Exosomal Manf originated from endothelium regulated osteoclast differentiation by down- regulating NF-κB signaling pathway

doi:10.21203/rs.3.rs-5024812/v1

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Exosomal Manf originated from endothelium regulated osteoclast differentiation by down- regulating NF-κB signaling pathway

https://doi.org/10.21203/rs.3.rs-5024812/v1

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Background

Endothelium-derived exosomes has been reported to enhanced osteogenesis. However, the role of endothelial exosomes on osteoclastgenesis is still unknown.

Methods

Human umbilical vein endothelial cells (HUVECs) were used to isolate exosomes. PBS or HUVEC-Exos were used to treat RAW 264.7 cells. Then, the preconditioned RAW 264.7 cells were subjected to TRAP staining and RT-qPCR assays. In vivo, we constracted osteoporosis mice model. PBS or HUVEC-Exos were injected through tail vein after ovariectomy surgery. Bone mass was assessed by micro-CT and TRAP staining. Furthermore, we conducted RNA sequencing and found the genes that were differentially expressed.

Results

Osteoclast differentiation was inhibited by endothelium-derived exosomes in this study. Moreover, HUVEC-Exos demonstrated a specific action on bones to promote in vivo bone resorption. Furthermore, exosomal Manf promoted bone resorption via down-regulating NF-κB signaling, and HUVEC-Exos Manf inhibited osteoclast differentiation in vivo.

Conclusion

HUVEC-exosomal Manf suppressed osteoclastogenesis via down-regulating NF-κB signaling.

endothelium

exosome

osteoclast

NF-Κb

Manf

Low bone mass, the breakdown of the bone's microstructure, and increased bone fragility leading to fractures are the hallmarks of osteoporosis (OP), a disease that affects the entire bone [1]. Osteoporosis primarily affects older individuals and postmenopausal women [2, 3]. With the population aging, osteoporosis-induced fractures not only present challenges for patients and their families but also impose a significant burden on society and healthcare systems[4]. The processes of osteoblast-mediated bone production and osteoclast-mediated bone resorption are in a state of dynamic balance [5]. However, excessive osteoclast activation can cause bone loss, which in turn causes osteoporosis. Originating from the haematopoietic lineage of monocytes or macrophages, osteoclasts possess the ability to adhere to the bone matrix and break down it by secreting lytic enzymes and acids [6]. Thus, inhibition of osteoblast differentiation can delay bone loss caused by osteoporosis.

Extracellular vesicles range from 30–200 nm in diameter and are known as exosomes [7]. They comprise a plethora of active constituents, including proteins, nucleic acids, and growth factors, which facilitate intercellular communication[8]. Additionally, exosomes are highly stable and exhibit low immunogenicity[9]. These characteristics render exosomes a promising therapeutic modality for various diseases. Vascular endothelial cell-derived exosomes have been shown to target bone and block osteoclast activity, which delays the onset of osteoporosis [10]. However, the mechanism of osteoclast differentiation by umbilical vein vascular endothelial stem cells remains unclear.

Thus, it was demonstrated that Exos released from Human umbilical vein endothelial cells (HUVEC-Exos) played an important role in osteoclast differentiation. Furthermore, HUVEC-Exos were found to inhibited osteoclastgenesis in vitro and in vivo. Therefore, the present investigation primarily focused on the impact of HUVEC-Exos on bone resorption.

Cell culture

HUVECs were obtained from Cyagen Biotechnology (USA) and were subjected to culturing Dulbecco's Modified Eagle's Medium (DMEM high glucose, Sigma, USA) rich in glucose. In the medium, penicillin-streptomycin (1%, Gibco, USA) and bovine serum (10%, FBS; VivaCell, Shanghai, China) were added to form a complete medium. RAW 264.7 cells were acquired from the American Type Culture Collection (USA), which were then cultured in a complete DMEM medium. Both cell lines were cultivated at 37°C with 5% CO₂.

Exosome isolation

Exosome isolation and characterization were performed following the previously reported method [11]. Briefly, HUVEC cells were cultivated in complete media with EV-free FBS (SBI) for over 24 h. The supernatant containing HUVEC-Exos was subjected to centrifugation at 3,000 rpm for 40 min, followed by 20,000 rpm for 60 min. The supernatant was filtered out and subsequently centrifuged at 120,000 × g for 70 min and rinsed with PBS. Finally, suspended HUVEC-Exos in PBS were kept at -4°C for future use.

