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Exosomal transfer of osteoclast-derived miRNAs to chondrocytes contributes to osteoarthritis progression

Abstract

Osteoarthritis (OA) is a prevalent aging-related joint disease lacking disease-modifying therapies. Here, we identified an upregulation of circulating exosomal osteoclast (OC)-derived microRNAs (OC-miRNAs) during the progression of surgery-induced OA in mice. We found that reducing OC-miRNAs by Cre-mediated excision of the key miRNA-processing enzyme Dicer or blocking the secretion of OC-originated exosomes by short interfering RNA-mediated silencing of Rab27a substantially delayed the progression of surgery-induced OA in mice. Mechanistically, the exosomal transfer of OC-miRNAs to chondrocytes reduced the resistance of cartilage to matrix degeneration, osteochondral angiogenesis and sensory innervation during OA progression by suppressing tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-3. Furthermore, systemic administration of a new OC-targeted exosome inhibitor (OCExoInhib) blunted the progression of surgery-induced OA in mice. We suggest that targeting the exosomal transfer of OC-miRNAs to chondrocytes represents a potential therapeutic avenue to tackle OA progression.

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Fig. 1: Elevated OC-miRNAs in osteoclasts and circulating exosomes in early-stage OA.
Fig. 2: Genetic knockdown of OC-miRNAs retards the progression of ACLT-induced OA in mice.
Fig. 3: Blockage of OC-exosomes dampens the progression of ACLT-induced OA in mice.
Fig. 4: OC-exosomes promote chondrocyte catabolism, endothelial cell angiogenesis and axon innervation in vitro.
Fig. 5: OC-derived exosomal miRNAs inhibit TIMP-2 and TIMP-3 to reduce the resistance of cartilage to matrix degeneration, angiogenesis and sensory innervation in vitro.
Fig. 6: OC-derived exosomal miRNAs reduce the resistance of cartilage to matrix degeneration, angiogenesis and sensory innervation in OA.
Fig. 7: OCExoInhib treatment blunts OA progression in mice after ACLT.

Data availability

All miRNA qPCR array data (raw Ct values) can be freely downloaded and reproduced from https://github.com/TMBJ-HKBU/NATAGING_A00041_qPCRArray/. All data supporting the findings of this study are available within the article and its Extended Data files or are available from the corresponding author upon reasonable request.

Code availability

All code for the analytic software is available from the corresponding author upon reasonable request.

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Acknowledgements

We thank technical staff (Y. S. Cheung, W. K. Chan, S. Lee and C. L. Chan) from Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases, Hong Kong Baptist University for providing technical support on micro-CT, confocal imaging and flow cytometry analysis. We thank Guangzhou Biogene Biotechnology Co., Ltd. for providing technical support on mouse genotyping and AAV packaging. This work was supported by the funds from National Key R&D Program of China (2018YFA0800804 to L.C.) and National Natural Science Foundation Council of China (81702189 to J. Liu; 81803374 to J. Lu., 81802187 to G.H., 81703049 to F.L. and 81700780 to C.L.), the Theme-based Research Scheme from the Research Grants Council of Hong Kong (T12-201/20-R to A.L.), the General Research Funds from the Research Grants Council of Hong Kong (12102914 to G.Z., 12101117 to G.Z., 12136616 and 12103519 to J. Liu, 12101018 to F.L., 12102120 to Y.Y., 12102518 and 12100719 to A.L. and 14112915 to B.-T.Z.), the Basic and Applied Basic Research Fund from Department of Science and Technology of Guangdong Province (2019B1515120089 to G.Z.), the Natural Science Fund of Guangdong Province (2018030310355 to G.H.), the Shenzhen Science and Technology innovation fund (JCYJ20180302174121208 to J. Lu.), the Interdisciplinary Research Clusters Matching Scheme of Hong Kong Baptist University (RC-IRCs/17-18/02 to G.Z.; RC-IRCs/17-18/04 and RC-IRMS/15-16/01 to A.L.), the Inter-institutional Collaborative Research Scheme from Hong Kong Baptist University (RC-ICRS/19-20/01 to A.L.)

