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Abstract Abnormal subchondral bone remodeling plays a pivotal role in the progression of osteoarthritis (OA). Here, we analyzed subchondral bone samples from OA patients and observed a significant upregulation of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) specifically in subchondral bone osteoclasts. Notably, we found a strong correlation between UCHL1 expression and osteoclast activity in the subchondral bone during OA progression in both human and murine models. Conditional UCHL1 deletion in osteoclast precursors exacerbated OA progression, while its overexpression, mediated by adeno-associated virus 9, alleviated this process in male mice. Mechanistically, RANKL stimulates UCHL1 expression in osteoclast precursors, subsequently stabilizing CD13, augmenting soluble CD13 (sCD13) release, and triggering an autocrine inhibitory effect on the MAPK pathway, thereby suppressing osteoclast formation. These findings unveil a previously unidentified negative feedback loop, RANKL-UCHL1-sCD13, that modulates osteoclast formation and presents a potential therapeutic target for OA.

Abstract Infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA), but the underlying mechanism remains unclear. Here, it is find that IPFP from OA patients can secret small extracellular vesicles (sEVs) and deliver them into articular chondrocytes. Inhibition the release of endogenous osteoarthritic IPFP‐sEVs by GW4869 significantly alleviated IPFP‐sEVs‐induced cartilage destruction. Functional assays in vitro demonstrated that IPFP‐sEVs significantly promoted chondrocyte extracellular matrix (ECM) catabolism and induced cellular senescence. It is further demonstrated that IPFP‐sEVs induced ECM degradation in human and mice cartilage explants and aggravated the progression of experimental OA in mice. Mechanistically, highly enriched let‐7b‐5p and let‐7c‐5p in IPFP‐sEVs are essential to mediate detrimental effects by directly decreasing senescence negative regulator, lamin B receptor (LBR). Notably, intra‐articular injection of antagomirs inhibiting let‐7b‐5p and let‐7c‐5p in mice increased LBR expression, suppressed chondrocyte senescence and ameliorated the progression of experimental OA model. This study uncovers the function and mechanism of the IPFP‐sEVs in the progression of OA. Targeting IPFP‐sEVs cargoes of let‐7b‐5p and let‐7c‐5p can provide a potential strategy for OA therapy.

Abstract Inflammatory cytokines-induced activation of the nuclear factor κB (NF-κB) pathway plays a critical role in the pathogenesis of osteoarthritis (OA). Circular RNA (circRNA) has been identified as important epigenetic factor in numerous diseases. However, the biological roles of inflammation-related circRNAs in regulating OA pathogenesis remain elusive. Here, we revealed circRNA expression profiles in human primary chondrocytes with interleukin-1β (IL-1β) stimulation by circRNA sequencing. We identified a highly upregulated circRNA, termed as circNFKB1 in inflamed chondrocytes and osteoarthritic cartilage. As a circRNA derived from exon 2–5 of NFKB1, circNFKB1 is located in both cytoplasm and nucleus of chondrocytes. Furthermore, knockdown of circNFKB1 inhibited extracellular matrix (ECM) catabolism and rescued IL-1β impaired ECM anabolism whereas ectopic expression of circNFKB1 significantly promoted chondrocytes degradation in vitro. Moreover, intraarticular injection of adenovirus-circNFKB1 in mouse joints triggered spontaneous cartilage loss and OA development. Mechanistically, circNFKB1 interacted with α-enolase (ENO1), regulated the expression of its parental gene NFKB1 and sustained the activation of NF-κB signaling pathway in chondrocytes. Therefore, this study highlights a novel ENO1-interacting circNFKB1 in OA pathogenesis, and provides valuable insights into understanding the regulatory mechanism of NF-κB signaling in chondrocytes and a promising therapeutic target for the treatment of OA.