Exosome identification

The properties of HUVEC-Exosomes, including shape, particle size, and markers, were determined by utilizing transmission electron microscopy (TEM), western blotting (WB), and nanoparticle tracking analysis (NTA), which were performed as previously described[11].

Western blot experiment

The Beyotime protein extraction kit was employed to extract the total proteins from the collected cells. The equal volume of protein samples was loaded, separated using SDS-PAGE, and then moved onto polyvinylidene difluoride (PVDF) membranes measuring 0.22-µm in diameter. The membranes underwent an overnight treatment with specific antibodies after being blocked with 5% skim milk. The primary antibodies employed were GAPDH (1:1000), HSP70 (1:1000), CD9 (1:1000), and CD63 (1:1000), all sourced from Abcam. After that, the membranes were exposed to secondary antibodies labeled with horseradish peroxidase (HRP) for an hour at the ambient temperature.

Osteoclast differentiation in vitro

In a 96-well plate, the RAW 264.7 cells were cultivated (1 × 10⁵ cells/well) to stimulate osteoclast differentiation. Following this, the cultured cells in complete DMEM media were supplemented with 30 ng/mL M-CSF (PeproTech) and 100 ng/mL soluble RANKL (PeproTech) for 7 days, with the medium being changed every 2 days. On Day 7, TRAP staining was conducted via a commercial kit (Sigma). Quantitive positive TRAP staining cells analysis was used by ImageJ software.

Real-time PCR

The extractraction of total RNA from cells was carried out after their harvesting and treatment with Trizol reagent. The concentration of RNA was determined while employing a Thermo Fisher Scientific NanoDrop-2000 spectrophotometer. Subsequently, a stem-loop reverse transcriptase primer kit (Ribobio, Guangzhou, China) was used to reverse-transcribe miRNA samples. SYBR Prime Script kit (Takara Bio Inc., Shiga, Japan) was used to conduct a triplicate quantitative reverse transcription polymerase chain reaction (qRT-PCR). The relative expression variation was determined using the comparative Ct (2^{− ΔΔCt}) method. The primers used for real-time PCR are listed in Table 1.

Animals

The General Hospital of the Southern Theatre Command of the PLA, located in Guangzhou, China, provided the female C57BL/6 mice. Within the animal center's specific pathogen-free (SPF) facilities, each mouse was kept. The Experimental Animal Centre of the General Hospital of the Southern Theatre Command of the PLA, Guangzhou, China, approved all animal care procedures and tests performed in this study. We constructed the osteoporosis (OP) model by bilateral removal of mice ovaries after adaptive feeding for one week. Following this, OVX mice were injected with HUVEC-Exos or PBS intravenously via the tail vein once weekly for 6 weeks (n = 8 in each group).

Micro-CT experiment

The SkyScan1178 apparatus and related software (Bruker MicroCT, Kontich, Belgium) were used to perform micro-CT scanning and femur analysis. With an energy setting of 35 kV and an intensity of 220 mA, images with a voxel size of 14 microns were acquired. DataViewer and CTVox were used to execute the two- and three-dimensional reconstructions, respectively. As the region of interest (ROI), the trabecular bone at the distal femur's metaphysis under the growth plate was selected. Using CTAn software, the following parameters were found: trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular bone volume percentage (BV/TV), and bone mineral density (BMD).

TRAP-Staining in vivo

Samples of femur bones from different groups were kept for 24 h in ice-cold 4% paraformaldehyde. For three weeks, bone specimens were decalcified with 10% ethylene diamine tetraacetic acid (EDTA). Following a previous methodology, bone samples were sectioned into 10-µm slices and stained for TRAP [12].

Statistical analysis

GraphPad Prism 8 was used to examine the data, which were then presented as the mean ± standard deviation. It used a one-way analysis of variance or the Student's t-test to assess group data. When the p-value was less than 0.05, the data was deemed statistically significant.

Features of HUVEC-Exos.

