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Deer Antler ASCs Exosomes Ameliorate Osteoarthritis via miR-140/MMP13 Axis-Mediated Dual Modulation of Inflammation and Cartilage Regeneration

ABSTRACT: Osteoarthritis (OA) is a progressive degenerative joint disorder characterized by chondrocyte dysfunction and extracellular matrix degradation. While stem cell therapies have been constrained by immunological rejection risks, their derived extracellular vesicles (Exos) emerge as a promising alternative therapeutic approach. Antler, a remarkable regenerative organ with extraordinary regenerative capacity and chondrogenic potential, offers a unique biological resource. Antler Stem Cells (ASCs) provide an innovative extracellular vesicle source (ASC-Exos) that presents a compelling therapeutic strategy for addressing OA's complex pathogenesis. To investigate the therapeutic effects and mechanism of ASC-Exos in OA treatment. ASCs characteristics were confirmed through immunofluorescence and tri-lineage differentiation, while exosomes were validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. A rat OA model was established to evaluate therapeutic effects of ASC-Exos via different administration routes. Small RNA sequencing identified key extracellular vesicle components, while dual-luciferase reporter assays confirmed regulatory interactions between miR-140 and metalloproteinase-13(MMP13). Stable miR-140 overexpressing ASCs were generated through lentiviral transduction. An IL-1β-stimulated chondrocyte OA model was employed to assess the impact of miR-140-engineered ASC-Exos (miR-140-Exos) on cellular proliferation, migration, and apoptosis. The therapeutic potential was further evaluated in in vivo animal models. We successfully isolated and characterized ASCs and ASC-Exos. Intra-articular injection of ASC-Exos demonstrated superior cartilage repair efficacy. The critical extracellular vesicle component miR-140 was identified. In vitro studies revealed that miR-140-Exos significantly enhanced chondrocyte proliferation and migration while reducing cellular apoptosis. Dual-luciferase assays confirmed miR-140 directly targets MMP13, with MMP13 overexpression partially reversing miR-140-mediated cellular effects. In vivo investigations demonstrated that miR-140-modified ASC-Exos effectively inhibited COL II degradation and suppressed NLRP3 elevation, thereby providing anti-inflammatory benefits and promoting cartilage regeneration. miR-140-Exos represent an innovative therapeutic approach for osteoarthritis, effectively promoting cartilage regeneration and alleviating inflammation through MMP13 inhibition, presenting a novel therapeutic strategy for OA treatment.

ORGANISM(S): Cervus nippon

PROVIDER: GSE306940 | GEO | 2025/09/03

REPOSITORIES: GEO

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Project description:OBJECTIVE: To analyze genome-wide changes in chondrocyte gene expression in a surgically induced model of early osteoarthritis (OA) in rats, to assess the similarity of this model to human OA, and to identify genes and mechanisms leading to OA pathogenesis. METHODS: OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression. Expression patterns of selected dysregulated genes, including Col2a1, Mmp13, Adamts5, Ctsc, Ptges, and Cxcr4, were validated by real-time polymerase chain reaction, immunofluorescence, or immunohistochemistry 2, 4, and 8 weeks after surgery. RESULTS: After normalization, comparison of OA and sham-operated samples showed 1,619 differentially expressed probe sets with changes in their levels of expression >/=1.5-fold, 722 with changes >/=2-fold, 135 with changes >/=4-fold, and 20 with changes of 8-fold. Dysregulated genes known to be involved in human OA included Mmp13, Adamts5, and Ptgs2, among others. Several dysregulated genes (e.g., Reln, Phex, and Ltbp2) had been identified in our earlier microarray study of hypertrophic chondrocyte differentiation. Other genes involved in cytokine and chemokine signaling, including Cxcr4 and Ccl2, were identified. Changes in gene expression were also observed in the contralateral knee, validating the sham operation as the appropriate control. CONCLUSION: Our results demonstrate that the animal model mimics gene expression changes seen in human OA, supporting the relevance of newly identified genes and pathways to early human OA. We propose new avenues for OA pathogenesis research and potential targets for novel OA treatments, including cathepsins and cytokine, chemokine, and growth factor signaling pathways, in addition to factors controlling the progression of chondrocyte differentiation. Experiment Overall Design: OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees, 28 days post-surgery. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression.

