Exosomal miR-423-5p Derived from Cerebrospinal Fluid Pulsation Stress-Stimulated Osteoblasts Improves Angiogenesis of Endothelial Cells via DUSP8/ERK1/2 Signaling Pathway

Exosomes secreted from osteoblasts (OBs) can regulate the angiogenesis of endothelial cells (ECs); however, whether cerebrospinal fluid pulsation (CSFP) stress, a special mechanical stimulation, can influence the cell's communication in the context of angiogenesis remains unknown. In this study, the effect of exosomes derived from CSFP stress-stimulated OBs on facilitating the angiogenesis of ECs was investigated. First, OBs were cultured in a CSFP bioreactor, and exosomes derived from OBs were isolated and identified. Cell Counting Kit 8 assay, transwell migration assay, wound healing migration assay, and tube formation assay were conducted to assess the effects of CSFP stress-stimulated OBs-derived exosomes (CSFP-Exos) on the angiogenesis of ECs. Then high-throughput RNA sequencing was used to determine the miRNA profiles of Non-CSFP stress-stimulated OBs-derived exosomes (NCSFP-Exos) and CSFP-Exos, and the luciferase reporter gene assay was performed to confirm the binging of miR-423-5p to DUSP8. In addition, the Matrigel plug assay was performed to explore whether exosomal miR-423-5p has the same effects in vivo. Our results suggested that CSFP-Exos can promote the angiogenesis of ECs, and miR-423-5p was enriched in CSFP-Exos. Moreover, miR-423-5p could promote the effect of angiogenesis via directly targeting dual-specificity phosphatase 8 (DUSP8), which inhibited the ERK1/2 signaling pathway. In conclusion, exosomal miR-423-5p derived from CSFP stress-stimulated OBs could promote the angiogenesis of ECs by the DUSP8/ERK1/2 signaling pathway.


Introduction
Bone tissue engineering has great potential to repair the vertebral lamina defect.During artificial laminae reconstruction, blood vessel growth and osteogenesis are tightly interconnected biological processes [1,2].The growth of blood vessels is a prerequisite for bone formation, providing oxygen, nutrients, and cells [3,4], and many studies have focused on exploring the effect of mechanical factors on facilitating angiogenesis.Studies showed that pulsatile mechanical stimulation could not only promote the differentiation of mesenchymal stem cells (MSCs) into osteoblasts (OBs) and endothelial cells (ECs) [5,6] but also induced OBs to secrete angiogenic factors and related chemokines and promote the proliferation and migration of ECs [7].Previously, we reported that cerebrospinal fluid pulsation (CSFP) stress could promote the vascularization [8], osteogenesis [9], and remodeling of tissue-engineered laminae (TEL) [10], and in another study, we showed that exosomes derived from OBs could effectively promote the proliferation, migration, and tube formation of ECs [11].However, whether CSFP stress can regulate angiogenesis through exosomes derived from OBs is still unknown.
Exosomes, extracellular vesicles ranging from 30 to 150 nm in size, act as key mediators of intercellular communication by delivering proteins, mRNAs, and miRNAs to recipient cells [12,13].Exosomes can be considered as a kind of biological material in the field of regeneration medicine for its excellent cellular internalization and biocompatibility.Numerous studies have indicated that exosomes derived from different cell types can facilitate the angiogenesis of ECs, in which exosomal miRNAs play a crucial role [14][15][16].Based on previous studies, we aimed to investigate whether exosomal miRNAs derived from CSFP stressstimulated OBs can promote the angiogenesis of ECs.
In this study, we identified that CSFP stress can stimulate the secretion of OBs-derived exosomal miR-423-5p, and exosomal miR-423-5p can be transferred to ECs, thereby promoting angiogenesis by targeting DUSP8.In addition, we demonstrated that exosomal miR-423-5p mediated angiogenesis by regulating the ERK1/2 signaling pathway.To the best of our knowledge, our study is the first time to show CSFP stress can stimulate the secretion of OBs-derived exosomal miR-423-5p which promotes angiogenesis and elucidate the underlying mechanism.

Exosomes Isolation, Purification, and Characterization.
According to the manufacturer's protocol for the Exo quick reagent (Invitrogen, USA, Cat 4478359), exosomes were isolated with some adjustments.Briefly, 15 mL MC3T3-E1 cell culture medium was centrifuged at 3,000 x g for 10 min to eliminate cells and filtered with a 0.22 µm filter to discard dead cells and debris.The supernatant was further ultracentrifuged at 4,000 x g for 60 min at 4°C using 100 kDa ultrafiltration filter devices (Millipore, USA, Cat UFC9100).After adding an equal volume of the Total Exosome Isolation and mixing well, the mixture was incubated at 4°C overnight.Then, the mixture was centrifuged at 12,000 x g for 60 min at 4°C, and the supernatant was discarded.Exosomes were collected from the final pellet and then resuspended by 1x phosphate-buffered saline (PBS, Servicebio, China, Cat G4202-100ML) and stored at −80°C until use.The exosomes were identified by western bolting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM).The BCA Protein Assay Kit (Thermo, USA, Cat 23225) was used for the quantitative analysis of exosomes.

