Exosomes derived from bone marrow mesenchymal stem cells promote angiogenesis via transfer of miR-21-5p after cerebral ischemia in mice

Hui Hu Zhejiang Chinese Medical University xiaowei Hu Zhejiang Chinese Medical University lin Li Zhejiang Chinese Medical University Jingjing Gu Zhejiang Chinese Medical University Yan Fang Zhejiang Chinese Medical University Yan Yang Zhejiang Chinese Medical University Jiadong Xu Zhejiang Chinese Medical University Lisheng Chu (  chulisheng@21cn.com ) Zhejiang Chinese Medical University https://orcid.org/0000-0001-5149-5881


Introduction
Stroke is one of the leading causes of death and disability worldwide [1]. Up to now, the only recommended drug for treatment of acute cerebral ischemia is tissue-type plasminogen activator (tPA), which works as a thrombolytic agent. However, the narrow therapeutic time window and potentially fatal hemorrhage severely limited the clinical application of tPA [2]. Therefore, novel effective strategies designed to improve functional recovery after ischemic stroke are urgently needed. A growing number of researches have indicated that stem cell-based therapy represents a new approach for the treatment of ischemic stroke [3][4][5]. Mesenchymal stem cells (MSCs) can be obtained easily and expanded rapidly in vitro, which make it an ideal candidate for cell-based therapy [6]. A variety of studies have demonstrated that transplanted MSCs could promote neurogenesis and angiogenesis, thus facilitate the recovery from stroke [7,8]. As for the underlying mechanism of these functions, it was initially believed that MSCs homed to ischemic tissues and differentiated to replace injured cells [9,10]. Nevertheless, following researches reported that the engraftment and differentiation of MSCs into impaired sites are at a poor level and transient [11,12]. Until now, it is proposed that MSCs exert their bene cial effects mainly by secreting paracrine factors, such as trophic factors and extracellular microvesicles (EVs) [13,14].
Exosomes (Exos) are small EVs sized approximately 30-150 nm in diameter [15]. They are secreted by a diversity of cell types, and contain biological molecules, including proteins, lipids, mRNAs and microRNAs (miRs) [16]. The bene cial effects of MSCs-derived Exos on stroke have been particularly studied, but the mechanisms of action are still not fully clari ed [17,18]. Recent studies have shown that Exos transfer miRs to the recipient cells in the brain to affect the nervous and vascular systems, and ultimately alleviate cerebral ischemic injury [19][20][21].
MiRs are small single-stranded noncoding RNAs that bind to target mRNAs and suppress proteins expression by inhibiting translation or degrading mRNAs [22]. They are engaged in many physiological and pathological processes, such as cell proliferation, migration, differentiation, metabolism and apoptosis [23,24]. Increasing evidence has demonstrated that miRs are important regulators of angiogenesis. MiR-21-5p notably promoted local neovascularization in myocardial infarction model [25].
However, it is not clear whether BMSC-Exos promote angiogenesis after cerebral ischemia through transfer of miR-21-5p.
In this study, we rstly studied the putative bene cial effects of BMSC-Exos on the neurological function and angiogenesis with cerebral ischemia, and then further con rmed whether these proangiogenic effects are related to the miR-21-5p transferred by BMSC-Exos.

Animals
Male Sprague-Dawley rats weighing 80-100 g (3 weeks old) and male ICR mice weighing 25-30 g (8-10 weeks old) were obtained from SIPPR/BK Laboratory Animal (Shanghai, China). The animals were kept at stable temperature (22 ± 2 °C) and humidity (60 ± 5%) condition, with 12-h light/dark cycle and free access to water and food. All animal care and experimental procedures were approved by the Zhejiang Chinese Medical University Animal Welfare Ethics Committee (reference number: ZSLL-2017-058), and performed in accordance with the NIH guidelines for the Care and Use of Laboratory Animals [29].

