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. 2022 Sep 5;11(17):2766.
doi: 10.3390/cells11172766.

Extracellular Vesicle-Mediated miR-150-3p Delivery in Joint Homeostasis: A Potential Treatment for Osteoarthritis?

Huan Wang  1 , Jun Shu  2 , Chengfei Zhang  3 , Yang Wang  4 , Rongxing Shi  5 , Fan Yang  1 , Xuezhang Tang  1
Affiliations

Extracellular Vesicle-Mediated miR-150-3p Delivery in Joint Homeostasis: A Potential Treatment for Osteoarthritis?

Huan Wang et al. Cells. .

Abstract

Background: The disruption of joint homeostasis is a critical event during the process of joint injury in osteoarthritis (OA). As regulatory molecules, microRNAs (miRNAs) can be released from secretory cells and delivered to recipient cells through extracellular vesicles (EVs), thereby playing an important role in regulating joint homeostasis. We hypothesized that the fibroblast-like synoviocytes (FLSs) in healthy joints could release EVs enriched in miRNAs that can maintain joint homeostasis by regulating the signal transduction pathways in the joints, whereby the articular cartilage (AC) is protected from degeneration, and OA progression is delayed.

Methods: Via high-throughput sequencing and qPCR, we found that miR-150-3p was enriched in the circulating EVs in healthy rats. Next, we established an in vitro cell model in which chondrocytes were cultured with (i) FLSs transfected with miR-150-3p mimics or (ii) EVs released by FLSs (FLS-EVs) inside the healthy synovial membrane (SM). The transportation mechanism from FLSs to chondrocytes was studied using the EV inhibitor GW4869, and the FLSs were transfected with a miR-150-3p mimic or inhibitor. To assess the therapeutic effect of miR-150-3p-carrying EVs (EVs-150) in vivo, healthy FLS-derived EVs (H-FLS-EVs) were injected into the tail vein of rats with OA at various stages of the pathogenesis and evaluated for the progression of OA.

Results: The chondrocytes could uptake fluorescent-labeled miR-150-3p mimics and FLS-EVs, and GW4869 suppressed this uptake. The overexpression of miR-150-3p could significantly reduce the concentrations of pro-inflammatory cytokines in the cell culture medium and the expression of the miR-150-3p target T cell receptor-interacting molecule 14 (Trim14), as well as the innate immune-related factors, including nuclear factor kappa B (NF-κB) and interferon-β (IFN-β). Similarly to the in vitro findings, the miR-150-3p level in the serum EVs was significantly upregulated among the EV-treated rats. In the AC of the OA rat model injected with H-FLS-EVs, the joint degeneration was suppressed, and Type II collagen (COLII) and aggrecan (ACAN) were significantly upregulated, whereas the innate immune-related factors Trim14, NF-κB, and IFN-β were downregulated compared with the levels in the untreated OA rats. Notably, the suppression of joint degeneration was more significant when H-FLS-EVs were administered at the early stages of OA rather than the late stages.

Conclusion: H-FLS-EVs protect chondrocyte function and maintain joint homeostasis by modulating the innate immune response by suppressing the Trim14/NF-κB/IFNβ axis. These effects are achieved through the EV-mediated transport of miR-150-3p from the FLSs to the chondrocytes. Our findings show that EV-mediated miR-150-3p can be used to suppress OA, thus providing a novel therapeutic strategy. Additionally, the EV-mediated miR-150-3p transport may also serve as a potential biomarker in the diagnosis, treatment, and prognosis of OA.

Keywords: extracellular vesicles; innate immune response; joint homeostasis; miR-150-3p; osteoarthritis.

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Conflict of interest statement

The authors have declared that there are no competing interest.