To explore the function of HUVEC-Exos in osteoclast differentiation, TEM, NTA, and WB were performed to identify HUVEC-Exos. HUVEC-Exos showed a distinctive cup-shaped morphology measuring 90–150 nm, as shown by TEM and NTA (Fig. 1A, 1B). The expression of specific protein markers HSP70, CD9, and CD63 in HUVEC-EXos was confirmed by WB analysis (Fig. 1C). The results indicate that the Exos were successfully isolated and purified.

HUVEC-Exos inhibited differentiation of osteoclast in vitro

To investigate the impact on osteoclast differentiation of exosomes produced from HUVEC, The HUVEC-derived exosomes were initially tagged with the Dio, a lipophilic dye. This demonstrated the effective transport of HUVEC-Exos to RAW264.7 cells (Fig. 2A). After that, we treated RAW 264.7 cells with PBS or HUVEC-Exos in the same volume. We found that HUVEC-Exos enhanced TRAP staining (Fig. 2B, 2C). Furthermore, in comparison to the negative control, HUVEC-Exos decreased the expression of Ctsk and Nfatc1. Thus, osteoclast differentiation was inhibited by HUVEC-Exos.

HUVEC-exos target bone in vivo.

PKH67-labelled HUVEC-Exos were injected intravenously into 8-week-old male C57BL/6J mice to investigate whether HUVEC-Exos may target bone in vivo. We evaluated the distribution of PKH67-exosomes by biophotonic imaging at 8 h after injections. The liver exhibited the highest fluorescence signals, whereas detectable signals were seen in the bone tissue (Fig. 3A). When combined, HUVEC-Exos may target bone in vivo.

Bone formation was promoted by HUVEC-exos in vivo.

To examine the function of HUVEC-derived exosomes in vivo, an osteoporosis (OP) mice model was generated to confirm whether HUVEC-Exos could relieve bone loss from osteoporosis. Then, we administered HUVEC-Exos or PBS into OVX mice. Micro-CT examination revealed a significant reduction in trabecular separation and increased trabecular bone volume, number, and thickness in OVX mice treated with HUVEC-Exos compared to the PBS group (Fig. 4A, 4B). Moreover, TRAP staining revealed that the HUVEC-Exos group had a lower osteoclast count than the PBS group (Fig. 4C, 4D). In a conclusion, HUVEC-Exos could promote bone formation in vivo.

HUVEC-exosomal Manf inhibited osteoclast differentiation by targeting NF-κB signaling pathway

To investigate the molecular mechanism through which HUVEC-exos relieve bone loss, we conducted RNA sequencing and statistical analysis of the genes that were differentially expressed. We found that Manf was greater in HUVEC-Exos group than in PBS group (Fig. 5A). Moreover, on day 7, Manf expression was higher in RAW264.7 cells treated with HUVEC-Exos in comparison to the corresponding negative controls (Fig. 5B). Then, to determine the role of Manf in vitro, we treated RAW 264.7 cells with Manf-siRNA or negative control (N.C.). We found that Manf-siRNA inhibited TRAP staining in comparsion with its negative control (Fig. 5C, 5D). Furthermore, Ctsk and Nfatc1expression were increased by Manf-siRNA in comparison with negative control (Fig. 5E).

Furthermore, Manf has been reported to fuction through NF-κB pathway. Here, we found that Manf inhibited endogenous levels of P65 protein expression (Fig. 5F,5G). Taken together, HUVEC-exosomal Manf inhibited osteoclast differentiation

HUVEC-exosomal Manf relieved bone loss from osteoporosis

In order to examine Manf's potential as a treatment for osteoporosis, Manf was ultrasonically encapsulated into exosomes generated from HUVEC. The OP mouse model was given HUVEC-exosomal Manf or PBS (negative control) every two weeks for a total of eight weeks. Mice administered HUVEC-exosomal Manf had a significant increase in trabecular bone volume, thickness, and number, along with a decrease in trabecular separation when compared to their negative controls (Fig. 6A, 6B). Moreover, TRAP staining revealed that the number of osteoclasts were lower in HUVEC-exosomal Manf group than in negative control group (Fig. 6C,6D). Thus, the above data indicates that HUVEC-exosomal Manf suppresses osteoclast differentiation in vivo and alleviates bone loss caused by osteoporosis.

