1, Shujie Zhao1, Fanqi Kong1, Jiaxing Wang1, Zhimin Zhou1,2, Tao Xu1, Fangyi Gong1, Yifan Huang1, Changjiang Gu1, Xuan Zhao1, Jianling Bai3, Feng Wang4, Wene Zhao4, Le Zhang4, Xiaoyan Li4, Guoyong Yin 1,* Jin Fan1,* and Weihua Cai1*

Affiliation: 1Department of Orthopaedics, the First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, China 2

Department of Orthopaedics, Zhongda Hospital Southeast Univeristy, Nanjing,

Jiangsu, 210009, China 3

Department of Biostatistics, School of Public Health, Nanjing Medical University,

Nanjing, Jiangsu, 211166, China 4

Department of Analytical & Testing Center, Nanjing Medical University, Nanjing,

Jiangsu, 211166, China

#

Author contribution: These authors contributed equally to this work.

*

Corresponding authors: Weihua Cai, Jin Fan & Guoyong Yin

E-mail: [email protected]; [email protected]; [email protected].

1

ABSTRACT: Increasing evidence has suggested that paracrine mechanisms might be involved in the underlying mechanism of mesenchymal stem cells (MSCs) transplantation, and exosomes are an important component of this paracrine role. However, MSCs are usually exposed to normoxia (21% O2) in vitro but experience large differences in oxygen concentration in the body under hypoxia. Indeed, hypoxic precondition of MSCs can enhance their paracrine effects. The main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (Hypo-Exos) exhibit greater effects on bone fracture healing than those under normoxia (Exos). Using in vivo bone fracture model and in vitro experiments including cell proliferation assay, cell migration assay and so on, we confirmed that Hypo-Exos administration promoted angiogenesis, proliferation and migration to a greater extent when compared to Exos. Furthermore, utilizing a series in vitro and in vivo gain and loss of function experiments, we confirmed a functional role for exosomal miR-126 in the process of bone fracture healing. Meanwhile, we found that knockdown of hypoxia inducible factor 1 (HIF-1α) resulted in a significant decrease of miR-126 in MSCs and exosomes, thereby abolishing the effects of Hypo-Exos. In conclusion, our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing.

Statement of significance Studies have confirmed that transplantation of exosomes exhibit similar therapeutic effects and functional properties to directly-transplanted stem cells but have less significant adverse effects. However, during in vitro culture conditions, MSCs are usually exposed to normoxia (21% O2) which is very different to the 2

oxygen concentrations found in the body under natural physiological conditions. Our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126 and the SPRED1/Ras/Erk signaling pathway. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing.

KEYWORDS: exosomes, hypoxia, miR-126, SPRED1, HIF-1α, angiogenesis, bone fracture

1. INTRODUCTION It is estimated that approximately 15 million new fractures occur in the United States each year, caused mainly by car accidents, sports injuries, or work accidents [1, 2]. Furthermore, delayed or non-healing occurs in a significant proportion of these patients (10–15%). Both delayed and non-healing requires additional prolonged or repeated treatment, and represents a significant impact on both the cost of treatment and quality of life [3, 4]. Bone fracture repair is a complex process of utilizing endogenous regenerative potential to restore original bone structure with promoting an increase in mineralized tissues[5] . To date, a number of related studies have been conducted on several major regulatory factors involved in bone fracture healing[6, 7]. Among these modulatory processes, angiogenesis is recognized to have an important role in bone metabolism [8]. During bone fracture healing, endothelial cells invade the cartilage in the growth plate area to provide nutrients and serve as a new scaffold for bone formation. During angiogenesis, a 3

number of processes are involved including proliferation and migration of endothelial cells, formation of capillaries and stabilization of mesenchymal stem cells (MSCs) [9]. However, fracture treatment has progressed slowly in recent years [1, 10]. It has been demonstrated that transplantation of MSCs exhibit therapeutic effects in several models of disease, including the promotion of osteogenesis and angiogenesis in the stabilized fracture model [11-13]. However, there remains limitations and challenges that cannot be ignored when transplanting MSCs directly into target tissues [14]. For example, in ischemic tissues, the survival rate of transplanted stem cells has proven to be extremely low. Moreover, other related risks limit the underlying application of transplanting MSCs in the clinic to aid bone fracture healing including: immune rejection, cell dedifferentiation, and tumor formation [15, 16]. Recent studies looking at the role of MSCs in tissue regeneration have shown that paracrine mechanisms might be involved in the underlying mechanism of action of MSCs in the treatment of several diseases and exosomes may play an important role in this process [17]. Exosomes are important components of the paracrine secretion of cells and are originated from invagination of endosomal membranes. They are derived from multivesicular bodies with a diameter of 50 to 150 nm [18-20] and are released into the extracellular space through fusion with the plasma membranes, while protecting their contents from degradation. They participate in the transport of biochemicals such as cytokines, mRNAs, miRNAs, and proteins and as a result play an essential crucial role in intercellular communication through transfer of genetic material [21, 22]. Of note, the specific surface ligands of exosomes ensure that it binds to target cells and deliver their contents, ultimately regulating specific biological functions. It has been confirmed that transplantation of exosomes exhibit similar therapeutic effects and functional properties to directly-transplanted stem cells but have less adverse effects seen when transplanting stem cells directly [22-24]. A recent study has shown that exosomes derived from MSCs could promote angiogenesis in a model of myocardial 4

infarction [25]. Similarly, it has been shown that exosomes derived from human umbilical cord MSCs (HucMSCs) could alleviate rat hepatic ischemia-reperfusion injury [26]. Our recent studies have shown that exosomes derived from stem cells could enhance functional recovery after spinal cord injury by inhibiting neuronal cell apoptosis and promoting autophagy [27, 28]. Indeed, the first patient has been successfully treated with MSC-exosomes in Graft versus Host Disease (GvHD) [29]. The concentration of oxygen has been recognized to be vitally important in the process of proliferation, differentiation and self-renewal of MSCs [30]. However, during in vitro culture conditions, MSCs are usually exposed to normoxia (21% O2) which is very different to the oxygen concentrations found in the body under natural physiological conditions. In fact, a large proportion of MSCs exist in a hypoxic environment (2%–8% O2 or even lower) in the body[31]. A recent study isolated exosomes from MSCs which were grown in media similar to that found in peripheral arterial disease (0% FBS, 1% O2) and found that these exosomes contained a number of pro-angiogenic factors that may be beneficial to ischemic tissues [32]. A different study, using an infarcted heart model, found that exosomes derived from MSCs after hypoxic treatment, exhibited increased vascularization, lower apoptosis rates of cardiomyocytes and increased recruitment of cardiac progenitor cells [33]. Our previous study also showed that ischemic hypoxia preconditioning could suppress cell death in a model of ischemia-reperfusion injury in rats [34]. Indeed, hypoxic precondition of MSCs can significantly enhance their biological functions and activities, thereby improving the transplantation efficacy of MSCs in the treatment of various disease models [35, 36]. However, it is still unclear whether MSCs under hypoxic conditions can promote bone fracture healing and whether such enhancement is mediated by exosomal signaling. Recent studies have focused on exosomal contents including proteins and RNAs and attempted to determine their underlying mechanisms in the treatment of various diseases [37, 38]. However, the miRNA in exosomes derived from MSCs under hypoxia and the underlying mechanisms by which these contribute to bone fracture 5

healing in vivo remains unknown. It has been demonstrated that exosomal miRNAs could exert their regulatory effects on target cells and this could represent a new way of intracellular messages communication [37, 39]. Because treatment using hypoxia preconditioning can improve the therapeutic effects of MSCs and modulate specific miRNA expression, we attempted to confirm a role for hypoxia treatment in the enhancement of exosome bio-activity through the regulation of miRNAs and bone fracture healing. Using a miRNA microarray, miR-126 was found to be enriched in Hypo-Exos and promoted endothelial cell proliferation, angiogenesis and migration in vivo and in vitro. Correspondingly, our results demonstrated that knockdown of miR-126 in Hypo-Exo (miR-126KD-Hypo-Exos) could abolish the beneficial effects seen with Hypo-Exos. In this study, we demonstrated that miR-126-enriched exosomes released from HIF-1α-activated HucMSCs, promoted endothelial cell proliferation,

angiogenesis

and

migration

by suppressing

the

activity of

Sprouty-related EVH1 domain-containing protein 1 (SPRED1), thereby activating the Ras/Erk pathway. This finding indicated a mechanism of action for HucMSCs-derived exosomes under hypoxia and provided a promising therapeutic target for bone fracture healing.

2. MATERIALS AND METHODS 2.1 Cell culture and hypoxia treatment. Human umbilical cord samples (n=21) were obtained with permission from parents after healthy neonatal deliveries and informed consent was confirmed by all patients prior to this study. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University and conducted in accordance with the ethical principle of the World Medical Association Declaration of Helsinki, and local legislation. Primary cultures of HucMSCs were established according to previous studies [40, 41]. Briefly, cords were rinsed twice with phosphate-buffered saline (PBS) supplemented with penicillin and streptomycin (pen/strep; Gibco, Carlsbad, CA) followed by removal of cord vessels. They were then rinsed and divided into 10 mm3 6