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Authors

Contributions

J. Liu, A.L. and G.Z. jointly supervised the project and wrote most of the manuscript. J. Liu, X.W., J. Lu, L.D., H.Z. and C.Z. performed the majority of the experiments, analyzed data and prepared the manuscript. G.H. collected and analyzed the clinical samples. Z.Z and D.L. assisted in the animal breeding and sample collection. F.L., C.L. and Y.Y. provided technical instructions. B.-T.Z. and L.C. provided their professional expertise.

Corresponding authors

Correspondence to Jin Liu, Aiping Lu or Ge Zhang.

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The authors declare no competing interests.

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Peer review information Nature Aging thanks Mary Goldring, Michiel Pegtel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Characterization for bone marrow-derived OCs and serum OC-exosomes.

(a) QPCR analysis of the mRNAs of Ctsk and Acp5 in osteoclasts (OSCAR+CTR+OCs), OC precursors (OSCAR+CTR-OCPs) and non-osteoclastic cells (OSCAR-CTR-cells) isolated from mouse hindlimb bone marrow cells by magnetic activated cell sorting (MACS). (b) Representative TRAP staining images and the number of TRAP+ multinucleated cells in the culture of the aforementioned OCs, OCPs and non-OCs. Scale bars: 100 μm. c, Nanoparticle tracking analysis showing the size distribution of the serum exosomes. (d) Representative transmission electronic microscope (TEM) images of serum exosomes. Scale bars: 200nm. (e) Western blot analysis of the exosome markers CD63, CD9, TSG101, ALIX, the osteoclast-specific markers OSCAR and CTR, and the cytoplasma proteins GM130 and cytochrome C in serum exosomes. g, Gating strategy for characterization of the circulating OC-exosomes by flow cytometry analysis. The circulating exosomes were harvested from the serum, purified by the CD63 magnetic beads and visualized by the FITC staining. The CD63+ fraction was further gated for sorting the OC-exosomes co-expressing OSCAR (PE) and CTR (APC). (g) Representative TEM images of serum OC-derived exosomes expressing OSCAR and CTR (indicated by red arrows). Scale bars: 200nm. (h) QPCR analysis of sera exosomal miR-223-3p and miR-34a-5p from ACLT and Sham mice at 2 weeks after surgery, respectively. (i) The exosome quantitation (per 106 cells) in cell culture supernatant of OCs differentiated from bone marrow monocyte/macrophage (BMMs) from ACLT and Sham mice at 2w after surgery, respectively. Data are shown as mean ± s.d. All experiments were performed in at least biologically independent triplicates in each group. The exact p values are shown in figures. In a & h, Two-way analysis of variance (ANOVA) with either Turkey’s (a) or Bonferroni’s (h) multiple comparisons test was used for statistical analysis. In b, One-way ANOVA with Turkey’s multiple comparisons was used for statistical analysis. In i, student’s t-test was used for statistical analysis.

Source data

Extended Data Fig. 2 Characterization of OCmiR-KD mice.

(a) Representative genotyping image of the Dicerfl/fl mice, Rosa-YFP mice and Dicerfl/fl; Rosa-YFP mice. (b) Representative confocal fluorescent micrographs of YFP expression after Cre-mediated recombination in TRAP+ OCs in the cryosection of subchondral bone from the Dicerfl/fl; Rosa-YFP mice administered with Cre-expressing plasmid encapsulated within the OC-TDS. (c) Gating strategy for the detection of Cre-mediated recombination in OCs in vivo by flow cytometry analysis. The dead cells were excluded by 7-aminoactinomycin D (7-AAD) staining, while the live cells were gated for identifying the OCs with the co-expression of OSCAR (PE) and CTR (APC). The Cre-mediated recombination in OCs was further confirmed by the YFP expression in OCs. (d) Microscopic images of multinucleated osteoclast formation with TRAP staining in BMMs with or without infection of Cre-expressing lentivirus (e) The exosome quantitation (per 106 cells) in cell culture supernatant of OCs differentiated from BMMs with or without infection of Cre-expressing lentivirus. (f-g) Western blot analysis of cellular protein expression of Rab27a and GAPDH (f) and exosomal protein expression of CD63 and CD9 (g). All scale bars: 100 μm. Data are shown as mean ± s.d. All experiments were performed in biologically independent triplicates in each group. The exact p values are shown in figures. Student’s t-test was used for statistical analysis.

