Murine Model Of Osteoarthritis Research Articles

Carbonic anhydrase 2 is important for chondrocyte function and metabolic homeostasis

ObjectivesAberrant chondrocyte metabolism significantly contributes to cartilage degeneration and osteoarthritis (OA) genesis. However, the mechanisms driving the metabolic shift in OA chondrocytes remain unclear. Interestingly, carbonic anhydrase 2 (CA2) is implicated in metabolic regulation, and its expression dramatically increases in OA chondrocytes, but its exact role and mechanism are poorly understood. This study investigates the mechanistic role of CA2 in chondrocyte metabolic homeostasis under inflammatory conditions. MethodsRNA-seq was performed on CA2-deficient C28/I2 cells to identify pathways affected by the loss of CA2 function. We examined CA2's impact on chondrocyte metabolism, anabolism, and catabolism using C28/I2 cells and primary chondrocytes under normoxia and hypoxia and in a model of inflammatory OA. ResultsRNA-seq revealed enrichment of glycolysis, apoptosis, and TNF signaling pathways in CA2-deficient cells. Under hypoxia, CA2 expression increased 10-fold in a HIF-1α-independent manner. Knockdown of CA2 reduced extracellular lactate production, increased ADP/ATP ratio, impaired glycolysis, reduced glycolytic capacity, and lowered expression of glycolysis rate-limiting enzymes but did not disrupt pHi and ROS production. CA2 deficiency altered chondrocyte anabolic and catabolic equilibrium by affecting PI3K/AKT and RELA/p65 signaling. Chondrocyte migration was impeded, proliferation suppressed, and the cell cycle arrested at G0/G1 in cells lacking CA2. Forced expression of CA2 stabilized chondrocyte metabolism and restored cellular functions. ConclusionsOur research uncovered a novel mechanistic role for CA2 in regulating chondrocyte energy metabolism and inflammation, underscoring its potential as a critical mediator in OA pathogenesis. Further research using a murine model of experimental OA is warranted to capture the functional implications of CA2.

IRF1 regulation of ZBP1 links mitochondrial DNA and chondrocyte damage in osteoarthritis

BackgroundZ-DNA binding protein 1 (ZBP1) is a nucleic acid sensor that is involved in multiple inflammatory diseases, but whether and how it contributes to osteoarthritis (OA) are unclear.MethodsCartilage tissues were harvested from patients with OA and a murine model of OA to evaluate ZBP1 expression. Subsequently, the functional role and mechanism of ZBP1 were examined in primary chondrocytes, and the role of ZBP1 in OA was explored in mouse models.ResultsWe showed the upregulation of ZBP1 in articular cartilage originating from OA patients and mice with OA after destabilization of the medial meniscus (DMM) surgery. Specifically, knockdown of ZBP1 alleviated chondrocyte damage and protected mice from DMM-induced OA. Mechanistically, tumor necrosis factor alpha induced ZBP1 overexpression in an interferon regulatory factor 1 (IRF1)-dependent manner and elicited the activation of ZBP1 via mitochondrial DNA (mtDNA) release and ZBP1 binding. The upregulated and activated ZBP1 could interact with receptor-interacting protein kinase 1 and activate the transforming growth factor-beta-activated kinase 1-NF-κB signaling pathway, which led to chondrocyte inflammation and extracellular matrix degradation. Moreover, inhibition of the mtDNA-IRF1-ZBP1 axis with Cyclosporine A, a blocker of mtDNA release, could delay the progression of DMM-induced OA.ConclusionsOur data revealed the pathological role of the mtDNA-IRF1-ZBP1 axis in OA chondrocytes, suggesting that inhibition of this axis could be a viable therapeutic approach for OA.

A standardized approach to evaluation and reporting of synovial histopathology in two surgically induced murine models of osteoarthritis

ObjectiveSynovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for reporting of synovial histopathology in mouse models of OA. MethodsCoronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T-Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations. Inter-reader agreement of each feature score was determined. ResultsThere was acceptable to very good agreement when using 3-4 individual readers. After DMM and PMX, expected medial predominant changes in hyperplasia and cellularity were observed, with fibrosis noted at 12 weeks post-PMX. Synovial changes were consistent from section to section in the mid-joint area. When comparing stains, H&E and T-blue resulted in better agreement compared to Saf-O stain. ConclusionsTo account for the pathologic and anatomic variability in synovial pathology and allow for a more standardized evaluation that can be compared across studies, we recommend evaluating a minimum set of 3 pathological features at standardized anatomic areas. Further, we suggest reporting individual feature scores separately before relying on a single summed “synovitis” score. H&E or T-blue are preferred, inter-reader agreement for each feature should be considered.