pieces and attached to the substrate of culture plates and cultured in Dulbecco’s Modified Eagle’s Medium/low glucose (DMEM; Hyclone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco Laboratory, Grand Island, NY) and 1% pen/strep at 37oC with 5% CO2. Media was replaced every three days after plating. The cells were passaged into new flasks for further expansion when they reached 80% confluency. Only HucMSCs from passage 3–5 were used for further experiments. HucMSCs were cultured at 37oC, 5% CO2, 21% O2 or at 1% O2, 94%N2 and 5% CO2 using an oxygen control incubator (Heal Force, Shanghai, China) in exosome-depleted FBS containing (System Biosciences, Mountain View, CA, USA) media for 48 hours. For HucMSC identification, Alizarin Red, Oil Red O and Alcian Blue stains were utilized to identify osteogenic, adipogenic and chondrogenic differentiation respectively. The HucMSCs at passage 3–5 were cultured in OriCellTM osteogenic, adipogenic or chondrogenic differentiation media (Cyagen, Guangzhou, China) respectively. For chondrogenesis, pellet culture was used. For identification of HucMSCs markers, flow cytometry was performed using fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated antibodies (human anti CD34, anti-CD44, anti-CD45, anti-CD73, anti-CD90, anti-CD105, anti-CD133 and anti-CD151, BD Biosciences Pharmingen, San Jose, CA). PE-IgG1 and FITC-IgG1 isotypic immunoglobulins were used as isotype controls. Fluorescence signals were sorted using a flow cytometer (FACSCalibur, BD Biosciences, USA) and the results were analyzed using FlowJo software. The human umbilical vein endothelial cell (HUVEC) line was purchased from American Type Culture Collection (ATCC; Manassas, VA). Cell lines were cultured in DMEM/high glucose media contain 10% FBS and 1% pen/strep. The human fetal osteoblastic 1.19 (hFOB 1.19) cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in medium composed of 1:1 DMEM/Ham’s F-12 supplemented with 15% FBS and 1% pen/strep and 0.3 mg/ml G418 (Sigma). 2.2 Exosome isolation and identification. After HucMSCs reached 80% confluency 7

culture media was replaced with an exosome-depleted FBS for an additional 48 h and cultured under normoxic or hypoxic conditions. The media was then collected and centrifuged at 300g for 10 min, followed by centrifugation at 2,000 × g for 10 min at 4°C. After centrifugation, a 0.22-μm sterile filter (SteritopTM Millipore, Burlington, MA) was used to filter the cell supernatant from the whole cells and cellular debris. The filtered supernatant was then applied to the upper compartment of an Amicon Ultra-15 Centrifuge Filter Unit (Millipore) followed by centrifuging at 4,000 × g until the volume was reduced to nearly 200 μL in the upper compartment. After this, the ultra-filtered supernatant was washed twice with PBS and re-filtered to another 200 μL. To purify the exosomes, the liquid was loaded onto the top of a 30% sucrose/D2O cushion in a sterile Ultra-ClearTM tube (Beckman Coulter, Asphalt, CA, USA) and centrifuged at 100,000 × g for 60 min at 4°C using an optima L-100 XP Ultracentrifuge (Beckman Coulter). The fraction containing HucMSCs-Exos was recovered using an 18-G needle, then diluted in PBS, and centrifuged at 4,000 × g and at 4°C in a centrifugal filter unit until the final volume reached 200 μL. Exosomes were either stored at -80°C or used immediately for downstream experiments. A Nanosight LM10 System (Nanosight Ltd, Navato, CA) was applied to analyze the distribution of vesicle diameters from the Exos and Hypo-Exos. The morphology of the acquired exosomes under normoxia and hypoxia was observed under a transmission electron microscope (TEM; Tecnai 12; Philips, Best, The Netherlands). Western blotting was used to determine specific exosome surface markers such as TSG101, CD9, CD63, and CD81. HucMSCs-Exo protein concentration was determined using a bicinchoninic acid protein assay (BCA; Thermo Fisher Scientific, Waltham, MA). Absorbance was read at 562 nm using a microplate reader (ELx800; Bio-Tek Instruments, Inc., Winooski, VT). 2.3 Exosome uptake by HUVECs. Fluorescent labeling of Exos and Hypo-Exos was performed according to the manufacturer’s instructions. Briefly, 4 mg/mL Dil solution (Molecular Probes, Eugene, OR, USA) was added to PBS containing exosomes and 8

incubated. Excessive dye from labeled exosomes was removed by ultracentrifugation at 100,000 × g for 1 h at 4oC. Exosome pellets were then washed three times by resuspending the pellet in PBS with a final wash and resuspension in PBS. These Dil-labeled exosomes were co-cultured with HUVECs for 24 h, and then the cells were washed with PBS and fixed in 4% paraformaldehyde. The uptake of Dil-labeled Exos and Hypo-Exos by HUVECs was then observed by laser confocal microscopy (Zeiss, Oberkochen, Germany, LSM 710) and the fluorescence intensity of Dil was measured with ZEN lite software (Blue Edition, Zeiss, Oberkochen, Germany) at different time points within the two groups. 2.4 Femoral fracture model and X-ray imaging. The animal experiments in this study conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were conducted according to the guidelines of the Nanjing Medical University (NJMU) Institutional Animal Care and Use Committee (IACUC). The femoral fracture model was performed as previously described[42, 43]. Briefly, mice (10–12 weeks of age) were anesthetized and a 10-mm incision was made. Then a Kirschner’s wire was inserted into the femoral marrow cavity through the patellar tendon. A mid-diaphyseal fracture was created by using bone forceps. The mice were then randomly assigned to three groups: PBS, Exos or Hypo-Exos (n=8/group). Next Exos or Hypo-Exos (200 μg of total protein of exosomes precipitated in 200 μL of PBS) or an equal volume of PBS (200 μL) were injected immediately near the fracture followed by wound closure and suture. After this, the mice were injected with buprenorphine daily post-surgery for three days to control pain levels. We used an X-ray system (MX-20, Faxitron, USA) to evaluate the callus seven days after the fracture. Finally, the femurs were harvested, fixed with 4% paraformaldehyde for 24 h, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 21–28 days, and embedded with paraffin. 2.5 Microcomputed tomography imaging (micro-CT). The Kirschner’s wire was firstly removed, and then the femora were fixed in 4% paraformaldehyde for 24 h. The femora were scanned using a micro-CT system (SkyScan 1172, Bruker, Belgium) 9

at a resolution of 18 µm at the following settings 50 kV and 200 µA. Three-dimensional structures were constructed and the bone morphometric parameters (mineralized CV/TV) analyzed using a CT-Analyser (CTAn, Bruker, Belgium). 2.6 Micro-CT analysis of angiogenesis at the fracture sites. Micro-CT system (SkyScan 1172, Bruker, Belgium) was used to assess vascularity. Vascular networks at the cortical bone junction and around the fractures were examined using micro-CT analyses combined with perfusion of a contrast agent. Briefly, blood vessels were first rinsed with normal saline containing heparin and 4% paraformaldehyde (PFA). Then, using MICROFIL® injection compound (Flow Tech, Inc., Carver, Massachusetts) contrast media, a radio-opaque silicone rubber compound containing lead chromate was perfused via the heart. After perfusion, the fractured femur was removed and scanned using the micro-CT system. The samples were subsequently decalcified for 10 days using a 10% EDTA solution. After complete decalcification, the samples were scanned again to visualize only the vascularization within the callus tissue. Three-D reconstructions were made using NRecon software (Ver. 1.6.9.4, Bruker, Kontich, Belgium). 2.7 Immunofluorescence staining. Sections were incubated with anti-CD31 (Abcam, Cambridge, UK) and anti-Endomucin (Emcn; Santa Cruz, CA, USA) or anti-Ki-67 (Abcam, Cambridge, UK) antibody for double immunofluorescent staining overnight at 4oC, followed by Alexa Fluor 488- and Alexa Flour 594-conjugated goat secondary antibodies (1:200, Jackson ImmunoResearch, USA) for 1 h at room temperature. After triple washing with PBS, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) and fluorescent images were acquired using a fluorescence microscope (AxioVertA1 and ImagerA2). 2.8 MiRNA/siRNA transfection. LV2 vector containing miR-126 inhibitor (miR-126-inhibitor) and a negative control (NC) with empty LV2 were constructed into lentiviral vectors (GenePharma, Shanghai, China). We infected MSCs grown to 40%-50% confluence by using lentiviral vectors at an appropriate multiplicity of 10

infection (MOI=50). Small interfering RNA targeting human SPRED1 (siSPRED1), Ago2 (siAgo2) and HIF-1α (siHIF-1α), with their scrambled control siRNAs (siNC) were purchased from GenePharma (Shanghai, China). Lipofectamine 3000 reagent (Invitrogen) was used for transfection according to the manufacturer’s instructions. 2.9 Real-time RT-PCR. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from cells and exosomes. Complementary DNA (cDNA) was synthesized using a Reverse Transcription System (Toyobo, Osaka, Japan) and real-time PCR was performed with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7900 fast real-time PCR system (Applied Biosystems, Carlsbad, USA). Expression levels were normalized to the internal controls (GAPDH or U6) and the relative expression levels were evaluated using the 2-ΔΔCT method. The specific primers for miR-126, miR-855-5p, miR-146b, miR-223, U6, SPRED1, HIF-1α, Ago2, ALP, COL1A1, OCN, VEGF and GAPDH were purchased from RiboBio Co, Ltd. (Guangzhou, China). The primer sequences are listed in Supplementary Table 1. 2.10 Exosomal miRNA microarray assay. The microRNA arrays for Exos and Hypo-Exos were performed by OE Biotech Company (Shanghai, China). Three samples were processed for each type of exosome. The fragmentation mixtures were hybridized to an Agilent-Human microRNA array 21.0 (8*60K, Design ID:070156). For microarray analysis, the Affymetrix (Santa Clara, CA, USA) miRNA 4.0 platform was employed. The sample labeling, microarray hybridization and washing were performed based on the manufacturer’s instructions (Agilent Technologies Inc., Santa Clara, California, USA). Differentially expressed miRNAs were identified using a fold change cut off value of  1.5 set for both up- and down-regulated genes. 2.11 Cell Counting Kit-8 (CCK8) assay. A CCK8 assay (Dojindo, Kumamoto, Japan) was used for the cell proliferation assay. In brief, HUVECs were seeded in a 96-well plate at a density of 2000 cells/100 μL of medium/well, and co-cultured with PBS, Exos or Hypo-Exos at a concentration of 100 μg/mL. To this, 10 μL of CCK8 solution in fresh culture medium was added every 24 h and incubated for 2 h at 37°C, and the 11