Cell culture
BMSCs were isolated based on our previously described method [30] BMSC-Exos isolation, puri cation and identi cation BMSC-Exos were isolated and puri ed by differential ultracentrifugation method [31]. Briefly, upon reaching 70-80% confluence, BMSCs were rinsed three times with PBS and cultured in fresh medium containing 10% Exos-free FBS medium (SBI Biosciences, CA, USA). The supernatants were collected after an additional 48 h incubation, and sequentially centrifuged at 300 × g and 2000 × g for 10 min to remove the dead cells. Then, the supernatant was centrifuged at 10,000 × g for 30 min at 4°C to clear the residual cellular debris and filtered with a 0.22-μm filter (Millipore, MA, USA). Afterwards, the ltrates were ultracentrifuged at 4°C and 100,000 × g for 2 h, followed by washing with PBS and ultracentrifuged at 100,000 × g for 2 h. At last, the pelleted Exos were resuspended in 100 μL PBS and quanti ed by total proteins using a Micro BCA Protein Assay kit (Thermo Scienti c, IL, USA), then stored at -80°C.
For identification of Exos, the morphology, particle concentration, size distribution, and speci c surface markers (CD9, CD63 and TSG101) of isolated Exos were detected by transmission electron microscope (TEM, Hitachi, Japan), nanoparticle tracking analysis (NTA), and western blot analysis respectively.

Focal cerebral ischemia model in mice
Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as previously described [32]. Brie y, male ICR mice were intraperitoneally anesthetized with 10% chloral hydrate at a nal dose of 350 mg/kg. Then, a tip blunted and poly-L-lysine coated 6-0 nylon mono lament suture was advanced approximately 10 mm distal to the right carotid bifurcation to occlude the origin of the MCA. After 60 min of ischemia, the suture was carefully and slowly withdrawn. Sham-operated mice received identical surgery without inserting a suture. During the surgery, the rectal temperature of mice was maintained at 37°C with a homeothermic pad.

BMSC-Exos administration and BrdU labeling
To evaluate the therapeutic effects of BMSC-Exos, mice were allocated to four cohorts: sham operation group, MCAO group, 25 μg BMSC-Exos group and 50 μg BMSC-Exos group, and then were administered with Exos in 100 μL PBS or PBS alone by tail vein injection at 24 h after ischemia. To observe cell proliferation, mice were administered 50 mg/kg of 5-bromo-2-deoxyuridine (BrdU, Sigma-Aldrich, MO, USA) by initial intraperitoneal injection 24 h after ischemia, followed by daily consecutive injection for up to14 days.

Neurological function evaluation
The neurological de cit score was evaluated 1, 3, 7, and 14 days after stroke according to the Zea Longa score [33]: 0, no de cit; 1, failure to fully extend left forepaw; 2, circling to the left; 3, paresis to the left; 4, depressed level of consciousness and no spontaneous walking. The corner test was carried out as previously described [34]. Brie y, two 30 cm×20 cm×1 cm boards were attached to each other at an angle of 30° and with a small opening between the two boards. A mouse was placed into the central square facing the corner. When both sides of the vibrissae were stimulated by boards, the mouse then reared forward and upward, after which it turned back to face the open end. Each mouse was tested for ten trials, and the selected turning sides were recorded.

Infarct volume assessment
Mice were euthanized by overdose anesthesia with 10% chloral hydrate, and brains were moved and frozen immediately at -20°C for approximately 5 min, and then dissected into 1 mm-thick coronal slices. The slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) for 15 min, and xed with 4% paraformaldehyde for 24 h. The infarct volume was evaluated by Image J software.

Immuno uorescence staining
Mice were transcardially perfused with normal saline followed by 4% paraformaldehyde solution for 10 min at 14 days after MCAO. Brains were xed overnight at 4°C, and then soaked in 30% sucrose solution. The brains were frozen and cut into 10-μm-thick frozen sections (Leica, Wetzlar, Germany). BrdU/von Willebrand factor (vWF) was detected by double immuno uorescence staining as described in our previous study [30].

MiR-21-5p inhibitor transfection
The miR-21-5p inhibitor and the negative control (NC) were synthesized by RiboBio (Guangzhou Ribobio, Guangzhou, China). BMSCs at 80% con uence were transfected with 100 nM miR-21-5p inhibitor or NC, which were performed using Lipofectamine 2000 and Opti-MEM medium (Invitrogen, CA, USA) according to the manufacturer's protocol. After 6 h, the transfection mixture was replaced by DMEM/F12 containing 10% exosome-free FBS. Conditioned medium of transfected cells was collected and centrifuged as described above.

Scratch wound healing assay
HUVECs were seeded into 6-well plates (5×10 5 cells/well) for con uence, followed by scratching with 200 μL pipette tip when reaching 90% con uence. Then, 2 mL of serum-free RPMI-1640 medium supplemented with Exos was added to each well. Images were captured at 0 h and 24 h after wounding.