Intraarticular treatment with integrin α10β1-selected mesenchymal stem cells affects microRNA expression in experimental post-traumatic osteoarthritis in horses.

Osteoarthritis (OA) remains a major cause of lameness in horses, which leads to lost days of training and early retirement. Still, the underlying pathological processes are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that serve as regulators of many biological processes including OA. Analysis of miRNA expression in diseased joint tissues such as cartilage and synovial membrane may help to elucidate OA pathology. Since integrin α10β1-selected mesenchymal stem cell (integrin α10-MSC) have shown mitigating effect on equine OA we here investigated the effect of integrin α10-MSCs on miRNA expression. Cartilage and synovial membrane was harvested from the middle carpal joint of horses with experimentally induced, untreated OA, horses with experimentally induced OA treated with allogeneic adipose-derived MSCs selected for the marker integrin α10-MSCs, and from healthy control joints. miRNA expression in cartilage and synovial membrane was established by quantifying 70 pre-determined miRNAs by qPCR. Differential expression of the miRNAs was evaluated by comparing untreated OA and control, untreated OA and MSC-treated OA, and joints with high and low pathology score. A total of 60 miRNAs were successfully quantified in the cartilage samples and 55 miRNAs were quantified in the synovial membrane samples. In cartilage, miR-146a, miR-150 and miR-409 had significantly higher expression in untreated OA joints than in control joints. Expression of miR-125a-3p, miR-150, miR-200c, and miR-499-5p was significantly reduced in cartilage from MSC-treated OA joints compared to the untreated OA joints. Expression of miR-139-5p, miR-150, miR-182-5p, miR-200a, miR-378, miR-409-3p, and miR-7177b in articular cartilage reflected pathology score. Several of these miRNAs are known from research in human patients with OA and from murine OA models. Our study shows that these miRNAs are also differentially expressed in experimental equine OA, and that expression depends on OA severity. Moreover, MSC treatment, which resulted in less severe OA, also affected miRNA expression in cartilage.

DNA demethylation of promoter region orchestrates SPI-1-induced ADAMTS-5 expression in articular cartilage of osteoarthritis mice.

Osteoarthritis (OA) is one of the most prevalent joint diseases in aged people and characterized by articular cartilage degeneration, synovial inflammation, and abnormal bone remodeling. Recent advances in OA research have clearly shown that OA development is associated with aberrant DNA methylation status of many OA-related genes. As one of most important cartilage degrading proteases in OA, a disintegrin and metalloproteinase with thrombospondin motifs subtype 5 (ADAMTS-5) is activated to mediate cartilage degradation in human OA and experimental murine OA models. The pathological factors and signaling pathways mediating ADAMTS-5 activation during OA development are not well defined and have been a focus of intense research. ADAMTS-5 promoter is featured by CpG islands. So far there have been no reports concerning the DNA methylation status in ADAMTS-5 promoter during OA development. In this study, we sought to investigate DNA methylation status in ADAMTS-5 promoter, the role of DNA methylation in ADAMTS-5 activation in OA, and the underlying mechanisms. The potential for anti-OA intervention therapy which is based on modulating DNA methylation is also explored. Our results showed that DNA methyltransferases 1 (Dnmt1) downregulation-associated ADAMTS-5 promoter demethylation played an important role in ADAMTS-5 activation in OA, which facilitated SPI-1 binding on ADAMTS-5 promoter to activate ADAMTS-5 expression. More importantly, OA pathological phenotype of mice was alleviated in response to Dnmt1-induced DNA methylation of ADAMTS-5 promoter. Our study will benefit not only for deeper insights into the functional role and regulation mechanisms of ADAMTS-5 in OA, but also for the discovery of disease-modifying OA drugson the basis of ADAMTS-5 via modulating DNA methylation status.

Prokineticin System Is a Pharmacological Target to Counteract Pain and Its Comorbid Mood Alterations in an Osteoarthritis Murine Model.