Figures

Figure 1
Figure 1
Construction and validation of a rat model of knee OA. (A) Establishment of OA model in rats by ALCT method. (a) The hair around the knee was removed to expose the surgical incision site. (b) After disinfection, the skin and various subcutaneous tissues were sequentially incised using a scalpel. (c) The knee joint capsule was exposed laterally. (d) The anterior cruciate ligament was cut. (e) The muscles, fascia, and skin were sequentially sutured. (B) The screening flow for the differentially available miRNAs between the circulating EVs of the control (healthy rats) and the model (OA rats) groups. The circulating EVs were extracted from serum using differential centrifugation, and TEM, NTA, and WB were used to identify the EVs. Then, miRNA sequencing was performed on these EVs. (C) Morphological observation. After the skin and subcutaneous tissues surrounding the knee, the knee joints were removed layer by layer. (a) The skin on the knee surface was opened. (b) Then, the joint cavity was opened, and the SM was exposed. (D) Histological observation. The entire knee joints were stained using HE.
Figure 2
Figure 2
EVs were isolated using differential centrifugation and identified using TEM, NTA, and WB. (A) TEM observation of the EV morphology. (B) NTA measurement of the EV particle distribution and concentration. (C) WB analysis of the EV markers CD9, CD63, and HSP70.
Figure 3
Figure 3
RNA sequencing analyzed DE miRNAs in EVs between the control (C) and model (M) groups. (A) Volcano plot of differentially present miRNAs. Gray indicates the miRNAs present at similar levels between the two groups; red indicates the significantly higher presence of miRNAs; and green indicates the significantly lower presence of miRNAs; X-axis is log2 Fold Change, and Y-axis is −log10 p-value. p < 0.05, |log2 fold change| > 2. (B) Fourteen miRNAs were significantly differentially depicted, including twelve upregulated and two downregulated miRNAs. (C) Heatmap of the DE miRNAs. Red and blue indicate high and low levels, respectively. (D) Gene ontology analyses were performed. (E) qPCR validation of the miR-150-3p enrichment in the EVs. Three independent experiments indicate the data as mean ± SEM (n = 3). * p < 0.05.
Figure 4
Figure 4
Labeling of FLSs in SM, and cell viability detection for isolated FLSs and chondrocytes. (A) FLSs and chondrocytes obtained from SM and AC of rats, respectively. (B) Observation of FLSs and chondrocytes using light microscopy. (C) FCM was used to determine the viability of FLSs and chondrocytes. (D) Vimentin IHC staining of SM. (E) Vimentin IF staining of SM.
Figure 5
Figure 5
Characterization of the synoviocytes and chondrocytes. (A) Vimentin IF staining of FLSs: (a) test cells; (b) isotype controls. (B) The surface markers were analyzed using FCM: (a) the synoviocyte surface markers of CD90 (FLSs), CD68 (macrophages), and CD3 (T cells); (b) the MSC surface markers of CD29 and CD105. (C) Characterization of the chondrocytes using toluidine blue staining. (D) Characterization of the chondrocytes with COLII immunofluorescence analysis: (a) test cells; (b) isotype controls.
Figure 6
Figure 6
The ability of exosomes to transport miRNAs. miRNA transport in the transwell-based in vitro OA model. (a) FLSs were transfected with a miR-150-3p mimic labeled with Cy3 (red fluorescence) and co-cultured with chondrocytes. (b) FLSs were treated with Cy3 alone. (c) GW4869 inhibition of EV formation.
Figure 7
Figure 7
The identification FLS–EVs and uptake of FLS–EVs by chondrocytes. (A) TEM observation of the EV morphology. (B) NTA measuring of the EV particle distribution and concentration. (C) WB analysis of the EV markers CD9, TSG101, and HSP70. (D) Representative immunofluorescence images of chondrocytes at various time points, showing the uptake of PKH67 (green)-labeled EVs. Hoechst (blue) staining was performed to label the nuclei.
Figure 8
Figure 8
The mechanism underlying the effect of EVs-150 on chondrocyte repair. (A) qPCR analysis of the miR-150-3p expression in chondrocytes treated with H-FLS–EVs or GW4869. (B) qPCR analysis of the COLII and ACAN mRNA levels in chondrocytes upon H-FLS–EV treatment. (C) The COLII and ACAN protein levels in chondrocytes using WB upon H-FLS–EV treatment. (D) ELISA-based quantitation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the chondrocyte culture medium. (E) qPCR analysis of Trim14, NF-κB, and IFN-β mRNA levels. (F) WB analysis of Trim14, NF-κB, and IFN-β protein levels. The data are expressed as mean ± SEM from three independent experiments. **** p < 0.00001, *** p < 0.0001, ** p < 0.01, * p < 0.05.
Figure 9
Figure 9
Validation of the miR-150-3p target and the regulatory effect of miR-150-3p on the innate immune signaling and joint homeostasis. (A) A miR-150-3p binding site on Trim14 and validation of miR-150-3p targeting Trim14 through the dual luciferase assay. (B,C) COLII, ACAN, Trim14, NF-κB, and IFN-β protein levels were analyzed using WB. (D) qPCR analysis of the Trim14, NF-κB, IFN-β, COLII, and ACAN mRNA levels in chondrocytes after miR-150-3p interference. (E) ELISA-based quantitation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the chondrocyte culture medium. The data are expressed as mean ± SEM from three independent experiments. **** p < 0.00001, *** p < 0.0001, ** p < 0.01, * p < 0.05.
Figure 10
Figure 10
In vivo tracking and role of H-FLS–EVs in the joint homeostasis. (A) In vivo EV tracking using live imaging. (B) Lequesne index of rats’ behavior. (C) ELISA-based quantitation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in serum (a) and synovial fluid (b). (D) Histological analysis of EV-treated AC using Masson’s staining. The data are expressed as mean ± SEM from three independent experiments. **** p < 0.00001, *** p < 0.0001, ** p < 0.01, * p < 0.05; ns, not significant.
Figure 11
Figure 11
Regulatory effect of EVs-150 on AC protection in vivo. (A) Observation of the levels and localization of COLII and ACAN in the AC using IF staining. (B) qPCR analysis of the miR-150-3p expression in the serum EVs after EV treatment. (C) qPCR analysis of the COLII and ACAN mRNA levels in the AC after EV treatment. (D) The COLII and ACAN protein levels were analyzed in the AC using WB after EV treatment. The data are expressed as mean ± SEM from three independent experiments. **** p < 0.00001, *** p < 0.0001, ** p < 0.01, * p < 0.05.
Figure 12
Figure 12
Regulatory effect of EVs-150 on the Trim14/NF-κB/IFN-β axis in vivo. (A) The levels and localization of Trim14 and NF-κB in the AC were observed through IF staining. (B) qPCR analysis of the Trim14, NF-κB, and IFN-β mRNA levels in the AC after EV treatment. (C) After EV treatment, the Trim14, NF-κB, and IFN-β protein levels in the AC were analyzed using WB. The data are expressed as mean ± SEM (n = 3) from three independent experiments. **** p < 0.00001, *** p < 0.0001, ** p < 0.01, * p < 0.05.
Figure 13
Figure 13
Therapeutic mechanisms of FLS-derived EVs-150. EVs released by the FLSs in the healthy SM were collected, and these EVs could deliver miR-150-3p to chondrocytes. When OA occurs, Trim14 is recruited to the IKK complex by binding to NEMO, a large multi-unit complex that consists of two catalytic subunits (IKKα and IKKβ) and the regulatory subunit IKKγ (which is also known as NEMO, an essential modulator of NF-κB expression). IKK promotes the phosphorylation of IκBα and p65 as well as NF-κB activation and IFN-β expression. After the immune response was activated, the primary pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were expressed, and the various cytokines and chemokines were synthesized and released. Some of these inflammatory mediators were detected in the joint tissues and synovial fluid of OA rats. These mediators can accelerate chondrocyte degradation and metabolism, thereby injuring the joint and causing a series of clinical symptoms. EVs-150 from healthy FLSs could regulate the mRNA level of the target gene Trim14, thereby effectively inhibiting the activation of the positive feedback loop formed by the Trim14/NF-κB/IFN-β axis of the innate immune response, consequently exerting a suppressive effect on the OA processes indicated above.
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References