Transwell migration assay
Transwell migration assay was carried out using 24-well chambers (8 μm, Corning, NY, USA). 500 μL of the RPMI-1640 containing 1% FBS was added into the lower chamber, and HUVECs (6×10 4 cells/well) suspended in 100 μL FBS-free medium were seeded in the upper chamber in the presence or absence of Exos. After 8 h for migration, nonmigratory cells were removed from the top of the insert membrane using humidi ed cotton swabs. The migrated cells at the bottom surface of membrane were xed in 4% paraformaldehyde and stained with 0.1% crystal violet. The migrated cells were imaged and counted at 5 random elds. Three duplicates were set in each group.

Tube formation assay
After thawed overnight at 4 °C, 50 μL per well matrigel matrix (BD Biosciences, CA, USA) was added into precooled 96-well plates and incubated at 37°C to polymerize for 30 min. Next, HUVECs (2 × 10 4 cells/well) in FBS-free RPMI-1640 containing Exos were seeded onto matrigel-coated plates. Capillary-like tubular structures were captured after 6 h incubation. The total tube lengths from ve random microscopic elds were calculated using Angiogenesis Analyzer Image J software.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA from brain tissues of ischemic boundary region or cells was extracted using miRNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was produced from the total RNA using Mir-X TM miRNA First-strand Synthesis Kit (TaKaRa, Dalian, China). Subsequently, the product from reverse transcription was ampli ed with the SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) on an iQ5 real-time PCR detection system (Bio-Rad, CA, USA). Relative expression levels of miRNA were calculated by the 2 −ΔΔCt and were normalized to U6. Each sample repeated 3 times and at least three samples obtained from independent experiments were examined. All primers used in this study are listed in Table 1.  (1:1000; Santa Cruz, CA, USA) and GAPDH (1:1000; Santa Cruz, CA, USA), followed by incubation with corresponding secondary antibodies at room temperature for 1 h. Signals were visualized by enhanced chemiluminescence detection kit (Millpore, CA, USA). Relative expression levels were normalized to levels of GAPDH.

Statistical analysis
Data were analyzed by SPSS software (version 25.0, SPSS, IL, USA) and presented as mean ± standard deviation (SD). Neurological de cit and corner test data were analyzed by nonparametric Kruskal-Wallis H test. One-way analysis of variance (ANOVA) (Student-Newman-Keuls) statistical differences between groups was used for analyzing all other data. P<0.05 was considered statistically signi cant.
As presented in Fig. 2a , the BMSC-Exos had a typical spherical or cup-shaped morphology. NTA revealed a bell-shaped curve of BMSC-Exos size distribution with a peak at approximately 108.4 nm, and an average concentration of the particles was 4.8 ×10 7 particles/ml (Fig. 2b). Additionally, the isolated BMSC-Exos expressed exosomal markers CD9, CD63 and TSG101 without the expression of nonexosome marker protein GAPDH (Fig. 2c). These properties indicated that the BMSC-derived particles were Exos.

BMSC-Exos ameliorated ischemic brain injury in mice
To determine whether BMSC-Exos improved neurological function recovery, the neurological de cit score and the corner test were performed at 1, 3, 7 and 14 days after MCAO in mice. The results demonstrated that BMSC-Exos decreased the neurological de cit score and right-turn number in dose-related manner ( Fig. 3a-b). Results of TTC staining showed that BMSC-Exos signi cantly reduced infarction volume on 3 days after ischemia. At 14 days after ischemia, apparent atrophy of the ischemic hemisphere was observed in all groups of mice, except the sham-operated group. Moreover, the cerebral atrophy in the BMSC-Exos group was markedly reduced compared to the MCAO group. In addition, compared with the 25 μg BMSC-Exos group, the cerebral atrophy was signi cantly reduced in the 50 μg BMSC-Exos group ( Fig. 3c-d).
BMSC-Exos promoted angiogenesis in the ischemic boundary region Microvessel density was estimated by BrdU/vWF immuno uorescent staining at 14 days after MCAO.
Signi cantly increased number of BrdU + /vWF + cells was observed in the MCAO and BMSC-Exos groups compared to the sham group. Furthermore, microvessel density in the BMSC-Exos group increased more signi cantly than which in MCAO group (Fig. 4a-b).