optical density (OD) value at 450 nm wavelength was determined using a microplate reader (ELx800, Bio-Tek, USA). 2.12 5-Ethynyl-2’-deoxyuridine (EDU) assay. Cell proliferation was also measured using the EDU assay kit (RiboBio) according to the manufacturer's instructions. Briefly, cells were seeded into 24-well plates at a density of 2.0 × 104 cells/well and cultured for 24 h before the administration of EDU (50 mM). After this, Apollo and DNA stains were added. Finally, proliferation images were acquired and analyzed by fluorescence microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany). Also, an iClickTM EDU Andy Flour 647 Flow Cytometry Assay Kit (Genecopoeia, Germantown, MD) was used to measure cell proliferation according to the manufacturer’s instructions. The percentage of EDU positive cells was determined using a flow cytometer (FACS-Calibur; BD Biosciences). 2.13 Tube formation assay. To assess the possible angiogenic properties of Exos and Hypo-Exos, HUVECs were seeded at a density of 2.0 × 104 cells/well in a Matrigel-coated 96-well plate (Matrigel; BD Biosciences, San Jose, CA). Briefly, 96-well plates were covered with 50 μL of Matrigel using a pre-cooled tip at 4°C. After gelling, the Matrigel was then coated with 100 μL of a HUVEC suspension treated with PBS or 100 μg/mL of either Exos or Hypo-Exos for an additional 30 min at 37°C. Tube formation capacity was assessed by observing under an optical microscope (Nikon, Tokyo, Japan), the polygonal structures formed 6 h after plating the cells onto the Matrigel. Total tube length was carefully measured by randomly selecting five fields per well with the use of Image J software (National Institutes of Health, Bethesda, MD, USA). 2.14 Migration assay. Transwell assay was used to analyze the effect of Exos and Hypo-Exos on HUVEC migration ability. Briefly, 2 × 104 of HUVEC cells were seeded into the upper chamber of a 24-well transwell plate (Corning, NY, USA; pore size: 8 µm) after which 600 µL/well medium treated differently were added to the lower chamber. After co-incubation for 24 h, cells from the upper surface of the filter membrane were wiped away with a cotton swab. Cells that migrated to the lower 12

surface of the filter membrane were stained with 0.5% Crystal Violet for 1 min. Migratory activity was assessed by observing the stained HUVEC cells under an optical microscope (Nikon, Tokyo, Japan). A scratch wound assay was also performed to assess cell migration capability. In brief, HUVECs were seeded into 6-well plates at a density of 2 × 105 cells/well and then grown to 100% confluency. After this, the confluent cell layer was scratched using a sterile 200 µL pipette tip. After carefully washing the cells three times with PBS, PBS or 100 µg/mL Exos or Hypo-Exos were added and images were recorded at 0, and 12 h after scratching. 2.15 Luciferase reporter assay. The 3'-UTR sequence of the mutated SPRED1 sequence and the predicted target site were inserted into the pmir-GLO-promoter vector (Promega, Madison, WI, USA). They were termed pmirGLO-SPRED1 WT and pmirGLO-SPRED1-MUT respectively. HEK293T cells transfected with miR-NC or miR-126KD were seeded into 96-well plates and co-transfected with 100 ng of pmirGLO-SPRED1-WT or pmirGLO-SPRED1-MUT. In the next experiment, WT or mutated HIF-1α-binding site reporter was obtained by Obio Technology (Shanghai, China) and was transfected into HEK293T cells. Luciferase activity in the transfected cells was measured with a dual luciferase reporter assay system (Promega, Madison, Wisconsin, USA). The relative expression of firefly luciferase activity was normalized to Renilla luciferase activity. 2.16 Chromatin immunoprecipitation (CHIP) assay. Briefly, cells were cross-linked with 1% formaldehyde and rinsed twice with PBS and collected after a 5 minute centrifugation at 800 × g and at 4°C. Then cells were incubated in lysis buffer (150 nM NaCl, 25 nM Tris pH 7.5, 1% Trition X-100, 0.1% SDS, 0.5% deoxycholate). The chromatin was sheared into 400bp segments using an Ultrasonic Cell Disruptor (Covaris, Waltham, MA, USA). HIF-1α was immunoprecipitated from the supernatant using an anti HIF-1α or anti-IgG antibody (Abcam). Precipitated DNA fragments were detected by PCR and enrichments were presented as percentage of the input (Forward:

5’-TCGTTCGTGCAGCGTGTGTAC-3’ 13

and

Reverse:

5’-GGGCAAGAGCAAGGATTAGG-3’). 2.17 Western blot analysis. Proteins were extracted from cells and treated with radio-immunoprecipitation assay (RIPA) lysis and extraction buffer (KeyGen Biotechnology, Nanjing, China). Protein concentration was determined using the BCA method. Equal amounts of protein were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore Corp., Burlington, MA), and incubated overnight at 4°C with primary antibodies (Abs) followed by blocking with bovine serum albumin (BSA, 5%, v/v). Membranes were then incubated for 120 min at room temperature with the secondary antibodies. Reacting bands were visualized using ECL reagent (Thermo Fisher Scientific), and the density of protein bands was semi-quantified using ImageJ. The primary antibodies used in the experiments

were

anti-CD9,

anti-CD81,

anti-TSG101

(1:500;

Santa

Cruz

Biotechnology, Santa Cruz, CA, USA); anti-Calnexin, anti-SPRED1, anti-PLK2, anti-IRS1, anti-HIF-1α, anti-Raf, anti-GAPDH (1:1000; Abcam, USA) and anti-Ras, anti-p-Raf, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2, anti-p-ERK1/2 (1:1000; Cell Signaling Technology, USA). 2.18 Statistical Analysis. All data are presented as mean ± SD at least three independent experiments. GraphPad software 7.0 and SPSS 19.0 were used for statistical analysis. We used the Student’s t-test for comparisons between two groups and one-way or two-way ANOVA with Bonferroni post hoc test for multiple comparisons. A value of P < 0.05 was considered statistically significant. * represented a P-value < 0.05, ** represented a P-value < 0.01, *** represented a P-value < 0.001.

3. RESULTS 3.1 Identification of HucMSCs. HucMSCs were isolated from freshly isolated human umbilical cords as described above. At passage 3, HucMSCs were identified by morphology and flow cytometry. Cells adopted a spindle-like shape after reaching 80%-90% confluent (Figure S1A). Alizarin Red staining, Oli Red O staining and 14

Alcian Blue staining were applied to identify the osteogenic, adipogenic and chondrogenic differentiation of HucMSCs respectively (Figure S1B). Flow cytometry analysis was applied to confirm that HucMSCs were positive for CD44, CD73, CD90, CD105 and CD151 but negative for CD34, CD45 or CD133 (Figure S1C).

3.2 Hypoxia promotes exosome release from HucMSCs. The content and function of exosomes is dependent upon the cell of origin, suggesting that intercellular communication through exosomes is a dynamic system, and can be adapted depending upon the conditions of the producing cell. Changes in oxygen concentration affect many of the distinctive characteristics of stem and progenitor cells and can deliver biological information by internalization in neighboring or distant cells. On this basis, we determined whether the hypoxic condition of HucMSCs could influence the exosomes they release. HucMSCs were seeded under normoxia and hypoxia (1% O2) respectively and exosomes were isolated from serum-free media after 48 h incubation, and were then analyzed using an electron microscope, nanoparticle tracking analysis (NTA) and western blot. As shown in Figure 1A and 1B, TEM revealed typically rounded nanoparticles ranging in size from 50 to 150 nm in diameter and NTA exhibited a similar size distribution (average 112.3nm VS 114.8nm) in both the normoxia and hypoxia groups. No morphological difference between the two groups was observed with regard to their size, shape, or electron density. Western blot revealed the presence of exosome surface markers including TSG101, CD9, CD63, and CD81 with an absence of Calnexin. Increased protein levels of TSG101, CD9, CD63, and CD81 were observed in exosomes after exposure to 1% O2 for 48 hours (Figure 1C and 1D). Moreover, the protein concentration of exosomes derived from hypoxic HucMSCs was significantly higher when compared to those from the normoxic controls (Figure 1E, P=0.012). Hypoxic conditioning also induced a significantly increased release of exosomes when compared to the normoxic control.

15

3.3 The differential uptake of exosomes by HUVECs is dependent upon oxygen status. To examine whether exosomes derived from normoxia or hypoxic conditions were taken up differentially by HUVECs, a Dil dye was used to label the exosomes and then co-cultured with target HUVECs for 24 h. Fluorescence microscopy was used to monitor the rate of exosomes uptake by HUVECs in real time. As shown in Figure 1F, the number of exosomes taken up by HUVECs was significantly higher in the hypoxia group when compared to the normoxic control group. Figure 1G demonstrates a clear statistically significant difference between the two groups after 12 h (P=0.000), suggesting that exosomes derived under hypoxic conditions are taken up more easily by endothelial cells.

3.4 Bone fracture healing is promoted in mice after transplantation with Hypo-Exos. Initially, we used H&E staining and X-ray analysis to evaluate bone formation during femoral fracture healing after exposure to PBS, Exo or Hypo-Exo groups (n=8 per group). As shown in Figure 2A and Figure S2A, a hard callus with bridging of the fracture gap was observed and the fracture gap was obvious on postoperative day 7 in all three groups. However, larger callus volumes were observed in the Exos group when compared to the PBS group in agreement to a previous report [44]. Moreover, a significant increase in the Hypo-Exos group was seen when compared to the Exos groups in callus volumes (Figure 2A and Figure S2A). Next, High resolution micro-CT scanning was performed and reconstructed to qualitatively evaluate callus volumes on post-operative day 7 (Figure 2B). Similar to the results obtained with the H&E histology and X-ray examination, transplantation of Hypo-Exos caused a significant increase in callus volume/tissue volume (CV/TV) (Figure 2C, P=0.000). As demonstrated above, we have found that Hypo-Exos could enhance bone healing. As known, both osteogenesis and angiogenesis are beneficial to bone fracture healing. Here we wanted to determine the relative importance of both after Hypo-Exos administration. We added equal quantities of PBS, Exos or Hypo-Exos to 16

osteoblasts or HUVECs and osteogenesis-related genes (ALP, COLA1 and OCN) in osteoblasts and an angiogenesis-related gene (VEGF) in HUVECs were examined using real-time PCR. Results indicated that the mRNA expression levels of ALP, COLA1 and OCN did not show any difference between the three groups. However, the expression of VEGF exhibited a remarkable increase in expression after Hypo-Exos treatment (Figure S3, P=0.000). These results indicated that Hypo-Exos administration may contribute to bone healing primarily through angiogenesis rather than osteogenesis. Therefore, we performed quantitative vascular microCT analyses to evaluate neovascularization at day 7 in mice. Representative reconstructions indicated increased callus vascularity in the Hypo-Exos group when compared to the Exo group, with the mice in the Hypo-Exos group displaying a marked increase in vessel volume and number (Figure 2D and 2E, P=0.000). Recent studies have identified a new specific vascular endothelium subtype known as type H characterized by high expression of endothelial markers CD31 and endomucin (EMCN) during angiogenesis[45, 46]. Therefore, immunofluorescence was applied to observe this vascular subtype surrounding the femoral fracture callus using double staining with CD31 and Emcn. As shown in Figure 2F and 2G, newly formed vessels were more abundant in the Hypo-Exos group, when compared to the PBS group or Exos group (P=0.000). To determine whether administration of Hypo-Exos could influence cell proliferation in vivo, we used the proliferation marker Ki67 and the endothelial cells marker CD31 to double stain the slices within the three groups. As shown in Figure 2H and 2I, increased cell proliferation in endothelial cells was exhibited in the Hypo-Exos group when compared to the other groups (P=0.000). Taken together, these studies suggested that transplantation of Hypo-Exos promoted angiogenesis and enhanced bone healing in vivo.