Exosome Internalization.
PKH-67 green fluorescent dye (Sigma, USA, Cat MKCK8884) was used for labeled exosomes derived from OBs according to the manufacturer's protocol.The labeled exosomes were incubated with bEnd.3 cells for 12 hr, and the uptake was quantified by immunofluorescence.
2.6.Wound Healing Assay.bEnd.3 cells were seeded in sixwell plates and cultured overnight.The separate wounds were scratched by a 200 μL pipet tip, and 1x PBS was utilized to wash cells.Subsequently, cells were cultured with a serumfree medium.The images of migrated cells were photographed using phase-contrast microscopy at 0 and 24 hr.2.7.Cell Migration Assay.Cell migration was detected using a 24-well transwell plate (8 μm polycarbonate filter, Corning, USA, Cat 3422).Cells were seeded in the plate at a cell density of 3 × 10 5 cells/mL into the upper chamber with 100 μL per chamber, while 600 μL of DMEM containing 10% FBS was added into the lower chamber.After incubation for 24 hr, cells on the upper surfaces of upper chambers were removed with cotton-tipped swabs, and cells migrated to the lower surface were fixed in formaldehyde for 20 min and then stained with crystal violet staining solution (Beyotime, China, Cat Co121-100ml) and counted.
2.13.Statistical Analysis.Statistical analysis was conducted using GraphPad Prism version 9 for Mac (9.0a, USA).All data are presented as the means AE SD.Student's t-test and one-way ANOVA were conducted to compare the differences between two groups or more than two groups.* P <0:05 was considered to be statistically significant.Exosomes were isolated and purified as described above.TEM, NTA, and western blotting were performed to identify OBs-derived exosomes.As TEM images show, OBs-derived exosomes have a similar cup or sphere-shaped morphology (Figure 1(b)).The NTA analysis showed that the size of the particles of exosomes predominantly ranged from 48.25 to 149.25 nm (Figure 1(c)).Western blotting confirmed the expression of exosome surface makers, including CD63 and CD81 (Figure 1(d)).All these results suggested that these nanoparticles were exosomes.

Results
To demonstrate whether OBs-derived exosomes can be taken up by ECs, ECs were cocultured with PKH67-labeled exosomes for 12 hr and then visualized with fluorescence microscopy.The results showed PKH67-labeled exosomes surrounded the nuclei, indicating ECs can uptake OBsderived exosomes effectively (Figure 1(e)).

CSFP-Exos Promote Migration, Tube Formation, and
Proliferation of ECs.After coculturing ECs with CSPF-Exos, NCSPF-Exos, and an equal volume of PBS, ECs incubated with CSPF-Exos exhibited increased cell migration, which is detected by Transwell chamber migration assay and wound healing assay.The results showed both NCSFP-Exos and CSFP-Exos facilitated the migration and wound healing of ECs, and this facilitation of CSFP-Exos was greater than NCSFP-Exos (Figure 2(a)−2(d)).Tube formation assay was used to evaluate the capillary network formation of ECs.In the CSFP-Exos treated group, the cells demonstrated the strongest tube-forming ability (Figures 2(e) and 2(f)).Compared with the NCSFP-Exos treated group, the CCK8 assay suggested CSFP-Exos possessed a better effect on the proliferative abilities of ECs (Figure 2(g)).All the above evidence indicated that exosomes derived from OBs can promote the angiogenic ability of ECs.However, CSFP-Exos demonstrated a more excellent promotion.

