Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice

Altered mechanical loading is a widely suggested, but poorly understood potential cause of cartilage degeneration in osteoarthritis. In rodents, osteoarthritis is induced following destabilization of the medial meniscus (DMM). This study estimates knee kinematics and contact forces in rats with DMM to gain better insight into the specific mechanisms underlying disease development in this widely-used model. Unilateral knee surgery was performed in adult male Sprague-Dawley rats (n=5 with DMM, n=5 with sham surgery). Radio-opaque beads were implanted on their femur and tibia. 8 weeks following knee surgery, rat gait was recorded using the 3D²YMOX setup (Sanctorum et al. 2019, simultaneous acquisition of biplanar XRay videos and ground reaction forces). 10 trials (1 per rat) were calibrated and processed in XMALab (Knörlein et al. 2016). Hindlimb bony landmarks were labeled on the XRay videos using transfer learning (Deeplabcut, Mathis et al. 2019; Laurence-Chasen et al. 2020). A generic OpenSim musculoskeletal model of the rat hindlimb (Johnson et al. 2008) was adapted to include a 3-degree-of-freedom knee. Inverse kinematics, inverse dynamics, static optimization of muscle forces, and joint reaction analysis were performed. In rats with DMM, knee adduction was lower compared to sham surgery. Ground reaction forces were less variable with DMM, resulting in less variability in joint external moments. The mediolateral ground reaction force was lower, resulting in lower hip adduction moment, thus less force was produced by the rectus femoris. Rats with DMM tended to break rather than propel, resulting in lower hip flexion moment, thus less force was produced by the semimembranosus. These results are consistent with lower knee contact forces in the anteroposterior and axial directions. These preliminary data indicate no overloading of the knee joint in rats with DMM, compared with sham surgery. We are currently expanding our workflow to finite element analysis, to examine mechanical cues in the cartilage of these rats (Fig1G)

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA. Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CRE. ERT2. ;Piezo1. fl/fl. Piezo2. fl/fl. ) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria. Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology. Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis. PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks. PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A. Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA

Introduction and Objective. Osteoarthristis (OA) has been associated with many genes and yet the genetic basis for this disease has never formally been established. Recent realization that epigenetic changes could be the underlying pathological mechanisms has helped to explain many complex multifactorial diseases with no clear genetic cause. We therefore asked whether epigenetics could also play a role in OA. We have previously shown that the DNA epigenetic modification, specifically the hydroxymethylation on cytosine (5hmC), undergoes a fivefold increase on OA-associated genes which are activated at OA onset. In this study, we further uncovered a set of 5hmC-mediated gene targets and their mechanistic link to OA progression. Materials and Methods. We surgically induced OA on 4 to 6 months old Tet1−/− mice (Tet1tm1.1Jae, the Jackson laboratory) and wild-type littermates by performing destabilization of the medial meniscus (DMM) surgery. Joints were collected for histological assessment through blinded grading with the OARSI scoring system. Human articular chondrocytes were harvested from OA cartilage samples obtained during total knee arthroplasty or from grossly normal cartilage pieces obtained during notchplasty or debridement from patients undergoing anterior cruciate ligament (ACL) reconstruction with no history of OA symptoms, under approved Human subjects Institutional Review Board protocols. Bioinformatic analyses of RNA-sequencing and CCGG sequencing (reduced representation 5hmC profiling) were performed to identify TET1 target genes associated with OA progression. Several measurements were used to assess the effect of TET1 ablation on the phenotype of mouse cartilage tissue and human chondrocytes including, histological evaluation, and quantitative bone assessment by micro-CT imaging and multiplex cytokine analyses in the serum of mice in vivo (mouse 39-plex assay) and in the supernatant of human chondrocyte cultures (human 62-plex assay). Results. We used a mouse model with surgically induced OA and found that OA onset was accompanied by a gain of ∼40,000 differentially hydroxymethylated sites prior the notable histological onset of the disease. We additionally revealed that these changes are mediated by the ten-eleven-translocation enzyme 1 (TET1), since Tet1−/− mice lost 98% of 5hmC sites upon OA induction. Remarkably, Tet1−/− mice were protected from OA development including degeneration of the cartilage surface and osteophyte formation. Silencing of TET1 expression in human OA chondrocytes reduced the expression in a set of genes, which may represent the pathological gene targets that exacerbate OA including MMP3 and MMP13 and several inflammatory cytokines. Therefore, our study reveals the unexpected beneficial role of TET1 inhibition in blocking OA progression. In fact, intra-articular injections of a dioxygenases’ inhibitor, 2 hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Furthermore, treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss, implicating a therapeutic potential of TET inhibition in OA patients. Conclusions. Collectively, our study not only demonstrate the role of TET1 in OA; the 5hmC-mediated gene targets acting on multiple OA pathways were identified and can be modulated as therapeutic intervention to treat OA