3.5 Hypo-Exos promote proliferation, migration, and tube formation in HUVECs. To determine whether Hypo-Exos exert similar therapeutic effects to those seen in vivo, we cultured in vitro HUVECs with PBS, Exos or Hypo-Exos at a concentration 17

of 100 µg/mL. Proliferation of HUVECs was examined using CCK8 (Figure 3A) and EDU assays (Figure 3B-3D). As shown in Figure 3B and 3C, the results of the EDU assay demonstrated that the number of EDU-stained HUVECs was higher in both the Exos and Hypo-Exos groups when compared to the PBS control group (P=0.003). However, Hypo-Exos treatment caused an increased number of EDU-stained cells. Similar results were also found using the CCK8 assay (Figure 3A, P=0.000). These results demonstrated that incubation with Hypo-Exos resulted in a remarkable increase in proliferation of HUVECs. Tube lengths were counted after HUVECs began to form capillary tubes. As shown in Figure 3E and 3F, both Exos and Hypo-Exos could significantly enhanced tube formation at 6 h when compared to the PBS group (P=0.000). Furthermore, incubation with Hypo-Exos exhibited an enhanced tube formation capability when compared to the Exos group. Using transwell (Figure 3G and 3H) and Scratch assays (Figure 3I and 3J), we determined the effect of exosomes on HUVEC migration. These results demonstrated that both Exos and Hypo-Exos contributed to the migration of HUVECs when compared to the PBS group. However, Hypo-Exos treatment could greatly enhance the migration capability of HUVECs when compared to Exos alone (P=0.000). Collectively, these findings revealed that Hypo-Exos could promote a greater increase in proliferation, migration and tube formation when compared to both PBS and Exos treatment in vitro.

3.6 The miR-126 is upregulated in Hypo-Exos and transferred to HUVECs by exosomes. Both in vitro and in vivo analyses revealed that Hypo-Exos enhanced angiogenesis when compared to Exos. A number of previous studies have indicated that miRNAs are one of the main functional components of exosomes and may play a crucial role in cell communication and eventually regulate biological function. Argonaut-2 (Ago2) protein, a component of the RNA-induced silencing complex (RISC), is the key regulator of miRNA function by mediating the activity of miRNA-guided mRNA cleavage or by translational inhibition [47]. Previous studies 18

have demonstrated that exosomes contain Ago2 protein [48, 49]. Therefore, we transfected HucMSCs with siRNA-Ago2 and then exposed them to hypoxia and then collected the exosomes from the supernatant. The transfection efficiency was confirmed using real-time PCR (Figure 4A, P=0.000). As shown in Figure 4B, using the CCK8 assay, we found that exosomes derived from HucMSCs transfected with siRNA-Ago2 under hypoxia exhibited a lower cell proliferation when compared to those transfected with siRNA-NC (P=0.000). This result suggested that miRNAs from Hypo-Exos, may exert a biological functional and eventually contribute to bone fracture healing. Based on the above results, we then went on to isolated RNA from Exos and Hypo-Exos and performed microarray profiling of the miRNAs derived from the exosomes and compared them between the two groups. The miRNA microarray analysis (Figure 4C) showed that there were 94 miRNAs upregulated and 39 downregulated in the Hypo-Exos group when compared to the Exos group (≥1.5-fold, P < 0.05). Based on this miRNA profiling data, we went on to select the top five up-regulated miRNAs including: miR-126, miR-855-5p, miR-146b, miR-223 and miR-451, and validated their expression further using real-time PCR. As shown in Figure 4D, four miRNAs including miR-126, miR-855-5p, miR-146b, miR-223 from the five selected were significantly upregulated in Hypo-Exos when compared to Exos (P=0.000). Among them, miR-126 has been shown to have a positive effect on angiogenesis. Based on our microarray results and the previous findings, we concentrated on miR-126 and determined whether Hypo-Exos promoted angiogenesis by the transfer of miR-126. Therefore, in order to gain a mechanistic insight into the role of exosomally derived miR-126 in Hypo-Exos-induced angiogenesis in our bone fracture

model,

we

constructed

miR-126-knockdown

HucMSCs

using

a

lentiviral-based method and negative control (miR-NC). The transfection efficiency was confirmed using real-time PCR and is shown in Figure 4E (P=0.001). Next, exosomes were isolated from miR-NC-Hypo-MSC and miR-126KD-Hypo-MSC, respectively. As shown in Figure 4F, miR-126KD-Hypo-Exos caused a decrease in the 19

expression of miR-126 when compared to the miR-NC-Hypo-Exos (P=0.000). Furthermore, the miR-126 expression level in the target HUVECs in the miR-126KD-Hypo-Exos treatment group exhibited a dramatic decrease in expression when compared to the miR-NC-Hypo-Exos treatment group (Figure 4G, P=0.000). In addition, Cy3 labelled immunofluorescence was used to observe exosomal miR-126 (Figure 4H). Similar to our results seen using real-time PCR, the immunofluorescence data demonstrated that after treatment with miR-126KD-Hypo-Exos, miR-126 immunofluorescence

intensity

was

significantly

lower

than

that

of

miR-NC-Hypo-Exos. These data indicated that hypoxic MSCs-derived exosomal miR-126 can be transferred to HUVECs.

3.7 Knockdown of miR-126 inhibits Hypo-Exos-mediated proliferation, angiogenesis and migration in vitro and in vivo. Because we have demonstrated that hypoxic MSCs-derived miR-126 could be transferred to HUVECs, we determined whether miR-126 represented a biological messenger between hypoxic MSCs and HUVECs, and can regulate angiogenesis. Gain- and loss-of function analyses were utilized and two groups including HUVECs treated with miR-NC-Hypo-Exos and miR-126KD-Hypo-Exos

were

used.

Using

our

proliferation

assay,

miR-126KD-Hypo-Exos administration significantly attenuated HUVECs proliferation as evaluated by CCK8 (Figure 5A, P=0.000) and EDU assays (Figure 5B-5D, P=0.003) when compared to miR-NC-Hypo-Exos administration. Tube formation was then evaluated between the two groups. The results shown in Figure 5E and 5F demonstrate that treatment of HUVECs with miR-126KD-Hypo-Exos impaired their ability for vascular formation, when counting the total tube length (P=0.003). Using the migration assays, miR-126KD-Hypo-Exos administration could suppress the migration of HUVECs in both transwell (Figure 5G and H, P=0.002) and scratch assays (Figure 5I and J, P=0.000). These results indicated that knockdown of exosomal miR-126 could inhibit the ability for proliferation, angiogenesis and migration of HUVECs in vitro. 20

Next, High resolution micro-CT scanning was performed and showed that administration of miR-126KD-Hypo-Exos could cause a decrease in CV/TV (Figure 5K and 5L, P=0.002). Also, H&E staining confirmed this finding. (Figure S2B). Furthermore, immunofluorescence was applied to determine whether miR-126 knockdown could exhibit similar effects in vivo. By double staining with CD31 and Emcn, we found that miR-126KD-Hypo-Exos administration in vivo could significantly reduce the newly formed blood vessels when compared to the miR-NC-Hypo-Exos group (Figure 5M and 5N, P=0.000). Using Ki-67/CD31 double staining,

miR-126KD-Hypo-Exos

administration

could

significantly

suppress

endothelial cell proliferation in vivo (Figure 5O and 5P, P=0.02). Taken together, these results demonstrate an important function for miR-126 in Hypo-Exos-mediated proliferation, angiogenesis and migration.

3.8 Exosomal miR-126 regulates SPRED1 by directly targeting the 3'-UTR. To further investigate the potential mechanism of action of exosomal miR-126 in the Hypo-Exos promotion of endothelial cell proliferation, angiogenesis and migration, three online databases including TargetScan, PicTar and DIANA were independently used to search the predicted mRNA targets for miR-126. As shown in Figure 6A, SPRED1, PLK2 and IRS1 were predicated by all three databases. We then performed western blot to examine the expression levels of these three target genes after administration of miR-NC-Hypo-Exos and miR-126KD-Hypo-Exos in HUVECs. Figures 6B and 6C demonstrate that miR-126KD-Hypo-Exos treatment could significantly increase the protein expression level of SPRED1 (P=0.000). However, the other two predicted genes remained unchanged. Moreover, SPRED1 has been demonstrated to show a negative role in endothelial cell proliferation, angiogenesis and migration.[50] To verify that the SPRED1 3'UTR is a direct target for miR-126, we analyzed transfected HUVECs by luciferase reporter assay (Figure 6D). The relative luciferase activity was increased when downregulated miR-126 was co-transfected with SPRED1 WT luciferase construct, but not with the MUT (Figure 21

6E, P=0.002). Given the known functions of SPRED1 as a negative regulator of endothelial cell proliferation, angiogenesis and migration, along with our western blot and luciferase report assay results, we confirmed that SPRED1 was a target gene for miR-126.