Osteoarthritis (OA) is the most prevalent musculoskeletal disease characterized by multiple joint structure damages, including articular cartilage, subchondral bone and synovium, resulting in disability and economic burden. Bone marrow lesions (BMLs) are common and important magnetic resonance imaging (MRI) features in OA patients. Basic and clinical research on subchondral BMLs in the pathogenesis of OA has been a hotspot. New evidence shows that subchondral bone degeneration, including BML and angiogenesis, occurs not only at or after cartilage degeneration, but even earlier than cartilage degeneration. Although BMLs are recognized as important biomarkers for OA, their exact roles in the pathogenesis of OA are still unclear, and disputes about the clinical impact and treatment of BMLs remain. This review summarizes the current basic and clinical research progress of BMLs. We particularly focus on molecular pathways, cellular abnormalities and microenvironmental changes of subchondral bone that contributed to the formation of BMLs, and emphasize the crosstalk between subchondral bone and cartilage in OA development. Finally, potential therapeutic strategies targeting BMLs in OA are discussed, which provides novel strategies for OA treatment.

Abstract Introduction Osteoarthritis (OA) is the most common joint disorder in the world but there are no approved therapies to slow disease progression. Historically, OA has been considered a wear and tear joint disease and efforts to identify and develop disease modifying therapies have primarily focused on inhibiting cartilage degradation directly. However, increasing studies have suggested that inflammation is a key player of joint pathology in some OA patients and also a link between obesity and OA. Therefore, targeting inflammation in these patients could be a promising therapeutic strategy. Areas Covered In this review, we collate the latest evidence on the relationship between inflammation and OA which allows to propose the inflammatory phenotype of OA, and summarize major results of some emerging therapies targeting this phenotype in clinical trials which could implicate future directions for OA treatment. Expert Opinion The authors believe early diagnosis, phenotyping OA patients and precise therapies would expedite the development of DMOADs targeting OA inflammatory phenotype. In the future, well‐designed clinical trials including appropriate subgroups or phenotypes of patients according to underlying causes and mechanisms of OA are expected for investigational therapies.

A role for ferroptosis in articular cartilage destruction associated with rheumatoid arthritis (RA) has not been identified. We previously reported transient receptor potential melastatin 7 (TRPM7) expression was correlated with RA cartilage destruction. Herein, we further characterized a role for TRPM7 in chondrocyte ferroptosis. The expression of TRPM7 was found to be elevated in articular chondrocytes derived from adjuvant arthritis (AA) rats, human RA patients, and cultured chondrocytes treated with the ferroptosis inducer, erastin. TRPM7 knockdown or pharmacological inhibition protected primary rat articular chondrocytes and human chondrocytes (C28/I2 cells) from ferroptosis. Moreover, TRPM7 channel activity was demonstrated to contribute to chondrocyte ferroptosis by elevation of intracellular Ca2+. Mechanistically, the PKCα-NOX4 axis was found to respond to stimulation with erastin, which resulted in TRPM7-mediated chondrocyte ferroptosis. Meanwhile, PKCα was shown to directly bind to NOX4, which could be reduced by TRPM7 channel inhibition. Adeno-associated virus 9-mediated TRPM7 silencing or TRPM7 blockade with 2-APB alleviated articular cartilage destruction in AA rats and inhibited chondrocyte ferroptosis. Collectively, both genetic and pharmacological inhibitions of TRPM7 attenuated articular cartilage damage and chondrocyte ferroptosis via the PKCα-NOX4 axis, suggesting that TRPM7-mediated chondrocyte ferroptosis is a promising target for the prevention and treatment of RA.

Abstract Background Mesenchymal stem cells (MSCs) have been recognized as a promising tool for the treatment of various inflammatory disorders and autoimmune diseases. Stress conditions affect immune-mediated treatment and activate autophagy in MSCs. However, whether autophagy affects the MSC-mediated recruitment and differentiation of CD4+ T cells remains elusive. Methods MSCs were pretreated with 3-methyladenine (3-MA) and rapamycin to regulate autophagy, and then co-cultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of well-known chemokines were analyzed by quantitative real-time PCR. Enzyme-linked immunosorbent assays and western blot analysis were performed to detect C-X-C motif chemokine ligand 8 (CXCL8) and transforming growth factor (TGF)-β1 protein levels. An exogenous antibody and short hairpin RNA were used to regulate CXCL8 and TGF-β1 levels, which enabled us to evaluate how autophagy affected MSC-mediated CD4+ T cell migration and differentiation. Results 3-MA inhibited autophagy in MSCs, which was activated by rapamycin. Rapamycin increased the migration of CD4+ T cells, whereas 3-MA decreased their migration. Mechanistically, we found that autophagy strengthened CXCL8 secretion, and the addition of exogenous CXCL8 and an anti-CXCL8 antibody eliminated the difference of CD4+ T cell migration among groups. Further, the ratio of regulatory T (Treg) cells was increased in rapamycin-pretreated MSCs, but the ratio of T helper 1 (Th1) cells was decreased, while pretreatment of MSCs with 3-MA induced the opposite effect compared with the control group. TGF-β1 overexpression and knockdown using lentiviruses rectified the differences in the ratios of Treg and Th1 cells among the groups. Conclusion This study demonstrates that autophagy of mesenchymal stem cells mediates CD4+ T cell migration and differentiation through CXCL8 and TGF-β1, respectively. These results provide a potential new strategy for improving MSC-mediated therapy.