Source data

Extended Data Fig. 3 Effect of OC-exosome blockage on osteoblasts in mice after ACLT.

(a) Representative fluorescent images of the Runx2 (red) immunostaining at tibial articular cartilage and subchondral bone. (b) The number of Runx2+ cells at subchondral bone (SCB) area or growth plate (GP) area of mice from the indicated groups at 4w after ACLT. Scale bar: 100 μm. Data are shown as mean ± s.d. N=8 mice in each group. The exact p values are shown in figures. Two-way ANOVA with Turkey’s multiple comparisons test was used for statistical analysis.

Source data

Extended Data Fig. 4 The transfer of OC-exosomes to OA cartilage in vivo.

The representative confocal images of the PKH26-labelled OC-exosomes in tibial cartilage, subchondral bone and metaphysis area in ACLT mice or Sham mice at 2w after surgery. The mice were intravenously injected with OC-exosomes (20 μg per mouse) 3 days before euthanasia. Left: PKH26-labelled OC-exosomes indicated by red fluorescent signal. Middle left: Endomucin+ vessels indicated by green fluorescent signal. Middle right: Merged images with DAPI-stained nucleates (blue) and bright field. Right: 20x zoom-in of the cartilage layer. Dotted lines indicate the osteochondral junction. Arrows indicate the PKH26-labelled OC-exosomes at osteochondral junction and cartilage layer. Scale bar: 100 μm. All experiments were performed in biologically independent triplicates in each group.

Extended Data Fig. 5 OC-derived exosomal miR-214-3p promote cartilage matrix degeneration, angiogenesis and sensory axon innervation in vivo.

(a) Schematic diagram showing the strategy for osteoclast-targeted delivery of control ncRNA in Ctsk-Cre mice (WT, OCExo-Intact), osteoclast-specific miR-214 knockout mice (CKO, OCExo-Intact) and osteoclast-specific miR-214 knockin mice (CKI, OCExo-Intact) and osteoclast-targeted delivery of Rab27a siRNA in the CKI mice (OCExo-BLK). (b-c) QPCR analysis of the miR-214-3p in BM-OCs and serum exosomes (b) and the pri-miR-214 and miR-214-3p in cartilage (c) from the indicated mice. (d) The burrowing performance. (e) Left: Representative Safranin O staining images of tibial articular cartilage. Right: the OARSI scores. (f-g) Left: Representative fluorescent images of the Endomucin (EDMC, green) and CD31 (red) immunostaining (f) and CGRP (green) immunostaining (g) at tibial articular cartilage and subchondral bone. Right: the number of EDMChiCD31hi cells (f) and CGRP+ fibers (g) around the osteochondral junction. All scale bar: 100 μm. Data are shown as mean ± s.d. In b & c, N=4 mice per group. The exact p values are shown in figures. Two-way ANOVA with Bonferroni’s (h) multiple comparisons test was used for statistical analysis. In d-g, N=6 mice in each group. One-way ANOVA with Turkey’s multiple comparisons test was used for statistical analysis.

Source data

Extended Data Fig. 6 OC-derived exosomal miRNAs regulate TIMP2 and TIMP3 in chondrocytes in vitro.

(a) Representative confocal fluorescent micrographs showing the co-localization of FAM-labelled miR-214-3p mimics and PKH26-labelled OC-exosomes in chondrocytes. Scale bar: 100 μm. (b) QPCR analysis of the mRNA levels of TIMP1, 2 and 3 in IL-1β-stimulated chondrocytes with or without OCs co-culture. (c) Western blot analysis of the mRNA levels of TIMP1, 2 and 3 in IL-1β-stimulated chondrocytes with or without OCs co-culture. The aforementioned OCs were differentiated from BMMs with either Rab27a siRNA or ncRNA transfection. Data are shown as mean ± s.d. All experiments were performed in biologically independent triplicates in each group. The exact p values are shown in figures. Two-way ANOVA with Turkey’s multiple comparisons test was used for statistical analysis.

Source data

Extended Data Fig. 7 The development of OCExoInhib.