Differential miRNA Expression
Profile in CSFP Stress-Induced OBs-Derived Exosomes.Studies support that exosomal miRNAs are important regulators of biological activities [17,18].To identify the mechanism by which CSFP-Exos   Stem Cells International seq (Figure 3(c)).Among the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, "Ras signaling pathway," "Wnt signaling pathway," "Relaxin signaling pathway," "MAPK signaling pathway," and "cGMP-PKG signaling pathway," have been reported to be related to angiogenesis.Gene ontology (GO) enrichment analysis and KEGG enrichment analysis help us to know the gene ontology classification and to further explore the possible pathways in which targets were involved (Figure 4(d)-4(e)).
Results showed that CSFP-Exos could promote the angiogenesis of ECs, and miR-423-5p was enriched in CSFP-Exos, compared with NCSFP-Exos.Thus, we focused on miR-423-5p in the subsequent experiments.
After CSFP stress stimulation, miR-423-5p was packaged into exosomes and then transferred to bEnd.3 cells, yet it was unclear whether miR-423-5p was mainly transferred by exosomes.To examine the delivery method of OBs-derived exosomal miR-423-5p, Cy3-miR-423-5p mimic (red fluorescence) were used to transfer into OBs, and then coculture with ECs in a transwell plate (Figure 4(a)).The appearance of red fluorescence in ECs demonstrated that Cy3-miR-423-5p mimic was delivered from the OBs in the upper chamber to the recipient bEnd.3 seeded in the lower chamber (Figure 4(a)).The appearance of red fluorescence in OBs showed that the Cy3-miR-423-5p mimic (red fluorescence) was successfully transferred into OBs (Figure 4(b)).After coculture of OBs with bEnd.3 cells, a several-fold increase in OBs-derived miR-423-5p expression was detected by qRT-PCR.Additionally, the pretreatment of extracellular vesicle inhibitor GW4869 blocked exosome production of OBs; thus, the miR-423-5p expression in bEnd.3 was similar to coculture without OBs (Figure 4(c)).
Overall, our results suggested that miR-423-5p was mostly secreted in the form of exosomes.
To explore by which mechanism exosomal miR-423-5pregulated angiogenesis, we used TargetScan and miRDB databases to identify miR-423-5p target genes.After detailed sequence analysis, we found that 3′UTR of DUSP8 exhibited a putative binding site of miR-423-5p (Figure 4(d)).Dualluciferase reporter assay revealed that cotransfection of miR-423-5p mimic inhibited the activity of firefly luciferase reporter carrying the wild-type 3′UTR of DUSP8 (Figure 4(e)).In addition, we checked the DUSP8 level in transfected cells; treatment with miR-423-5p mimic significantly suppressed the expression of DUSP8 in bEnd.3, and cells transfected with miR-423-5p inhibitor exhibited a high level of DUSP8 (Figure 4(f)).The data indicated that exosomal miR-423-5p derived from OBs may promote angiogenesis by inhibiting DUSP8 in ECs.

Exosomal miR-423-5p Derived from OBs Promotes
Angiogenesis of ECs by DUSP8/ERK1/2 Signaling Pathway In Vitro.Transwell assay and wound healing assay were performed again to investigate the effect of exosomal miR-423-5p on the migration of ECs.Results indicated that exosomal miR-423-5p can distantly increase the migration of cells, and PD98059 weakened the facilitation but was still higher than the NC-Exos group (Figure 5(a)-5(d)).Besides, PD98059 withdrew the promotion in the proliferation of ECs compared with the Mimic-Exos group (Figure 5(e)).The tube formation assay showed PD98059 and also attenuated the effect of exosomal miR-423-5p in enhancing tube length and meshes number of ECs (Figures 5(f ) and 5(g)).Importantly, western blotting indicated the expression level of phosphorylated ERK 1/2 (pERK1/2) in ECs was significantly decreased after ECs were cocultured with exosomal miR-423-5p derived from OBs which were pretreated with miR-423-5p inhibitor.The expression level of pERK1/2 in the Mimic-Exos group was higher than in the NC-Exos group, but the ERK1/2 inhibitor could still attenuate the expression level of pERK1/2 in the Mimic-Exos group.All the evidence demonstrated that exosomal miR-423-5p derived from OBs could increase migration, proliferation, and tube formation of ECs by DUSP8/ERK1/2 signaling pathway.Stem Cells International group.These results suggested that exosomal miR-423-5p can also promote the angiogenesis of ECs by DUSP8/ERK1/2 signaling pathway in vivo.