    1. Sacitharan P.K. Biochemistry and Cell Biology of Ageing: Part II Clinical Science. Volume 91. Springer; Singapore: 2019. Ageing and Osteoarthritis; pp. 123–159. Subcellular Biochemistry. - PubMed
    1. Hawker G.A. Osteoarthritis is a serious disease. Clin. Exp. Rheumatol. 2019;37((Suppl. 120)):3–6. - PubMed
    1. Hermann W., Lambova S., Muller-Ladner U. Current Treatment Options for Osteoarthritis. Curr. Rheumatol. Rev. 2018;14:108–116. doi: 10.2174/1573397113666170829155149. - DOI - PubMed
    1. Mandl L.A. Osteoarthritis year in review 2018: Clinical. Osteoarthr. Cartil. 2019;27:359–364. doi: 10.1016/j.joca.2018.11.001. - DOI - PubMed
    1. Saviola G., Ferrari P., Niccolò E., Casabella A., Ghellere F., Bonazzi S., Lul A.-A., Comini L., Molfetta L. Use of clodronate for painful knee prosthesis in osteoarthritis patients: A 6-month pilot study. Minerva Med. 2020;111:551–559. doi: 10.23736/S0026-4806.20.06706-3. - DOI - PubMed

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Grants and funding

This project was supported by grants from the National Natural Science Foundation of China (Youth Program) (No.81804214), and the Beijing Excellent Talent Training Funding Project (No.2018000052580G469).