By successfully identifying exosomes from HUVEC supernatant cultures, this study showed that exosomes produced from HUVECs may target bone tissue and inhibit the production of osteoclasts in vivo as well as in vitro. Then, We found that HUVEC exosomes upregulate Manf expression in RAW264.7 cells by transcriptome sequencing. Subsequently, we found that Manf inhibited osteoclastgenesis through NF-κB Pathway. Manf was then loaded into HUVEC exosomes. Then, In vivo experiments, HUVEC-Exo^Manf rescued bone loss caused by osteoporosis. Our results showed that Manf could be delivered to bone tissue by HUVEC-Exo and was a potential treatment for osteoporosis.

OP is a skeleton disorder, mainly due to metabolic abnormalities, which result in decreased bone mass, changes in bone structure, and reduced bone quality.In the past, it was believed that the Over-activation of osteoclasts causes bone resorption to be greater than bone formation, resulting in a reduction in bone mass and the eventual development of osteoporosis.

Many studies have supported the role of exosomes as a bone-targeting bionanomaterial. Y. Hu et al. fused CXCR4(+) exosomes with antagomir-188 loaded liposomes to obtain bone-targeting hybrid nanoparticles(NPs)[14]. This nanoparticle enhanced osteogenesis and suppressed adipogenesis in BMSCs, thereby restoring the loss of trabecular bone that occurs with age in vivo. G. Zheng et al. grafted alendronate (ALN) onto polyethylene glycolated phospholipids and subsequently modified PL-derived exosomes(PL-exo) to achieve bone-targeted aggregation[15]. Bone-targeted PL-exo rescued bone loss in osteoporotic mice by promoting osteogenesis and angiogenesis. Furthermore, some exosomes are naturally bone-targeted. Exosomes derived from lung adenocarcinoma cells containing miR-328 could be enriched for bone tissue and promoted osteoclast formation by down-regulating Nrp-2 expression[16]. In a separate study, it was found that vascular endothelial cell-secreted exosomes (EC-Exos) had the ability to target bones and counteract osteoporosis by inhibiting osteoblastic differentiation through miR-155[17].

Developing targeted drugs is an important part of precision medicine[18]. Exosomes are natural carriers which offer several advantages, including high stability in blood, low immunogenicity, and direct drug delivery to cells[19–21]. Bone targeting with exosomes often requires surface modifications such as aptamers[22], CXCR4[14] and alendronate (ALN) modification. However, these modifications can significantly increase the economic cost. In contrast, our delivery of Manf, targeted to bone and using natural exosomes from HUVEC cells, could reduce economic costs.

Several limitations to the present study should be considered. First, the effectiveness of the results of this study has only been validated in mouse and cellular models. Other animal models must be used for additional validation. Second, the use of exosomes is limited by the difficulty of obtaining large quantities, the high cost of isolating them and the short shelf-life required.

HUVEC-exosomal Manf suppressed osteoclastogenesis via downregulating NF-κB signaling.

Competing interests

The authors declare that they have no conflicts of interest.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study was approved by the Medical Research Ethics Board of the General Hospital of Southern Theater Command of PLA (No. 20221202), which waived the need for written informed consent due to the retrospective nature of the study. All methods were carried out in accordance with relevant guidelines and regulations.

Funding

declaration

Author Contribution

Z.P and Y.W. wrote the main manuscript text and X.W. prepared figures 1-3. P.L. prepared figures 4-6. All authors reviewed the manuscript.

Acknowledgements

None.

Availability of Data and Material

The datasets generated and/or analysed during the current study are not publicly available due to patient privacy but are available from the corresponding author upon reasonable request.