3.9 Exosomal miR-126 promotes HUVECs proliferation, angiogenesis and migration by targeting SPRED1. To further explore the relationship between exosomal miR-126 and SPRED1, a series of in vitro rescue experiments were conducted. We transfected siSPRED1 and siNC into HUVECs and detected expression levels by real-time PCR (Figure 7A, P=0.000). In addition, the expressional level of SPRED1 was also detected in HUVECs after administration of miR-126KD-Hypo-Exos. Results demonstrated that HUVECs transfected with siSPRED1 showed a significantly lower expression of SPRED1 when compared to siNC after administration of miR-126KD-Hypo-Exos (Figure 7B, P=0.000). In the cell proliferation assays (CCK8 and EDU), we found that silencing SPRED1 could promote cell proliferation during co-treatment with miR-126KD-Hypo-Exos (Figure 7C, P=0.000; Figure 7D-E, P=0.004). Furthermore, tube formation (Figure 7F, P=0.006), transwell (Figure 7G, P=0.000) and scratch assays (Figure 7H, P=0.000) showed that inhibition of SPRED1 could remove the negative effects of miR-126KD-Hypo-Exos on the angiogenesis and migration of HUVECs. As a result of a series of rescue experiments, we have demonstrated that siSPRED1 in HUVECs can abolish the inhibitory role of miR-126KD-Hypo-Exos on cell proliferation, angiogenesis and migration. Therefore, we concluded that exosomal miR-126 promoted HUVEC proliferation, angiogenesis and migration by targeting SPRED1.

3.10 HIF-1α induces the enrichment of miR-126 in exosomes shed from hypoxic HucMSCs. Hypoxia inducible factor-1 is a key transcription factor mediating adaptive responses to hypoxia. Previous studies have demonstrated that HIF-1α is essential for the expression of miRNAs including miR-23a, miR-135b and miR-210 22

during hypoxia [51-53]. Therefore, we determined whether HIF-1α is necessary for the regulation of miR-126 in exosomes shed from hypoxic HucMSCs. Firstly, we demonstrated that the expression level of HIF-1α under hypoxia is much higher than that under normoxia (Figure 8A). We then performed siRNA transfection assays to determine whether miR-126 is regulated by HIF-1α in HucMSCs under hypoxia. As shown in Figure 8A, the efficiency of siRNA targeting of HIF-1α was confirmed by the detection of decreased HIF-1α protein levels using western blots (P=0.000). We found that downregulation of HIF-1α markedly reduced hypoxia-induced miR-126 expression in HucMSCs (Figure 8B, P=0.000). Similarly, the expression levels of miR-126 in exosomes were markedly lowered when hypoxic HucMSCs were transfected with HIF-1α siRNA (Figure 8C, P=0.000). By employing the JASPAR database

(http://jaspar.genereg.net/)

and

the

UCSC

genome

browser

tool

(http://genome.ucsc.edu/index.html), we speculated that HIF-1α may bind to the miR-126 promoter and activate its expression (Figure 8D). Therefore, we constructed the HIF-1α binding site reporter and transfected this into HEK293 cells and found that the HIF-1α binding site WT reporter had lower luciferase activity when compared to the

mutant

reporter

(Figure

8E,

P=0.002).

Correspondingly,

chromatin

immunoprecipitation (ChIP) assay also showed the binding of HIF-1α was enriched at the miR-126 promoter in HucMSCs after hypoxia stimulation, demonstrating a direct interaction between HIF-1α and miR-126 in response to hypoxia (Figure 8F, P=0.000). Therefore, HIF-1α induced the expression of miR-126 and its loading into exosomes.

3.11 Exosomal miR-126 promotes proliferation, angiogenesis and migration in HUVECs via the SPRED1/Ras/Erk pathway. It is known that SPRED1 is an intracellular inhibitor of the Ras/extracellular signal-regulated kinase (Erk) cascade and is involved in the regulation of several cellular processes including differentiation, survival, motility and the cell cycle [50]. For this reason, we assumed that Hypo-Exos exerted their inhibitory effects on proliferation, angiogenesis and migration via the SPRED1/Ras/Erk pathway in HUVECs. We performed western blots to compare the 23

expression of SPRED1 and major Ras/Erk pathway members. As expected, when treated with Hypo-Exos, the expression level of SPRED1 was significantly lower. Consequently, the downstream effectors of Ras including p-Raf, p-MEK1/2 and p-ERK1/2

were

greatly

increased.

In

contrast,

when

treated

with

miR-126KD-Hypo-Exos, SPRED1 expression levels were significantly higher than when treated with miR-NC-Hypo-Exos, and the downstream Ras/Erk pathway was suppressed (Figure 9A and 9B). At the same time, no significant changes were observed in the expression levels of Ras, Raf, MEK1/2 or ERK1/2 in the five groups. Taken together, our results indicated that Hypo-Exos suppressed SPRED1 by directly targeting its 3'-UTR, and thereby activating the Ras/Erk pathway.

4. DISCUSSION With the development of regenerative medicine, stem cell transplantation has been considered as a viable option to treat various refractory clinical diseases. These cells have great potential as a therapy due to their ability to self-replicate, differentiate, and regulate hematopoietic and immune cells [12, 54, 55]. However, it has been reported that the majority of transplanted MSCs are trapped by the lung or liver after intravenous administration and as a result, only 1% reach the target tissue [56]. Furthermore, in age related disorders, the biological function of MSCs is remarkably reduced [57]. As a result, several challenges remain to be overcome before these cells can be used for clinical applications Previous studies have indicated that the therapeutic effects displayed by transplanted MSCs may be due to their paracrine mechanisms [17, 20, 21]. Mesenchymal stem cell derived exosomes have been shown to have therapeutic effects in several diseases (osteonecrosis, liver/renal failure, traumatic brain/spinal cord injury, myocardial infraction, ischemic diseases, and chronic cutaneous wounds) when compared to the administration of MSCs directly [27, 58-60]. As well as a potential therapy exosomes are potentially also able to overcome the limitations seen with direct MCS transplantation. As known, the oxygen concentration in vitro and in 24

vivo under physiological conditions are very different to those seen in culture (21% O2 under normoxia). These conditions do not mimic real microenvironments in vivo. Moreover, some studies have shown that MSCs under hypoxia were able to enhance their biological function and increase their therapeutic effects [30, 35]. Because the hypoxic microenvironment is a prominent feature of various inflammatory and diseased tissues, these interactions must be evaluated after both normoxic and hypoxic cell conditioning. Taken together, we established a stable bone fracture model in mice and hypothesized that: (1) exosomes derived from MSCs could overcome the restrictions seen with direct MSC administration; (2) whether exosomes derived from MSCs under hypoxic conditions could exert a greater therapeutic effect when compared to those under normoxia; (3) the underlying mechanism of action of Hypo-Exos in the promotion of bone fracture healing. Among the existing sources of MSCs, the human umbilical cord represents an economical, productive, feasible, and universal source and studies have reported the application of exosomes derived from HucMSCs in various of diseases. For example, recent studies have shown that exosomes derived from HucMSCs could protect against

cisplatin

induced

renal

oxidative

stress,

alleviate

rat

hepatic

ischemia-reperfusion injury, prevent scar formation during wound healing and promote angiogenesis in a rat myocardial infarction model [25, 26, 61, 62]; Additionally, we found a recent study by Zhang et al demonstrating a role for HucMSCs derived exosomes in the promotion of angiogenesis in a bone fracture model [44]. However, the authors could not confirm the underlying mechanism of this process. Furthermore, MSCs after bone fracture are under extreme hypoxia and it is not possible to recreate such conditions in an in vitro setting. In this study, we performed a series of experiments in vivo and in vitro in order to verify our hypothesis. The results of TEM and NTA exhibited no morphological differences between the Exos and Hypo-Exos groups with regard to their size, shape, or electron density. However, further studies revealed that hypoxic conditions could promote exosome release from HucMSCs and that Hypo-Exos can be more easily taken up by HUVECs. 25

In our preliminary in vitro and in vivo studies, we demonstrated that transplantation of HucMSC-Exos could promote cell proliferation, angiogenesis and migration and these therapeutic effects were increased in Hypo-Exos. We also tried to determine the underlying mechanism that contributed to the differences between the two groups. In our next series of experiments, we analyzed miRNA content, biological effects, and pro-angiogenesis properties of Hypo-Exos in vitro and in a mouse model of bone fracture. Our results showed that (1) administration of Hypo-Exo after bone fracture increased angiogenesis and enhanced bone fracture healing; (2) hypoxic MSC-derived Exos were enriched with miR-126; (3) Hypo-Exo-derived miR-126 caused profound pro-angiogenesis, pro-proliferative and pro-migratory effects via suppressing the expression of SPRED1 and hence activation of the Ras/Erk pathway. (4) HIF-1α is the key transcription factor in regulating the expression of miR-126 and its pro-angiogenesis ability. It is recognized that HIF-1α is a pivotal transcription factor that mediates hypoxic adaptive responses [63]. As a transcriptional factor, HIF-1α recognizes and binds to hypoxia-responsive elements (HREs) to activate the transcriptional activity of target genes. Under normoxia, HIF-1α is hydroxylated by prolyl hydroxylase to undergo proteasomal degradation, whereas hypoxia inhibits prolyl hydroxylase activity, thereby enabling HIF-1α to remain intact [64]. Accumulated HIF-1α can then translocate to the nucleus where it transactivates target genes. Previous studies have demonstrated that HIF-1α is essential for the expression of miRNAs including miR-23a, miR-135b and miR-210 under hypoxic conditions [51-53]. Analysis of the miR-126 promoter using the JASPAR core database revealed a putative HREs in the promoter sequence of miR-126, suggesting that HIF-1α might modulate miR-126 expression through this promoter sequence. In our next set of experiments, we knock downed HIF-1α in HucMSCs and found a remarkable decrease in the expression of miR-126 as well as the expression of miR-126 in encapsulated released exosomes. Further studies including dual luciferase reporter and ChIP assays confirmed a direct interaction between HIF-1α and miR-126. 26