Project description:Targeting MMP13 directly by RNA interference (RNAi) silenced MMP13 in a murine post-traumatic osteoarthritis (PTOA) and was found to have broad effects on overall joint health, including both the articular cartilage and surrounding hard and soft tissues. To further characterize the global impacts of targeted MMP13 inhibition, the mechanical loading mouse model was applied with samples were harvested at 4 weeks to capture an intermediate stage of disease. The nanoString nCounter Inflammation panel was used to broadly quantify gene expression of the knee joint samples (articular cartilage, meniscal, and synovial tissue). Abstract: Osteoarthritis (OA) is a debilitating and prevalent chronic disease, but there are no approved disease modifying OA drugs (DMOADs), only pharmaceuticals for pain management. OA progression, particularly for post-traumatic osteoarthritis (PTOA), is associated with inflammation and enzymatic degradation of the extracellular matrix. In particular, matrix metalloproteinase 13 (MMP13) breaks down collagen type 2 (CII), a key structural component of cartilage extracellular matrix, and consequently, matrix degradation fragments perpetuate inflammation and a degenerative cycle that leads to progressive joint pathology. Here, we tested extracellular matrix-binding MMP13 RNA interference (RNAi) nanoparticles (NPs) as a DMOAD. The retention approach pursued here deviates from the convention of targeting specific cell types (e.g., through cell surface receptors) and instead leverages a monoclonal antibody (mAbCII) that targets extracellular CII that becomes uniquely accessible in OA-damaged cartilage. CII monoclonal antibody-functionalized nanoparticles carrying small interfering ribonucleic acid (siRNA) (mAbCII-siNPs) create an in situ NP depot for retention and potent activity within OA joints. The mAbCII-siNPs loaded with MMP13 siRNA (mAbCII-siNP/siMMP13) potently suppressed MMP13 expression (95% silencing) in TNFα-stimulated chondrocytes in vitro and had higher binding to trypsin-damaged porcine cartilage than control NPs. In an acute mechanical injury mouse model of PTOA, mAbCII-siNP/siMMP13 achieved 80% reduction in MMP13 expression (p = 0.00231), whereas control siNP/siMMP13 achieved only 55% silencing. In a more severe, longer-term PTOA model, weekly mAbCII-siNP/siMMP13 treatment provided significant protection of cartilage integrity (0.45+/-.3 vs 1.6+/-.5 on the OARSI scale; p=0.0013) and overall joint structure (1.3+/-.6 vs 2.8+/-.2 on the Degenerative Joint Disease scale; p=0.0418), including reduction of osteophyte formation (siMMP13 vs siNEG: 0.088+/-0.074 vs 0.47+/-0.107 mm femoral osteophyte outgrowth; p<0.0001). Multiplexed gene expression analysis of 254 inflammation-related genes showed that MMP13 inhibition suppressed clusters of genes associated with tissue restructuring, angiogenesis, innate immune response, and proteolysis. Finally, in benchmarking studies, intra-articular mAbCII-siNPs better reduced OA disease progression relative to either one time or weekly treatment with the clinical gold standard steroid methylprednisolone. Here, we introduce the concept of anchoring to unique local extracellular matrix signatures to sustain retention and increase delivery efficacy of biologics with intracellular activity and also validates the promise of MMP13 RNAi as a DMOAD in a clinically-relevant therapeutic context.

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Deer Antler ASCs Exosomes Ameliorate Osteoarthritis via miR-140/MMP13 Axis-Mediated Dual Modulation of Inflammation and Cartilage Regeneration

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