BMSC-Exos increased VEGF/VEGFR and Ang-1/Tie2 protein expression after ischemia in mice
To test whether BMSC-Exos promote angiogenesis via upregulating the expression of VEGF, VEGFR2, Ang-1 and Tie2, the expression levels of these proteins were detected by western blot analysis. Compared with the sham group, the expression levels of VEGF, VEGFR2, Ang-1 and Tie2 were increased in the MCAO group and BMSC-Exos group. Furthermore, the expression levels of these proteins were increased more obviously in BMSC-Exos group compared to the MCAO group (Fig. 4c-d).

BMSC-Exos increased miR-21-5p expression after ischemia in mice
To study the changes of speci c miRs expression levels after stroke, the expression of several candidate miRs, including Let-7i-5p miR-21-5p miR-22-3p and miR-486 were detected by qRT-PCR at 14 days after MCAO. The expression levels of these miRs were increased in the BMSC-Exos group compared to the MCAO group. In particular, miR-21-5p expression levels increased approximately 22-fold ( Fig. 5a-d).
HUVECs uptake BMSC-Exos To con rm that BMSC-Exos could be internalized into HUVECs, BMSC-Exos were labeled with PKH67 and then were added to HUVECs for 12 h incubation. Fluorescence images showed that PKH67 uorescence were incorporated into the cytoplasm of HUVECs (Fig. 6a). This result revealed that our puri ed BMSC-Exos had been taken up and transferred to HUVECs.

BMSC-Exos promoted HUVECs angiogenesis by transferring miR-21-5p
Since endothelial cell proliferation, migration, and sprouting of are critical for angiogenesis, the effects of BMSC-Exos on HUVECs proliferation, migration and tube formation were studied. MTT assay, scratch wound healing assay, transwell assay and tube formation assay indicated that BMSC-Exos can enhance the above-mentioned capability of HUVECs (Fig. 6b-h). Moreover, the effects of BMSC-Exos were in a dose-related manner so 50 μg/mL was selected for the following experiments.

BMSC-Exos upregulated VEGF and VEGFR2 expression in HUVECs by transferring miR-21-5p
To examine the mechanisms of proangiogenic action of BMSC-Exos, the VEGF and VEGFR2 protein expression in HUVECs were detected by western blot analysis. Results showed that BMSC-Exos remarkably improved the expression levels of VEGF and VEGFR2. However, this effect was reversed by the miR-21-5p inhibitor (Fig. 8h-j). The above results revealed that BMSC-Exos enhances VEGF and VEGFR2 expression by transferring miR-21-5p into HUVECs.