These results indicated that under hypoxic conditions, increased HIF-1α was translocated to nucleus and bound to HRE in the miR-126 promoter region and was essential for the activation and expression of miR-126. Numerous studies have reported that exosomes derived from MSCs exert their biological functions on target cells by the delivery of specific miRNAs [37, 39]. A recent study showed that BMSCs-derived exosomes could modulate age-related insulin resistance via the transfer of functional exosomal miR-29b-3p and thereby inhibiting the expression of the target gene SIRT1 [65]. A separate study demonstrated that exosomes derived from human synovial MSCs could enhance tissue regeneration by directly delivering miR-140-5p. Furthermore, overexpressing miR-140-5p in MSCs-Exos could promote tissue regeneration and as a result prevent osteoarthritis when compared to non-modified MSC-Exos [66]. It has also been found that miR-193a-3p, miR-210-3p and miR-5100 could be transferred to lung cancer cells through BMSCs derived exosomes and could promote invasion by activating STAT3 signaling-induced epithelial-mesenchymal transition [67]. However, an unbiased analysis of the miRNA profile of mouse Hypo-Exos and a mechanistic study for the miRNA-mediated effects of promoting bone fracture healing have not been reported. Our miR-array experiments demonstrated that miR-126 was highly expressed in Hypo-Exos when compared to Exos, and that exosomal miR-126 could be transferred efficiently to the target endothelial cells after treatment with Hypo-Exos. Micro RNA-126 is known to be highly enriched in endothelial cells and plays an important role in angiogenesis [68, 69]. Previous studies have demonstrated that miR-126 could enhance maturation and stabilization of growing blood vessels by suppressing the p21-activated kinase 1 gene and regulating angiopoietin-1 signaling [70, 71]. Furthermore, miR-126 was found to play important roles in promoting angiogenesis during embryonic development by targeting PIK3R2 which is an inhibitor of angiogenic and cell survival signals in response to VEGF [72]. It has also been reported that downregulation of miR-126 could lead to delayed angiogenic sprouting, collapsed blood vessels, widespread hemorrhaging and partial embryonic lethality in 27

mice. They were also shown to exhibit a significantly reduced ability for angiogenesis after ischemia of the hind limb due to miR-126 [73]. Through a series of in vitro and in vivo experiments, we showed that knockdown of miR-126 in Hypo-Exos could abolished the favorable effects of Hypo-Exos in the process of bone fracture healing. Taken together, it is not surprising to conclude that Hypo-Exos enriched with miR-126 are pro-angiogenic, proliferative and cause migration all of which are essential for regeneration of new blood vessels after bone fracture. To better understand the underlying mechanism of exosomal miR-126, we next employed bioinformatic tools to identify the potential target gene of miR-126. We found that there are three consistent genes including SPRED1, PLK2 and IRS1 in the intersection of different databases. To verify the actual target gene in these three predicted genes, we evaluated the expression levels of the genes respectively in the miR-NC-Hypo-Exo and miR-126KD-Hypo-Exo groups. The results revealed that only the expression of SPRED1 was increased in the miR-126KD-Hypo-Exo group. As a result, we finally chose SPRED1 for further study among the candidate target genes. It has been previously reported that SPRED1 is an anti-angiogenesis gene which can significantly inhibit cell motility and Rho-mediated actin reorganization both necessary for migration and proliferation [74, 75]. In our study, we demonstrated that miR-126 was enriched in Hypo-Exos and could be transferred into HUVECs by exosomes, thereby suppressing the expression of SPRED1 and promoting the proliferation and migration of HUVECs. Through a series of rescue experiments, we showed that knockdown of SPRED1 in HUVECs could reverse the anti-angiogenesis effects caused by suppressing the expression of miR-126 in Hypo-Exos. Taken together these results showed that Hypo-Exos can act as biological vectors for the delivery of biologically functional miR-126 into recipient endothelial cells. Additionally, because there were a total of 94 miRNAs over expressed in Hypo-Exos and only the top five were selected for further study, we cannot rule out possible contributions from other miRNAs which may act alone or in combination in Hypo-Exos to produce therapeutic effects in the process of bone fracture healing. 28

Meanwhile, bone fracture healing is a complex process and we only investigated angiogenesis in this study. Thus, the precise mechanism of action of exosomes derived from hypoxic conditions in the promotion of bone fracture healing will be explored in our future studies. In this study, we were able to demonstrate that Hypo-Exos promote angiogenesis after bone fracture in mice and angiogenesis, migration and proliferation in vitro by transferring functional miR-126 to target HUVECs with subsequent down-regulation of SPRED1. These findings emphasize the fact that Hypo-Exos can stimulate endothelial repair by functionally influencing endothelial target cells.

5. CONCLUSIONS In conclusion, our study highlighted a mechanism by which cell-free Hypo-Exos promote bone fracture healing via exosomal miR-126 (Figure 9C). Taken together our results supported a mechanism whereby hypoxia mediates enhanced production of miR-126, possibly through the actions of HIF-1α. The enriched levels of exosomal miR-126 markedly improved therapeutic potential. Hypoxia preconditioning represents an effective and promising approach to optimize the therapeutic actions of MSC-derived exosomes for bone fracture healing. Hypo-Exos exert their pro-angiogenesis effect by transferring miR-126 to endothelial cells in a SPRED1/Ras/Erk pathway. DISCLOSURE The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was sponsored by the National Natural Science Foundation of China (Grant No.81974335), the Natural Science Foundation of Jiangsu Province (Grant No. BK20181490), the Six Talent Peaks Project in Jiangsu Province (Grant No. TD-SWYY-010)

and

the

Wu

Jieping

No.320-2745-16-117). 29

Medical

Foundation

(Grant

REFERENCES [1] Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nature reviews Rheumatology 2015;11:45-54. [2] Einhorn TA. Enhancement of fracture-healing. The Journal of bone and joint surgery American volume 1995;77:940-56. [3] Antonova E, Le TK, Burge R, Mershon J. Tibia shaft fractures: costly burden of nonunions. BMC musculoskeletal disorders 2013;14:42. [4] Tzioupis C, Giannoudis PV. Prevalence of long-bone non-unions. Injury 2007;38 Suppl 2:S3-9. [5] Ai-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. Journal of dental research 2008;87:107-18. [6] Seeman E. Bone modeling and remodeling. Critical reviews in eukaryotic gene expression 2009;19:219-33. [7] Teti A. Bone development: overview of bone cells and signaling. Current osteoporosis reports 2011;9:264-73. [8] Eriksen EF. Cellular mechanisms of bone remodeling. Reviews in endocrine & metabolic disorders 2010;11:219-27. [9] Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298-307. [10] Santolini E, West R, Giannoudis PV. Risk factors for long bone fracture non-union: a

30

stratification approach based on the level of the existing scientific evidence. Injury 2015;46 Suppl 8:S8-S19. [11] Gomez-Barrena E, Rosset P, Lozano D, Stanovici J, Ermthaller C, Gerbhard F. Bone fracture healing: cell therapy in delayed unions and nonunions. Bone 2015;70:93-101. [12] Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M. Repair of large bone defects with the use of autologous bone marrow stromal cells. The New England journal of medicine 2001;344:385-6. [13] Yanai T, Ishii T, Chang F, Ochiai N. Repair of large full-thickness articular cartilage defects in the rabbit: the effects of joint distraction and autologous bone-marrow-derived mesenchymal cell transplantation. The Journal of bone and joint surgery British volume 2005;87:721-9. [14] Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668-73. [15] Jeong JO, Han JW, Kim JM, Cho HJ, Park C, Lee N, Kim DW, Yoon YS. Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental

myocardial

infarction

and

diabetic

neuropathy.

Circulation

research

2011;108:1340-7. [16] Hass R, Kasper C, Bohm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell communication and signaling : CCS 2011;9:12. [17] Katsuda T, Kosaka N, Takeshita F, Ochiya T. The therapeutic potential of mesenchymal 31

stem cell-derived extracellular vesicles. Proteomics 2013;13:1637-53. [18] Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of cell biology 2013;200:373-83. [19] Simons M, Raposo G. Exosomes--vesicular carriers for intercellular communication. Current opinion in cell biology 2009;21:575-81. [20] Breakefield XO, Frederickson RM, Simpson RJ. Gesicles: Microvesicle "cookies" for transient information transfer between cells. Molecular therapy : the journal of the American Society of Gene Therapy 2011;19:1574-6. [21] Mathieu M, Martin-Jaular L, Lavieu G, Thery C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nature cell biology 2019;21:9-17. [22] Kourembanas S. Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annual review of physiology 2015;77:13-27. [23] Pitt JM, Andre F, Amigorena S, Soria JC, Eggermont A, Kroemer G, Zitvogel L. Dendritic cell-derived

exosomes

for

cancer

therapy.

The

Journal

of

clinical

investigation

2016;126:1224-32. [24] Phinney DG, Pittenger MF. Concise Review: MSC-Derived Exosomes for Cell-Free Therapy. Stem cells 2017;35:851-8. [25] Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H. Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. Journal of molecular medicine 2014;92:387-97. [26] Yao J, Zheng J, Cai J, Zeng K, Zhou C, Zhang J, Li S, Li H, Chen L, He L, Chen H, Fu H, 32

Zhang Q, Chen G, Yang Y, Zhang Y. Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate rat hepatic ischemia-reperfusion injury by suppressing oxidative stress and neutrophil inflammatory response. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2019;33:1695-710. [27] Liu W, Wang Y, Gong F, Rong Y, Luo Y, Tang P, Zhou Z, Zhou Z, Xu T, Jiang T, Yang S, Yin G, Chen J, Fan J, Cai W. Exosomes Derived from Bone Mesenchymal Stem Cells Repair Traumatic Spinal Cord Injury by Suppressing the Activation of A1 Neurotoxic Reactive Astrocytes. Journal of neurotrauma 2019;36:469-84. [28] Rong Y, Liu W, Wang J, Fan J, Luo Y, Li L, Kong F, Chen J, Tang P, Cai W. Neural stem cell-derived small extracellular vesicles attenuate apoptosis and neuroinflammation after traumatic spinal cord injury by activating autophagy. Cell death & disease 2019;10:340. [29] Kordelas L, Rebmann V, Ludwig AK, Radtke S, Ruesing J, Doeppner TR, Epple M, Horn PA, Beelen DW, Giebel B. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 2014;28:970-3. [30] Hu X, Wu R, Shehadeh LA, Zhou Q, Jiang C, Huang X, Zhang L, Gao F, Liu X, Yu H, Webster KA, Wang J. Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells. BMC genomics 2014;15:303. [31] Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell stem cell 2010;7:150-61. [32] Anderson JD, Johansson HJ, Graham CS, Vesterlund M, Pham MT, Bramlett CS, Montgomery EN, Mellema MS, Bardini RL, Contreras Z, Hoon M, Bauer G, Fink KD, Fury B, 33