Gopinath V. Osteoporosis. Med Clin North Am. 2023;107(2):213–25.
Eastell R, O'Neill TW, Hofbauer LC, Langdahl B, Reid IR, Gold DT, Cummings SR. Postmenopausal osteoporosis. Nat Rev Dis Primers. 2016;2:16.
Johnston CB, Dagar M. Osteoporosis in Older Adults. Med Clin North Am. 2020;104(5):873–84.
Salari N, Ghasemi H, Mohammadi L, Behzadi MH, Rabieenia E, Shohaimi S, Mohammadi M. The global prevalence of osteoporosis in the world: a comprehensive systematic review and meta-analysis. J Orthop Surg Res. 2021;16(1):20.
Chen X, Wang ZQ, Duan N, Zhu GY, Schwarz EM, Xie C. Osteoblast-osteoclast interactions. Connect Tissue Res. 2018;59(2):99–107.
Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337–42.
Pegtel DM, Gould SJ. Exosomes. In: Annual Review of Biochemistry, Vol 88. Volume 88, edn. Edited by Kornberg RD. Palo Alto: Annual Reviews; 2019: 487–514.
Colombo M, Raposo G, Théry C. Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles. In: Annual Review of Cell and Developmental Biology, Vol 30. Volume 30, edn. Edited by Schekman R, Lehmann R. Palo Alto: Annual Reviews; 2014: 255–289.
Yin K, Wang SH, Zhao RC. Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm. Biomark Res. 2019;7:8.
Song H, Li X, Zhao Z, Qian J, Wang Y, Cui J, Weng WZ, Cao LH, Chen X, Hu Y, et al. Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes. Nano Lett. 2019;19(5):3040–8.
Zhang H, Wang R, Wang G, Zhang B, Wang C, Li D, Ding C, Wei Q, Fan Z, Tang H, et al. Single-Cell RNA Sequencing Reveals B Cells Are Important Regulators in Fracture Healing. Front Endocrinol (Lausanne). 2021;12:666140.
Li B, Wang R, Huang X, Ou Y, Jia Z, Lin S, Zhang Y, Xia H, Chen B. Extracorporeal Shock Wave Therapy Promotes Osteogenic Differentiation in a Rabbit Osteoporosis Model. Front Endocrinol (Lausanne). 2021;12:627718.
Compston JE, McClung MR, Leslie WD. Osteoporosis. Lancet. 2019;393(10169):364–76.
Hu Y, Li X, Zhang Q, Gu Z, Luo Y, Guo J, Wang X, Jing Y, Chen X, Su J. Exosome-guided bone targeted delivery of Antagomir-188 as an anabolic therapy for bone loss. Bioactive Mater. 2021;6(9):2905–13.
Zheng G, Ma H-W, Xiang G-H, He G-L, Cai H-C, Dai Z-H, Chen Y-L, Lin Y, Xu H-Z, Ni W-F et al. Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling. J Nanobiotechnol 2022, 20(1).
Zhang CC, Qin JR, Yang L, Zhu ZY, Yang J, Su W, Deng HB, Wang ZQ. Exosomal miR-328 originated from pulmonary adenocarcinoma cells enhances osteoclastogenesis via downregulating Nrp-2 expression. Cell Death Discovery 2022, 8(1).
Song H, Li X, Zhao Z, Qian J, Wang Y, Cui J, Weng W, Cao L, Chen X, Hu Y, et al. Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes. Nano Lett. 2019;19(5):3040–8.
Manzari MT, Shamay Y, Kiguchi H, Rosen N, Scaltriti M, Heller DA. Targeted drug delivery strategies for precision medicines. Nat Reviews Mater. 2021;6(4):351–70.
El Andaloussi S, Maeger I, Breakefield XO, Wood MJA. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discovery. 2013;12(5):348–58.
Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478):640–.
Zeng WP, Wen ZB, Chen HL, Duan YY. Exosomes as Carriers for Drug Delivery in Cancer Therapy. Pharm Res. 2023;40(4):873–87.
Luo ZW, Li FX, Liu YW, Rao SS, Yin H, Huang J, Chen CY, Hu Y, Zhang Y, Tan YJ, et al. Aptamer-functionalized exosomes from bone marrow stromal cells target bone to promote bone regeneration. Nanoscale. 2019;11(43):20884–92.

No competing interests reported.

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You are reading this latest preprint version

Exosomal Manf originated from endothelium regulated osteoclast differentiation by down- regulating NF-κB signaling pathway

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods and materials

Cell culture

Exosome isolation

Exosome identification

Western blot experiment

Osteoclast differentiation in vitro

Real-time PCR

Animals

Micro-CT experiment

TRAP-Staining in vivo

Statistical analysis

Results

HUVEC-exosomal Manf inhibited osteoclast differentiation by targeting NF-κB signaling pathway

HUVEC-exosomal Manf relieved bone loss from osteoporosis

Discussion

Conclusion

Declarations

Funding

Author Contribution

Acknowledgements

Availability of Data and Material

References

Additional Declarations

Status:

Version 1