Discussion
In the present study, we rstly con rmed that systemic treatment of BMSC-Exos could improve cerebral ischemia injury and promote angiogenesis in mice. Simultaneously, BMSC-Exos increased proangiogenic proteins expression, including VEGF, VEGFR, Ang-1 and Tie-2, and upregulated miR-21-5p expression in the ischemic boundary regions. Then, we transfected miR-21-5p inhibitor into BMSCs and further con rmed BMSC-Exos improved HUVECs angiogenesis via transferring miR-21-5p in vitro.
Increasing evidence suggests that BMSCs exert their therapeutic effects through paracrine mechanisms including secretion of Exos [35,36]. Moreover, Exos not only show the same effects as BMSCs, but also show the advantages on targeted delivery, low immunogenicity, and high repairability [37]. BMSCs have been proven to secret Exos that show the potential for treatment of ischemic disease including ischemic stroke [38]. Recent studies showed that Exos derived from BMSCs promoted activation of microvascular endothelial cells and played an antiapoptotic role in cerebral ischemia/reperfusion injury [39]. In addition, BMSC-Exos could protect against myocardial ischemia reperfusion injury [40,41]. In this work, we found that BMSC-Exos could promote angiogenesis and improve ischemia/reperfusion injury in mice. Moreover, we also con rmed that BMSC-Exos promoted the proliferation, migration, and tube formation of HUVECs in vitro. Thus, our research presents a new evidence in favor of the concept that BMSC-Exos possess a potent proangiogenic activity.
Nowadays, accumulated studies have shown that angiogenesis is essential for brain tissue repair following stroke, and the promotion of angiogenesis is broadly recognized as a promising therapeutic strategy [42,43]. Angiogenesis is commonly de ned as a multi-step process, involving proliferation, sprouting, migration and tube formation from the pre-existing vasculature [44]. It is regulated by multiple proangiogenic factors, among which VEGF, VEGFR2, Ang-1, and Tie-2 play the most important roles. The rst step of angiogenesis is to remove the mural cells from the endothelial cells of established blood vessels, which results in an unstable blood vessel. Then, VEGF bonds to its receptor VEGFR2 and triggers downstream angiogenic signals, which lead to endothelial cells in these unstable blood vessel display different phenotypes: migratory tip cells or proliferating stalk cells [45,46]. In the presence of VEGF and other angiogenic factors, endothelial cells proliferate and migrate and then form new vascular tube [47].
However, the newly formed endothelial cell tubes are unstable due to the lack of pericytes. Ang-1 is one of the key angiogenic factors for the recruitment of the pericytes, and it act through the tyrosine kinase receptor Tie-2. The Ang-1/Tie-2 signal regulates maturation of newly formed vasculature, which eventually results in complex vascular network formation [48,49]. In this study, we found that BMSC-Exos could dramatically improve the levels of VEGF, VEGFR2, Ang-1, and Tie-2 in the peripheral zone of ischemic mice. Unfortunately, we could only nd the improved expression of VEGF and VEGFR2 in HUVECs after BMSC-Exos administration.
MiRs are a class of non-coding RNA that in uence nearly all fundamental biological processes, and are stable in Exos due to the prevention of RNase degradation [50]. To date, a considerable amount studies have reported miRs related to angiogenesis. MiR-22 could regulate endothelial angiogenesis, in ammation, and tissue injury by targeting vascular endothelial-cadherin [51]. MiR-486-5p released by adipose-derived stem cell-derived EV could mediate wound healing and promote angiogenesis [52]. MiR-21-5p could promote extracellular matrix degradation and angiogenesis in the unilateral anterior crossbite model [53]. More importantly, it has been reported that miR-486-5p, miR-22, miR-21-5p and Let-7i-5p were abundant in BMSC-Exos [28]. Thus, we detected the expression of Let-7i-5p, miR-21-5p, miR-22-3p, and miR-486 in ischemic boundary region of stroke mice and HUVECs. The qRT-PCR data indicated that BMSC-Exos up-regulated the expression of these miRs, among which miR-21-5p was the most signi cant one. Then, we transfected miR-21-5p inhibitor into BMSCs, the expression of miR-21-5p in BMSC-Exos was signi cantly decreased con rmed by qRT-PCR. More importantly, the proangiogenic effects of BMSC-Exos on HUVECs were greatly abolished by miR-21-5p inhibitor. Moreover, the expression levels of VEGF and VEGFR2 in HUVECs were also signi cantly reduced by exosomes derived from BMSCs transfected with miR-21-5p inhibitor. These results suggest that the therapeutic effects of BMSC-Exos on angiogenesis may depend on the transport of miR-21-5p. However, although our study found that BMSCderived exosomal miR-21-5p promoted angiogenesis in cerebral ischemia mice, the target genes of miR-21-5p and the detail mechanism need to be further investigated.

Conclusions
Our study indicates that BMSC-Exos can promote angiogenesis by transfer of miR-21-5p after stroke in mice. These ndings show that BMSC-Exos might be a promising candidate for stroke treatment, and that BMSC-Exos could be tailored to maximize therapeutic effect. Declarations data, manuscript writing; J.J.G., Y.Y.: administrative support, provision of study material, collection data; Y.F.: supervision, nancial support, data analysis and interpretation; J.D.X: data analysis and interpretation, manuscript writing; L.S.C.: conception and design, nancial support, provision of study material, manuscript writing, nal approval of manuscript, supervision.

Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University (reference number: ZSLL-2017-058), and performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.

Consent for publication
The authors declare that they consent to publication.

Competing interests
The authors declare no potential con icts of interest. Characterization of BMSCs. a P0 BMSCs showed short bar shapes and P3 BMSCs adopted a uniformly spindle-shaped population. Scale bars=200 μm. b Flow cytometric analysis of cell surface markers indicated that BMSCs expressed CD29 and CD90, but not for CD34 and CD45. BMSCs bone marrow mesenchymal stem cells, P0 passage 0, P3 passage 3 Figure 2 Identi cation of BMSC-Exos. a Cup-shaped morphology of puri ed BMSC-Exos (indicated with arrows) observed by TEM. Scale bar = 500 nm. b The particle size and particle concentration of BMSC-Exos analyzed by NTA. c Western blots of exosomal membrane markers CD9, CD63 and TSG101. BMSC-Exos bone marrow mesenchymal stem cell-derived exosome, TEM transmission electron microscope, NTA nanoparticle tracking analysis