Hendrix KJ, Chedin F, El-Andaloussi S, Hwang B, Mulligan MS, Lehtio J, Nolta JA. Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling. Stem cells 2016;34:601-13. [33] Zhu J, Lu K, Zhang N, Zhao Y, Ma Q, Shen J, Lin Y, Xiang P, Tang Y, Hu X, Chen J, Zhu W, Webster KA, Wang J, Yu H. Myocardial reparative functions of exosomes from mesenchymal stem cells are enhanced by hypoxia treatment of the cells via transferring microRNA-210 in an nSMase2-dependent way. Artificial cells, nanomedicine, and biotechnology 2018;46:1659-70. [34] Fan J, Zhang Z, Chao X, Gu J, Cai W, Zhou W, Yin G, Li Q. Ischemic preconditioning enhances autophagy but suppresses autophagic cell death in rat spinal neurons following ischemia-reperfusion. Brain research 2014;1562:76-86. [35] Hu X, Xu Y, Zhong Z, Wu Y, Zhao J, Wang Y, Cheng H, Kong M, Zhang F, Chen Q, Sun J, Li Q, Jin J, Li Q, Chen L, Wang C, Zhan H, Fan Y, Yang Q, Yu L, Wu R, Liang J, Zhu J, Wang Y, Jin Y, Lin Y, Yang F, Jia L, Zhu W, Chen J, Yu H, Zhang J, Wang J. A Large-Scale Investigation of Hypoxia-Preconditioned Allogeneic Mesenchymal Stem Cells for Myocardial Repair in Nonhuman Primates: Paracrine Activity Without Remuscularization. Circulation research 2016;118:970-83. [36] Hu X, Wu R, Jiang Z, Wang L, Chen P, Zhang L, Yang L, Wu Y, Chen H, Chen H, Xu Y, Zhou Y, Huang X, Webster KA, Yu H, Wang J. Leptin signaling is required for augmented therapeutic properties of mesenchymal stem cells conferred by hypoxia preconditioning. Stem cells 2014;32:2702-13. [37] Zhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S. Exosome and exosomal microRNA: 34

trafficking, sorting, and function. Genomics, proteomics & bioinformatics 2015;13:17-24. [38] Kilchert C, Wittmann S, Vasiljeva L. The regulation and functions of the nuclear RNA exosome complex. Nature reviews Molecular cell biology 2016;17:227-39. [39] Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM. Functional delivery of viral miRNAs via exosomes. Proceedings of the National Academy of Sciences of the United States of America 2010;107:6328-33. [40] Dong L, Pu Y, Zhang L, Qi Q, Xu L, Li W, Wei C, Wang X, Zhou S, Zhu J, Wang X, Liu F, Chen X, Su C. Human umbilical cord mesenchymal stem cell-derived extracellular vesicles promote lung adenocarcinoma growth by transferring miR-410. Cell death & disease 2018;9:218. [41] Ding Y, Cao F, Sun H, Wang Y, Liu S, Wu Y, Cui Q, Mei W, Li F. Exosomes derived from human umbilical cord mesenchymal stromal cells deliver exogenous miR-145-5p to inhibit pancreatic ductal adenocarcinoma progression. Cancer letters 2019;442:351-61. [42] Xu T, Luo Y, Kong F, Lv B, Zhao S, Chen J, Liu W, Cheng L, Zhou Z, Zhou Z, Huang Y, Li L, Zhao X, Qian D, Fan J, Yin G. GIT1 is critical for formation of the CD31(hi)Emcn(hi) vessel subtype in coupling osteogenesis with angiogenesis via modulating preosteoclasts secretion of PDGF-BB. Bone 2019;122:218-30. [43] Zhao SJ, Kong FQ, Cai W, Xu T, Zhou ZM, Wang ZB, Xu AD, Yang YQ, Chen J, Tang PY, Wang Q, Cheng L, Luo YJ, Zhou Z, Li LW, Huang YF, Zhao X, Yin GY, Xue MX, Fan J. GIT1 contributes to autophagy in osteoclast through disruption of the binding of Beclin1 and Bcl2 under starvation condition. Cell death & disease 2018;9:1195. 35

[44] Zhang Y, Hao Z, Wang P, Xia Y, Wu J, Xia D, Fang S, Xu S. Exosomes from human umbilical

cord

mesenchymal

stem

cells

enhance

fracture

healing

through

HIF-1alpha-mediated promotion of angiogenesis in a rat model of stabilized fracture. Cell proliferation 2019;52:e12570. [45] Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 2014;507:323-8. [46] Ramasamy SK, Kusumbe AP, Wang L, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 2014;507:376-80. [47] Meister G. Argonaute proteins: functional insights and emerging roles. Nature reviews Genetics 2013;14:447-59. [48] Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, Qiu L, Vitkin E, Perelman LT, Melo CA, Lucci A, Ivan C, Calin GA, Kalluri R. Cancer exosomes perform cell-independent

microRNA

biogenesis

and

promote

tumorigenesis.

Cancer

cell

2014;26:707-21. [49] Guduric-Fuchs J, O'Connor A, Camp B, O'Neill CL, Medina RJ, Simpson DA. Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types. BMC genomics 2012;13:357. [50] Wakioka T, Sasaki A, Kato R, Shouda T, Matsumoto A, Miyoshi K, Tsuneoka M, Komiya S, Baron R, Yoshimura A. Spred is a Sprouty-related suppressor of Ras signalling. Nature 2001;412:647-51. [51] Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting 36

factor-inhibiting HIF-1. Blood 2014;124:3748-57. [52] Li ZL, Lv LL, Tang TT, Wang B, Feng Y, Zhou LT, Cao JY, Tang RN, Wu M, Liu H, Crowley SD, Liu BC. HIF-1alpha inducing exosomal microRNA-23a expression mediates the cross-talk between tubular epithelial cells and macrophages in tubulointerstitial inflammation. Kidney international 2019;95:388-404. [53] Wang H, Bian S, Yang CS. Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1alpha. Carcinogenesis 2011;32:1881-9. [54] Assinck P, Duncan GJ, Hilton BJ, Plemel JR, Tetzlaff W. Cell transplantation therapy for spinal cord injury. Nature neuroscience 2017;20:637-47. [55] Copelan EA. Hematopoietic stem-cell transplantation. The New England journal of medicine 2006;354:1813-26. [56] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair--current views. Stem cells 2007;25:2896-902. [57] Xin Y, Wang YM, Zhang H, Li J, Wang W, Wei YJ, Hu SS. Aging adversely impacts biological properties of human bone marrow-derived mesenchymal stem cells: implications for tissue engineering heart valve construction. Artificial organs 2010;34:215-22. [58] Tao SC, Yuan T, Rui BY, Zhu ZZ, Guo SC, Zhang CQ. Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway. Theranostics 2017;7:733-50. 37

[59] Wang B, Yao K, Huuskes BM, Shen HH, Zhuang J, Godson C, Brennan EP, Wilkinson-Berka JL, Wise AF, Ricardo SD. Mesenchymal Stem Cells Deliver Exogenous MicroRNA-let7c via Exosomes to Attenuate Renal Fibrosis. Molecular therapy : the journal of the American Society of Gene Therapy 2016;24:1290-301. [60] Guo SC, Tao SC, Yin WJ, Qi X, Yuan T, Zhang CQ. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics 2017;7:81-96. [61] Zhou Y, Xu H, Xu W, Wang B, Wu H, Tao Y, Zhang B, Wang M, Mao F, Yan Y, Gao S, Gu H, Zhu W, Qian H. Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem cell research & therapy 2013;4:34. [62] Wang L, Hu L, Zhou X, Xiong Z, Zhang C, Shehada HMA, Hu B, Song J, Chen L. Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling. Scientific reports 2017;7:13321. [63] Choudhry H, Harris AL. Advances in Hypoxia-Inducible Factor Biology. Cell metabolism 2018;27:281-98. [64] Rosenberger C, Mandriota S, Jurgensen JS, Wiesener MS, Horstrup JH, Frei U, Ratcliffe PJ, Maxwell PH, Bachmann S, Eckardt KU. Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys. Journal of the American Society of Nephrology : JASN 2002;13:1721-32. [65] Su T, Xiao Y, Xiao Y, Guo Q, Li C, Huang Y, Deng Q, Wen J, Zhou F, Luo XH. Bone Marrow Mesenchymal Stem Cells-Derived Exosomal MiR-29b-3p Regulates Aging-Associated 38

Insulin Resistance. ACS nano 2019;13:2450-62. [66] Tao SC, Yuan T, Zhang YL, Yin WJ, Guo SC, Zhang CQ. Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model. Theranostics 2017;7:180-95. [67] Zhang X, Sai B, Wang F, Wang L, Wang Y, Zheng L, Li G, Tang J, Xiang J. Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT. Molecular cancer 2019;18:40. [68] Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, Richardson JA, Bassel-Duby R, Olson EN. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Developmental cell 2008;15:261-71. [69] Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, Ivey KN, Bruneau BG, Stainier DY, Srivastava D. miR-126 regulates angiogenic signaling and vascular integrity. Developmental cell 2008;15:272-84. [70] Zampetaki A, Kiechl S, Drozdov I, Willeit P, Mayr U, Prokopi M, Mayr A, Weger S, Oberhollenzer F, Bonora E, Shah A, Willeit J, Mayr M. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circulation research 2010;107:810-7. [71] Sessa R, Seano G, di Blasio L, Gagliardi PA, Isella C, Medico E, Cotelli F, Bussolino F, Primo L. The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85beta. Biochimica et biophysica acta 2012;1823:1925-35. [72] Zou J, Li WQ, Li Q, Li XQ, Zhang JT, Liu GQ, Chen J, Qiu XX, Tian FJ, Wang ZZ, Zhu N, 39

Qin YW, Shen B, Liu TX, Jing Q. Two functional microRNA-126s repress a novel target gene p21-activated kinase 1 to regulate vascular integrity in zebrafish. Circulation research 2011;108:201-9. [73] van Solingen C, Seghers L, Bijkerk R, Duijs JM, Roeten MK, van Oeveren-Rietdijk AM, Baelde HJ, Monge M, Vos JB, de Boer HC, Quax PH, Rabelink TJ, van Zonneveld AJ. Antagomir-mediated

silencing

of

endothelial

cell

specific

microRNA-126

impairs

ischemia-induced angiogenesis. Journal of cellular and molecular medicine 2009;13:1577-85. [74] Taniguchi K, Sasaki K, Watari K, Yasukawa H, Imaizumi T, Ayada T, Okamoto F, Ishizaki T, Kato R, Kohno R, Kimura H, Sato Y, Ono M, Yonemitsu Y, Yoshimura A. Suppression of Sproutys has a therapeutic effect for a mouse model of ischemia by enhancing angiogenesis. PloS one 2009;4:e5467. [75] Miyoshi K, Wakioka T, Nishinakamura H, Kamio M, Yang L, Inoue M, Hasegawa M, Yonemitsu Y, Komiya S, Yoshimura A. The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization. Oncogene 2004;23:5567-76.