Abstract Background The goals of this study were to explore the expression profiles and functional networks of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in mesenchymal stromal cells (MSCs) involved in regulating the function of monocytes and to clarify the mechanisms by which MSCs exert immunoregulatory effects on monocytes. Methods MSCs and CD14+ monocytes were separately isolated. The immunoregulatory effects of MSCs on monocytes were determined by flow cytometry. lncRNAs and mRNAs that were differentially expressed (DE) between the control group (MSCs only) and co-culture group (MSCs co-cultured with monocytes) were identified through high-throughput sequencing and bioinformatic analyses and were confirmed by qRT-PCR. Bioinformatic analyses were performed to identify the critical biological functions and signalling pathways involved in MSC-mediated monocyte regulation and to identify the functional networks formed between DE mRNAs and lncRNAs. Results MSCs showed a strong ability to induce monocyte migration but inhibited monocyte differentiation into M1 macrophages. A total of 145 DE lncRNAs and 768 DE mRNAs were identified between the control and co-culture groups. Significant fold changes in lncRNAs and mRNAs were confirmed by qRT-PCR. GO analysis demonstrated that DE mRNAs and lncRNAs were highly associated with terms related to binding and biological regulation. KEGG analysis revealed 122 significantly regulated pathways, including the cytokine-cytokine receptor pathway and chemokine signalling pathway. Interaction and co-expression networks were constructed for DE mRNAs and lncRNAs, and several key microRNAs were identified in the competitive endogenous RNA (ceRNA) network. Target genes of the DE lncRNAs were analysed to predict critical mRNA-lncRNA axes involved in the immunoregulatory function of MSCs. Conclusions Our research describes the lncRNA and mRNA expression profiles and functional networks involved in MSC-mediated regulation of monocytes. These results provide possible molecular mechanisms for the immunoregulatory function of MSCs and may help to elucidate possible molecular therapeutic targets in MSCs for the treatment of autoimmune diseases.

Background: Osteoarthritis (OA), a disease with whole-joint damage and dysfunction, is the leading cause of disability worldwide. The progressive loss of hyaline cartilage extracellular matrix (ECM) is considered as its hallmark, but its exact pathogenesis needs to be further clarified. MicroRNA(miRNA) contributes to OA pathology and may help to identify novel biomarkers and therapies against OA. Here we identified miR-214–3p as an important regulator of OA. Methods: qRT-PCR and in situ hybridization were used to detect the expression level of miR-214–3p. The function of miR-214–3p in OA, as well as the interaction between miR-214–3p and its downstream mRNA target (IKBKB), was evaluated by western blotting, immunofluorescence, qRT-PCR and luciferase assay. Mice models were introduced to examine the function and mechanism of miR-214–3p in OA in vivo. Findings: In our study, we found that miR-214–3p, while being down-regulated in inflamed chondrocytes and OA cartilage, regulated ECM metabolism and cell apoptosis in the cartilage. Mechanically, the protective effect of miR-214–3p downregulated the IKK-β expression and led to the dysfunction of NF-κB signaling pathway. Furthermore, intra-articular injection of miR-214–3p antagomir in mice joints triggered spontaneous cartilage loss while miRNA-214–3p agomir alleviated OA in the experimental mouse models. Interpretation: Decreased miR-214–3p activates the NF-κB signaling pathway and aggravates OA development through targeting IKKβ, suggesting miR-214–3p may be a novel therapeutic target for OA. Funding: This study was financially supported by grants from the National Natural Science Foundation of China (81,773,532, 81,974,342).