(a) TRAP staining analysis of the osteoclastogenesis of Raw264.7 cells treated with various exosome inhibitors at 0.5 μM. Scale bar: 100 μm. (b) MTT assay of the cell viability of Raw264.7 cells treated with Nexinhib20 or LJ001. (c) The synthetic route for d-Asp8-Nexinhib20 derivative conjugate. Reagents and conditions: (i) Br2, DCM, 0 °C to r.t., 8h; (ii) 1,2,4-triazole, Cs2CO3, acetonitrile, 80 °C, 4 h; (iii) 3-hydroxylbenzaldehyde, piperidine, toluene, 60 °C, 4 h; (iv) K2CO3, acetonitrile, 40 °C, 6 h; (v) LiOH, THF/H2O, 20 °C, 4h; (vi) N-Hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, DCM, 20 °C, 12h; (vii) Ethyldiisopropylamine, d-Asp8, DMF, 20 °C, 24h. (d) Exosome quantitation (per 106 cells) in cell culture supernatant of RANKL-induced Raw264.7 cells with the treatments indicated. (e) QPCR analysis of the mRNAs of Ctsk and Acp5 in RANKL-induced Raw264.7 cells with the treatments indicated. (f) The number of bone resorption pits in bone slices seeded with RANKL-induced Raw264.7 cells with the treatments indicated. (g) Molecular docking showing the interaction between Rab27a and Nexinhib20 (left panel), between Rab27a and LJ001 (middle panel), and between Rab27a and OCExoInhib (right panel), respectively. Red, yellow, blue and white ribbons: Rab27a. The binding surfaces are identified in gray. The structure of inhibitor is displayed by green ball-and-stick model and the structure of d-Asp8 is displayed by purple ball-and-stick model. (h) Representative biophotonic images of the distribution of the FAM-labelled LJ001 and OCExoInhib in organ levels. (i) Representative confocal images of the colocalization of FAM-labelled OCExoInhib and TRAP+ OCs (arrow indicated) at tibial subchondral bone surface. Dotted line indicates the osteochondral junction. Scale bar: 100 μm. Data are shown as mean ± s.d. All experiments were performed in biologically independent triplicates in each group. The exact p values are shown in figures unless the asterisks are annotated as follow. In a, ***: p < 0.001 vs Veh group. Two-way ANOVA (a & e) or One-way ANOVA (d & f) with Turkey’s multiple comparisons test was used for statistical analysis.

Source data

Extended Data Fig. 8 OCExoInhib treatment blunt the OA progression in mice after DMM.

(a) Schematic diagram showing the strategy for OCExoinhib treatment in C57BL/6 mice with DMM. (b) The burrowing performance. (c) Left: Representative Safranin O staining images of tibial articular cartilage. Right: the OARSI scores. (d-f) Left: Representative fluorescent images of the TIMP2 (green) immunostaining (d), the Endomucin (EDMC, green) and CD31 (red) immunostaining (e) or the CGRP (green) immunostaining (f) at tibial articular cartilage and SCB. Right: the number of TIMP2+ chondrocytes at cartilage layer (d), the number of EDMChiCD31hi cells (e) and CGRP+ fibers (f) around the osteochondral junction (white dotted line). All scale bars: 100 μm. Data are shown as mean ± s.d. N=8 mice per group. The exact p values are shown in figures unless the asterisks are annotated as follow. In b, ***: p < 0.001 DMM+Veh / DMM+OCExoInhib group vs Sham+Veh group, φφφ: p < 0.001 DMM+Veh group vs DMM+OCExoInhib group. Two-way ANOVA with Turkey’s multiple comparisons test was used for statistical analysis. In c-f, One-way ANOVA with Turkey’s multiple comparisons test was used for statistical analysis.

Extended Data Fig. 9 Schematic diagram of the osteoclast-chondrocyte crosstalk mediated by osteoclast-derived exosomal miRNA in OA.

Left side of the dotted line: an illustration of the health articular joint. Right side of the dotted line: an illustration of the articular joint with OA. The box illustrates the postulated exosomal miRNA-mediated osteoclast-chondrocyte crosstalk in OA.

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Liu, J., Wu, X., Lu, J. et al. Exosomal transfer of osteoclast-derived miRNAs to chondrocytes contributes to osteoarthritis progression. Nat Aging 1, 368–384 (2021). https://doi.org/10.1038/s43587-021-00050-6

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