Although osteoarthritis (OA) is characterized by articular cartilage damage, synovial inflammation is a prominent feature contributing to disease progression. In addition to synovial tissue resident macrophages, infiltrating macrophages and monocytes, their lineage precursors, may also contribute to pathological processes. In mice, peripheral blood monocytes may be categorized according to pro-inflammatory/classical and patrolling/non-classical subsets. The aim of this study was to identify profiles of peripheral blood monocyte subsets as well as different synovial macrophage phenotypes during disease development. OA was induced in knees of C57BL/6 mice by destabilization of the medial meniscus (DMM). Blood was harvested from the facial vein 7 days prior to and 1, 7, 14, 28, and 56 days post induction of OA. Separate mice were sham-operated as a control. Monocyte subsets and synovial macrophage populations were identified by flow cytometry. Levels of classical monocytes were significantly higher at day 14 (p<0.001) and day 28 (p=0.031) in peripheral blood of DMM-operated mice compared to control. Furthermore, the percentage of non-classical monocytes was significantly lower in DMM-mice at day 14 (p=0.026). At day 56 post OA-induction, an increase in total synovial macrophages (CD11b+F4/80+ cells) was observed between DMM and sham operated knees (p=0.021). The ratio between pro-inflammatory (CD11b+F4/80+CD86+) and tissue repair (CD11b+F4/80+CD206+) synovial macrophage subsets tended to be higher in DMM knees, however this finding was not statistically significant (p>0.05). In light of the present findings, further investigation is required to elucidate the relationship of peripheral blood monocyte subsets to synovial inflammation and features of OA pathogenesis

Osteoarthritis (OA) is no longer considered a cartilage-centric disease with remodelling of other joint tissues now recognized. While understudied, entheseal pathology is considered a secondary OA feature. A pivotal role for proteinase-activated receptor 2 (PAR2) in OA has been demonstrated previously in cartilage and subchondral bone at early time points, however the entheseal role of PAR2 has not been reported. OA was induced by destabilization of the medial meniscus (DMM) in wild type (WT) and PAR2 deficient (KO) animals. At 4 weeks and one year post surgery, knee joints were harvested for histological analysis. Medial collateral ligament (MCL) width was measured by 2D planimetry analysis. Immunohistochemistry was used to characterize the MCL and anterior cruciate ligament (ACL). Data were expressed as mean±SEM (n=4–6/group) and analysed using Student's t-test, with p<0.05 as the criterion of significance. MCL width increased between 4 weeks and 1 year in WT DMM (0.24 ±0.07 vs 0.40 ±0.008mm respectively, p<0.001). Interestingly, a significant reduction in MCL was observed in KO compared with WT at 1 year (0.23 ±0.005 vs 0.40 ±0.008mm respectively, p <0.001) post-DMM. Further characterization of DMM WT MCL and ACL at 4 weeks showed the presence of F4/80. +. cells in addition to IL-33 and histamine. At one year post-surgery, a cellular infiltrate was observed in MCL DMM WT but absent in KO mice. Histological evaluation revealed an absence of F4/80. +. cells but the presence of a PAR2. +. population, subsequently identified as hypertrophic-like chondrocytes (RUNX2) and chondrocytes-like cells (SOX9). Deletion of PAR2 affords long-term protection against ligament remodelling and demonstrates a critical role for this receptor in both OA joint pathology and ligament injuries. While PAR2 appears to be a credible therapeutic target in OA entheseal pathology, further understanding of the molecular mechanism regulated by this receptor will be required

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by inflammation, degradation of the articular cartilage and subchondral bone lesions, causing pain and decreased functionality. NF-κB pathway is involved in OA and, in most cases, its activation depends on the phosphorylation and degradation of IκBα, the NF-κB endogenous inhibitor that sequesters NF-κB in the cytosol. Under inflammatory stimuli, IκBα is degraded by the IKK signalosome and NF-κB moves into the nucleus, inducing the transcription of inflammatory mediator genes and catabolic enzymes. The IKK signalosome includes IKKβ and IKKα kinases, the latter shown to be pivotal in the OA extracellular matrix derangement. The current OA therapies are not curative and nowadays, the preclinical research is evaluating new structure-modifying pharmacological treatments, able to prevent or delay cartilage degradation. N-acetyl phenylalanine derivative (NAPA), is a derivative of glucosamine, a constituent of the glycosaminoglycans of cartilage and a chondroprotective agent. Previous in vitro studies showed the ability of NAPA to increase cartilage components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity and its nuclear migration. 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). Mice were divided into 3 groups:. -. DMM group: DMM surgery without NAPA;. -. DMM+NAPA group: DMM surgery with NAPA treatment;. -. NO DMM group: no DMM surgery. DMM surgery was performed in the right knee, according to Glasson SS [2], while the left knee did not undergo any surgery. Four weeks after surgery (mild-to-moderate OA), some animals received one intra-articular injection of NAPA (2.5 mM) and after 2 weeks, the animals were pharmacologically euthanized. The mice of the 1. st. group were euthanized 4 weeks after DMM and those of the 3. rd. group after 6 weeks from their arrival in the animal facility. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and subchondral bone microhardness. The injection of NAPA significantly improved cartilage structure, increased cartilage thickness (p<0.0005), reduced Chambers and Mankin scores (p<0.005), fibrillation index (p<0.005) and decreased MMP13 (p<0.05) and ADAMTS5, MMP10, and IKKα (p<0.0005) staining. The microhardness measurements did not shown statistically significant differences between groups. This study demonstrated the chondroprotective activities exerted by NAPA in vivo. NAPA markedly improved the physical structure of articular cartilage and reduced the amount of catabolic enzymes, and therefore of extracellular matrix remodeling. The reduction in OA grading and catabolic enzymes paralleled the reduction of IKKα expression. This further hints at a pivotal role of IKKα in OA development by regulating MMP activity through the control of procollagenase (MMP10) expression. We believe that the preliminary preclinical data, here presented, contribute to improve the knowledge on the development of disease modifying drugs since we showed the ability of NAPA of reverting the surgically induced OA in the widely accepted DMM model