Osteoporosis is a common systemic skeletal disorder resulting in bone fragility and increased fracture risk. It is still necessary to explore its detailed mechanisms and identify novel targets for the treatment of osteoporosis. Previously, we found that a lncRNA named GAS5 in human could negatively regulate the lipoblast/adipocyte differentiation. However, it is still unclear whether GAS5 affects osteoblast differentiation and whether GAS5 is associated with osteoporosis. Our current research found that GAS5 was decreased in the bones and BMSCs, a major origin of osteoblast, of osteoporosis patients. Mechanistically, GAS5 promotes the osteoblast differentiation by interacting with UPF1 to degrade SMAD7 mRNA. Moreover, a decreased bone mass and impaired bone repair ability were observed in Gas5 heterozygous mice, manifesting in osteoporosis. The systemic supplement of Gas5-overexpressing adenoviruses significantly ameliorated bone loss in an osteoporosis mouse model. In conclusion, GAS5 promotes osteoblast differentiation by targeting the UPF1/SMAD7 axis and protects against osteoporosis.

Abstract Background Interleukin-6 (IL-6) with IL-6 receptor (IL-6R) play an important role in the tissue regeneration in vivo, especially bone metabolism. Bone marrow -derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which are main origin of osteoblasts. However, the roles of IL-6 and IL-6R in the osteogenic differentiation of BM-MSCs are still unclear. Methods The expression of IL-6 and IL-6R was detected in BM-MSCs during osteogenic differentiation. The activation of the STAT3 pathway was assessed and its role in the osteogenic differentiation of BM-MSCs was determined using the specific inhibitor AG490. Exogenous IL-6/soluble IL-6R or antibodies against IL-6/IL-6R were used to confirm the mechanism by which the IL-6/IL-6R complex promotes the osteogenic differentiation. Results The levels of IL-6 and IL-6R, especially the level of membranous IL-6R but not that of soluble IL-6R, increased during osteogenic differentiation in BM-MSCs. The levels of IL-6 and IL-6R were positively correlated with the osteogenic potential of BM-MSCs. The STAT3 signaling pathway was activated during the osteogenic differentiation of BM-MSCs. AG490 markedly inhibited the activation of the STAT3 pathway and, subsequently, the osteogenic differentiation potential of BM-MSCs. Additionally, exogenous IL-6 and soluble IL-6R accelerated the osteogenic differentiation of BM-MSCs. In contrast, antibodies against IL-6 or IL-6R suppressed the osteogenic differentiation of BM-MSCs. Moreover, IL-6 and IL-6R were found to stimulate each other’s expression in BM-MSCs. Conclusions IL-6 and IL-6R levels increase during the osteogenic differentiation of BM-MSCs. These two molecules form a complex to activate the downstream STAT3 signaling pathway, which promotes osteogenic differentiation in BM-MSCs via an autocrine/paracrine feedback loop.

Extracellular vesicles (Evs) contain diverse functional proteins, mRNAs, miRNAs, and DNA fragments, are secreted by various types of cells, and play important roles in cellular communication. Here, we show for the first time that plasma Evs inhibited the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro and the level of inhibition was positively correlated with the plasma Evs concentration. Plasma Evs downregulated the expression of markers such as osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), and Osterix at mRNA levels required for osteogenic differentiation and reduced pSmad1/5/8 levels in MSCs. Furthermore, pSmad1/5/8 levels increased and MSCs underwent normal osteogenic differentiation after Evs-derived α2-HS glycoprotein (AHSG) function was inhibited with an anti-AHSG neutralizing antibody. However, the levels of pERK1/2, active β-catenin, and HES1 were not significantly altered. Therefore, we propose that as essential components of the extracellular microenvironment of MSCs, plasma Evs are taken up by MSCs and subsequently repress osteogenic differentiation through an AHSG-mediated decrease in pSmad1/5/8 levels. Our work identifies plasma Evs as novel regulators of MSC osteogenic differentiation.