40

Figure 1. Hypoxia promotes exosome release from HucMSCs. (A) Morphology of Exos and Hypo-Exos under TEM. (B) NTA analysis of Exos and Hypo-Exos revealed that exosomes from the two groups exhibit similar size ranges (50-150 nm). (C and D) Western blot analysis of exosomal proteins including TSG101, CD9, CD63 and CD81 (n=3, Student’s t-test). (E) Exosome protein concentration in the two groups using the BCA assay (n=3, Student’s t-test) (F) Uptake of the red fluorescence dye Dil labelled Exos and Hypo-Exos into HUVECs. (G) Statistical evaluation of fluorescence intensities in the two groups (n=3, two-way ANOVA). 41

Figure 2. Bone fracture healing is promoted in mice after transplantation of Hypo-Exos. (A) Representative radiograph images of the femur fracture model in mice on day seven post-fracture from PBS, Exos and Hypo-Exo exposed groups (n=8 mice per group). (B) Representative 3D images from micro-CT scanning of the femur fracture model in mice on day seven post-fracture from the different groups. 42

(C) The statistical analysis of mineralized callus volume/tissue volume (CV/TV, %) from micro-CT scanning (n=8, one-way ANOVA). (D) Representative 3D images vascular micro-CT scanning on day seven post-fracture. (E) Quantification of callus vascular parameters including vessel volume and vessel number (n=8, one-way ANOVA). (F) Representative immunostaining images of CD31 (green) and Emcn (red) in the callus tissues on day seven post-fracture. (G) Quantification of the vessel number of CD31+/Emcn+ blood vessels (one-way ANOVA). (H) Immunofluorescence staining of Ki-67 (red) and CD31 (green) in the callus tissues on day seven post-fracture. (I) Quantification of the positive Ki67+/CD31+ cells (one-way ANOVA).

43

Figure 3. Hypo-Exos promote proliferation, migration, and tube formation in recipient HUVECs in vitro. (A) Cell proliferation of HUVECs after PBS, Exos and Hypo-Exo administration as measured by CCK8 assay (n=4, two-way ANOVA). (B-D) Cell proliferation of HUVECs measured by EDU staining. Scale bar = 200 μm 44

(n=4, one-way ANOVA). (E) Representative images showing tube formation in HUVECs treated with PBS, Exos or Hypo-Exos. Scale bar = 200 μm. (F) Quantitative analysis of the tube formation assay. The values of total tube length were measured (n=4, one-way ANOVA). (G) Representative images showing migrated HUVECs using the transwell assay. Scale bar = 200 μm. (H) Quantitative analysis of the migrated cells (n=4, one-way ANOVA) (I) Representative images showing the migration ability of HUVECs at 12 h by scratch wound assay. Scale bar = 200 μm. (J) Quantitative analysis of the migration rate of HUVECs (n=4, one-way ANOVA).

Figure 4. miR-126 is up-regulated in Hypo-Exos and transferred by exosomes to HUVECs. (A) HucMSCs were transfected with siAgo2 and the transfection efficiency was evaluated using real-time PCR (n=3, Student’s t-test). (B) Decreased cell proliferation in HUVECs after administration of exosomes derived from HucMSC transfected with siAgo2 under hypoxia (n=4, two-way ANOVA). 45

(C) Heat map of the 94 up-regulated miRNAs and 39 down-regulated miRNAs with a ≥1.5-fold difference between Exos and Hypo-Exos. (D) Comparison of the top five elevated miRNAs including miR-126, miR-855-5p, miR-146b, miR-223 and miR-451 between Exos and Hypo-Exos using real-time PCR (n=3, Student’s t-test). (E) miR-126 knockdown in HucMSCs and the efficiency confirmed using real-time PCR (n=3, Student’s t-test). (F) The relative expression level of miR-126 in exosomes derived from hypoxic HucMSCs transfected with miR-NC (miR-NC-Hypo-Exos) or miR-126-inhibitor (miR-126KD-Hypo-Exos) (n=3, Student’s t-test). (G) Expression level of miR-126 in target HUVECs after administrating miR-NC-Hypo-Exos or miR-126KD-Hypo-Exos (n=3, Student’s t-test). (H) Representative images of Cy3-labeled exosomal miR-126 internalized by HUVECs after administration of miR-NC-Hypo-Exos or miR-126KD-Hypo-Exos.

46

Figure 5. Knockdown of miR-126 inhibits Hypo-Exos-mediated proliferation, angiogenesis and migration in vitro and in vivo. (A-J) The functional effects of miR-126KD-Hypo-Exos on cell proliferation, angiogenesis, and migration measured by CCK8, EDU, tube formation, transwell and scratch wound assays. Scale bar =200 μm (n=4, two-way ANOVA for CCK8 assay and Student’s t-test for other assays). (K) Representative 3D images from micro-CT scanning of the femur fracture model in mice on day seven post-fracture from mice administered with miR-NC-Hypo-Exos or miR-126KD-Hypo-Exos. Scale bar =1 mm (n=8 mice per group). (L) The statistical analysis of mineralized callus volume/tissue volume (CV/TV, %) from micro-CT scanning (n=8, Student’s t-test). (M) Representative immunostaining images of CD31 (green) and Emcn (red) in the callus

tissues

from

mice

administered 47

with

miR-NC-Hypo-Exos

or

miR-126KD-Hypo-Exos on day seven post-fracture. Scale bar =200 μm. (N) Quantification of the vessel number of CD31+/Emcn+ blood vessels. Scale bar =200 μm (Student’s t-test). (O) Immunofluorescence staining of Ki-67 (red) and CD31 (green) in the callus tissues from mice administered with miR-NC-Hypo-Exos or miR-126KD-Hypo-Exos on day seven post-fracture. Scale bar =100 μm. (P) Quantification of the positive Ki67+/CD31+ cells (Student’s t-test).

Figure 6. Exosomal miR-126 regulates SPRED1 by directly targeting the 3'-UTR. (A) Venn diagram showing the top 14 miR-126 targets identified by three different independent microRNA-target-predicting programs (TargetScan, DIANA and PicTar). (B and C) Western blot analysis of the expression levels of three predicted target genes

in

HUVECs

after

administration

of

miR-NC-Hypo-Exos

or

miR-126KD-Hypo-Exos (n=3, Student’s t-test). (D) The predicted miR-126 targeting sequence in the 3'-UTR of SPRED1. (E) Luciferase reporter assay was performed to confirm that SPRED1 is the target gene of miR-126 (n=6, Student’s t-test).

48

Figure 7. Exosomal miR-126 promotes HUVECs proliferation, angiogenesis and migration by targeting SPRED1. (A) Expression level of SPRED1 in HUVECs after transfection with siNC or siSPRED1 (n=3, Student’s t-test). (B) Expression level of SPRED1 in HUVECs after transfection with siNC or siSPRED1 followed by administration of miR-126KD-Hypo-Exos (n=3, Student’s t-test). (C-H) CCK8, EDU, Tube formation, transwell and scratch wound assays were utilized to verify the functional role of SPRED1 on cell proliferation, angiogenesis, and migration in HUVECs. Scale bar =200 μm (n=4, two-way ANOVA for CCK8 assay and Student’s t-test for other assays).

49

Figure 8. HIF-1α induces the enrichment of miR-126 in exosomes shed from hypoxic HucMSCs. (A) HucMSCs transfected with siHIF-1α or siNC were exposed to hypoxia for 48 h before harvesting. Western blot was used to detect the expression of HIF-1α. (n=3, one-way ANOVA). (B) Real-time PCR was used to evaluate the expression of miR-126 in HucMSCs (n=3, one-way ANOVA). (C) miR-126 expression in exosomes derive from HucMSCs cultured in hypoxia with siHIF-1α as assessed by real-time PCR (n=3, one-way ANOVA). (D) Bioinformatic analyses indicating binding of HIF-1α to the miR-126 promoter. (E) Relative luciferase activity was analyzed in HEK293 cells (n=6, Student’s t-test). (F) ChIP assay shows binding of HIF-1α to the miR-126 promoter is enhanced by hypoxia stimulation (n=6, one-way ANOVA).

50

Figure 9. Exosomal miR-126 promotes proliferation, angiogenesis and migration in HUVECs via SPRED1/Ras/Erk pathway. (A and B) The protein levels of SPRED1 and the Ras/Erk signaling members were identified by western blot (n=3, one-way ANOVA). (C) Schematic model of hypoxic exosomal miR-126 promotion of the proliferation, angiogenesis and migration of HUVECs. Hypoxia mediates enhanced production of miR-126, possibly through the actions of HIF-1α. The enriched exosomal miR-126 is then transferred to the target HUVECs where it exerts its biological actions and potential therapeutic effects via SPRED1/Ras/Erk pathway.

51

Graphical Abstract

52