ß-Ecdysone attenuates cartilage damage in a mouse model of collagenase-induced osteoarthritis via mediating FOXO1/ADAMTS-4/5 signaling axis.

BACKGROUND: ß-Ecdysone has been reported to perform a protective effect to prevent interleukin 1ß (IL-1ß)-induced apoptosis and inflammatory response in chondrocytes. In our study, the chondroprotective effects of ß-Ecdysone were explored in a mouse model of collagenase-induced osteoarthritis (OA). METHODS: Injection of collagenase in the left knee was implemented to establish a mouse model of OA. The histomorphological analysis was detected using safranine O staining. Serum pro-inflammatory cytokines were measured by ELISA assays. Protein expression in the femur and chondrocytes was analyzed using western blot. Chondrocyte apoptosis was evaluated by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining. RESULTS: Treatment of OA mice with ß-Ecdysone supplementation significantly inhibited the production of pro-inflammatory cytokines. Histologic examination exhibited that the degradation of proteoglycans and the loss of trabecular bone were observed in collagenase-injected mice. However, OA-like changes were attenuated by ß-Ecdysone administration in collagenase-injected mice. Both in vivo and in vitro models, nuclear forkhead box O1 (FOXO1) protein expression was significantly reduced in the femur of collagenase-treated mice and IL-1ß-stimulated chondrocytes. However, ß-Ecdysone treatment was able to rescue FOXO1 protein expression in the nucleus to inhibit the transcription and translation of a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4) and ADAMTS-5. CONCLUSION: The findings suggested that ß-Ecdysone functioned as a FOXO1 activator to protect collagenase-induced cartilage damage. FOXO1 might be a potential molecular target of ß-Ecdysone for the effective prevention and treatment of OA.

Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors.

OBJECTIVE: Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. METHODS: Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. RESULTS: Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. CONCLUSION: TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Effects of Anti-Inflammatory Agents on Expression of Early Responsive Inflammatory and Catabolic Genes in Ex Vivo Porcine Model of Acute Knee Cartilage Injury.

Objective Early intervention therapies targeting inflammation and cell death during the acute phase of cartilage injury have the potential to prevent posttraumatic osteoarthritis. The objective of this study was to investigate the effects of interleukin receptor antagonist protein (IRAP), hyaluronan (HA), dexamethasone (DEX), and mesenchymal stem cell (MSC) treatment on the expression of established genetic markers for matrix degradation, apoptosis, and inflammation in articular cartilage during the acute phase of injury. Design A custom impact device was used to create replicable injury ex vivo to intact porcine knee joint. One hour after impact, IRAP, HA, DEX, or MSCs was intra-articularly injected. At 8 hours postinjury, cartilage and meniscus samples were harvested for genetic expression analysis. Expression of miR-27b, miR-140, miR-125b, miR-16, miR-34a, miR-146a, miR-22, ADAMTS-4, ADAMTS-5, MMP-3, IL-1ß, and TNF-α was analyzed by real-time polymerase chain reaction. Results At 8 hours postinjury, expression of ADAMTS-4, ADAMTS-5, MMP-3, IL-1ß, and TNF-α in cartilage was significantly decreased in IRAP- and DEX-treated joints as compared to nontreated injured joints, whereas only IRAP upregulated expression of miR-140, miR-125b, miR-27b, miR-146a, and miR-22 in cartilage. HA and MSC treatments had no significant effects on catabolic and inflammatory gene expression in cartilage. However, HA treatment significantly upregulated expression of all miRNAs except miR-16. In addition, the treatments tested also exhibited significant influences on meniscus. Conclusions This study provides a valuable starting point for further research into potential targets for and efficacy of various early intervention strategies that may delay or prevent the progression of posttraumatic osteoarthritis after acute cartilage injury.

Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis.

OBJECTIVE: To downregulate the expression of leptin receptor functional isoform (Ob-Rb) on chondrocytes using lentiviral vector-mediated short-hairpin RNA (LV-shRNA) and to determine its effects on cartilage degeneration. METHOD: In vitro, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to select an optimal Ob-Rb LV-shRNA (LV-shRNA3) and to determine its effects on nine OA-related mediators in cultured rat chondrocytes. In vivo, an OA model was surgically induced in the right knees of rats, and LV-shRNA3, lentiviral vector-mediated non-targeting control sequence (LV-NTC) or phosphate buffered saline was injected into the joints. Osteoarthritis Research Society International (OARSI) scoring was performed to assess cartilage degeneration, and immunohistochemistry was performed to evaluate OA-related mediator expression in the above groups. RESULTS: Ob-Rb expression was significantly downregulated by LV-shRNA3 in cultured chondrocytes. In conjunction with Ob-Rb downregulation, the expression levels of pro-inflammatory mediators (TNF-α, IL-1ß and IL-6) and catabolic mediators (ADAMTS-5, MMP-9, NOS-2 and COX-2) were also significantly decreased, and the expression levels of anabolic type II collagen were significantly increased. The in vivo study results showed that OARSI scores were significantly decreased by LV-shRNA3. Immunohistochemistry analysis demonstrated that Ob-Rb expression levels on chondrocytes were significantly downregulated by LV-shRNA3. In conjunction with Ob-Rb downregulation, ADAMTS-5 and MMP-9 expression levels were also significantly decreased, and type II collagen expression levels were increased. CONCLUSION: These results indicate that LV-shRNA3-mediated Ob-Rb downregulation on chondrocytes inhibits cartilage degeneration in a rat model of OA, suggesting that Ob-Rb may be a novel target in the treatment of OA.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.