Discussion
This was the first time to reveal that CSFP stress-modulated OBs to ECs communication via exosomal miRNAs, which was related to the angiogenesis of ECs.First, we made sure that exosomes derived from OBs can be delivered into bEnd.3cells and CSFP-Exos demonstrated a strong ability to increase the angiogenesis of ECs.Second, we found that the stimulation of CSFP stress could lead OBs to secrete exosomal miR-423-5p to facilitate migration, proliferation, and tube formation of ECs.Finally, we proved that the effect of exosomal miR-423-5p on ECs was realized by targeting DUSP8 and regulating ERK1/2 signaling pathway.All the data indicated that exosomal miR-423-5p originating from CSFP stress-stimulated OBs could promote the angiogenesis of ECs via DUSP8/ERK1/2 signaling pathway.
Exosomes, common membrane-bound nanovesicles that are actively secreted by exocytosis, contain diverse biomolecules, such as proteins, mRNAs, and miRNA [19,20], which can mediate plenty of physiological and pathological processes in the recipient cells [21][22][23].Exosome secretion is a constitutive phenomenon, and both physiological and pathological processes can influence the exosomal surface molecules and the contents [24].Given our previous studies have already confirmed that CSFP, a special mechanical stimulation, could affect the angiogenesis and osteogenesis of TEL [9,10,25], we speculated that CSFP may have an effect on exosomes secretion that mediated cell communication between OBs and ECs.
Bone is a highly vascularized tissue, and mechanical stimulations regulate bone vascularization in the bone microenvironment through modulating all kinds of angiogenic mediators, which is essential for bone formation [26][27][28].Here, we found exosomal miR-423-5p acted as a mediator for OBs stimulated by CSFP to promote the angiogenetic capacity of ECs.In glioblastomas, miR-423-5p acted as an angiogenic promoter that contributed to the malignant phenotype by activating the AKT and ERK1/2 signaling pathways [29].What is more, hADSC-derived exosomal miR-423-5p was a critical miRNA for proangiogenic activity via targeting Sufu [30].These evidences indicated that miR-423-5p could affect the angiogenesis of ECs.Combined with our results of miRNA-seq, miR-423-5p mimic, miR-423-5p NC, and miR-423-5p inhibitor were used to investigate whether the miR-423-5p played a major role in CSFP-Exos facilitating the angiogenesis of ECs, and our later results proved that the angiogenetic capacity of ECs was significantly increased when miR-423-5p in CSFP-Exos was overexpressed.
DUSP8, a member of the DUSPs family, has been shown to be a physiological inhibitor of MAPK signaling and could regulate cell oxidative stress response, cell apoptosis, and the occurrence and development of diseases [31,32].In the metabolic homeostasis or cardiovascular system, DUSP8 could regulate the resistance to diet-induced obesity or cardiac ventricular remodeling by altering ERK1/2 signaling pathway.Here, the result demonstrated that miR-423-5p could inhibit the expression of DUSP8, thereby enhancing the angiogenesis of ECs.To further reveal the specific mechanism, biomarkers in ERK1/2 signaling pathway were detected.Data showed that the activity of ERK1/2 signaling pathway would be improved when miR-423-5p was overexpressed, while DUSP8 was inhibited.In addition, the inhibition of the ERK1/2 signaling pathway could obviously weaken the angiogenesis-promoting effect of miR-423-5p, suggesting the importance of ERK1/2 signaling pathway in promoting angiogenesis.
However, the study has several limitations.Angiogenesis can be regulated by multi kinds of factors, such as cytokines and chemokines [33], and exosomes carry various biomolecules, including protein, lipids, and nucleic acid.Hence, further studies are still needed to reveal the probable exosomal contents derived from OBs that can enhance angiogenesis.Besides, we haven't knocked down DUSP8 to further verify its effect on regulating angiogenesis and ERK1/2 signaling pathway.Despite the above shortcomings, our study clearly indicated that exosomal miR-423-5p derived from CSFP stress-stimulated OBs could enhance the angiogenetic ability of ECs via targeting DUSP8 and then activating ERK1/2 signaling pathway (Figure 7).

Conclusion
In conclusion, our study was the first time to reveal that exosomes derived from CSFP stress-stimulated OBs could promote the angiogenesis of ECs, and CSFP-Exos may exert facilitating vascular regeneration by transferring miR-423-5p to downregulate DUSP8 and activate ERK1/2 signaling pathway.These findings highlight the effect of exosomes derived from CSFP stress-stimulated OBs on facilitating the angiogenesis of ECs, which enriched our understanding of the interaction between angiogenesis and osteogenesis.

3. 1 .
Isolation, Characterization, and Internalization of OBs-Derived Exosomes.As shown by the ALP staining and Alizarin Red S staining (Figure 1(a)), MC3T3-E1 was cultured in an osteogenic induction medium for 21 days to get OBs and then cultured under the stimulation of CSFP for 7 days.

TABLE 1 :
List of the primer sequences.
mediate ECs migration, tube formation, and proliferation, miRNA Model of exosomal miR-423-5p derived from CSFP-induced OBs in promoting angiogenesis of ECs.CSFP induces the secretion of OBs-derived exosomal miR-423-5p.OBs-derived exosomal miR-423-5p can directly target DUSP8 and then regulate the ERK1/2 signaling pathway to promote the angiogenesis of ECs.