Osteoarthritis (OA) is the most prevalent joint disease associated with chronic pain. OA pain is often accompanied by mood disorders. We addressed the role of the Prokineticin (PK) system in pain and mood alterations in a mice OA model induced with monosodium iodoacetate (MIA). The effect of a PK antagonist (PC1) was compared to that of diclofenac. C57BL/6J male mice injected with MIA in the knee joint were characterized by allodynia, motor deficits, and fatigue. Twenty-eight days after MIA, in the knee joint, we measured high mRNA of PK2 and its receptor PKR1, pro-inflammatory cytokines, and MMP13. At the same time, in the sciatic nerve and spinal cord, we found increased levels of PK2, PKR1, IL-1β, and IL-6. These changes were in the presence of high GFAP and CD11b mRNA in the sciatic nerve and GFAP in the spinal cord. OA mice were also characterized by anxiety, depression, and neuroinflammation in the prefrontal cortex and hippocampus. In both stations, we found increased pro-inflammatory cytokines. In addition, PK upregulation and reactive astrogliosis in the hippocampus and microglia reactivity in the prefrontal cortex were detected. PC1 reduced joint inflammation and neuroinflammation in PNS and CNS and counteracted OA pain and emotional disturbances.

Evaluation of Cartilage Integrity Following Administration of Oral and Intraarticular Nifedipine in a Murine Model of Osteoarthritis.

Osteoarthritis (OA) ranks as the prevailing type of arthritis on a global scale, for which no effective treatments are currently available. Arterial hypertension is a common comorbidity in OA patients, and antihypertensive drugs, such as nifedipine (NIF), may affect the course of OA progression. The aim of this preclinical study was to determine the effect of nifedipine on healthy and OA cartilage, depending on its route of administration. In this study, we used the destabilization of medial meniscus to develop a mouse model of OA. Nifedipine was applied per os or intraarticularly (i.a.) for 8 weeks to both mice with OA and healthy animals. Serum biomarker concentrations were evaluated using the Luminex platform and alterations in the knee cartilage were graded according to OARSI histological scores and investigated immunohistochemically. Nifedipine treatment per os and i.a. exerted protective effects, as assessed by the OARSI histological scores. However, long-term nifedipine i.a. injections induced the deterioration of healthy cartilage. Lubricin, cartilage intermediate layer matrix protein (CILP), collagen type VI (COLVI), CILP, and Ki67 were upregulated by the nifedipine treatment. Serum biomarkers MMP-3, thrombospondin-4, and leptin were upregulated in the healthy groups treated with nifedipine, while only the levels of MMP-3 were significantly higher in the OA group treated with nifedipine per os compared to the untreated group. In conclusion, this study highlights the differential effects of nifedipine on cartilage integrity, depending on the route of administration and cartilage condition.

Injectable photocrosslinking spherical hydrogel-encapsulated targeting peptide-modified engineered exosomes for osteoarthritis therapy

Osteoarthritis (OA) is a common degenerative joint disease urgently needing effective treatments. Bone marrow mesenchymal stromal cell-derived exosomes (Exo) are considered good drug carriers whereas they have limitations such as fast clearance and low retention. This study aimed to overcome the limitations of Exo in drug delivery using multiple strategies. Novel photocrosslinking spherical gelatin methacryloyl hydrogel (GelMA)-encapsulated cartilage affinity WYRGRL (W) peptide-modified engineered Exo were developed for OA treatment and the performance of the engineered Exo (W-Exo@GelMA) loaded with a small inhibitor LRRK2-IN-1 (W-Exo-L@GelMA) was investigated in vitro and in vivo. The W-Exo-L@GelMA showed an effective targeting effect on chondrocytes and a pronounced action on suppressing catabolism and promoting anabolism in vitro. Moreover, W-Exo-L@GelMA remarkably inhibited OA-related inflammation and immune gene expression, rescuing the IL-1β-induced transcriptomic responses. With enhanced retention in the joint, W-Exo-L@GelMA demonstrated superior anti-OA activity and cartilage repair ability in the OA murine model. The therapeutic effect was validated in the cultured human OA cartilage. In conclusion, photocrosslinking spherical hydrogel-encapsulated targeting peptide-modified engineered Exo exhibit notable potential in OA therapy. Engineering Exo by a series of strategies enhanced the targeting ability and retention and cartilage-targeting and Exo-mediated drug delivery may offer a novel strategy for OA treatment.Clinical trial registration: Not applciable.Graphical

Sortilin Is Upregulated in Osteoarthritis-Dependent Cartilage Calcification and Associated with Cellular Senescence.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage calcification, loss of articular cartilage, bone changes, pain, and disability. Cartilage calcification is one hallmark of OA and is predominantly caused by basic calcium crystals formed due to an imbalance of the pyrophosphate pathway. Sortilin is a transmembrane protein that contributes to vascular calcification in atherosclerosis by externalizing alkaline phosphatase (ALP)-containing vesicles. Calcification in atherosclerosis and osteoarthritis has been associated with cellular senescence. The aim of this study was to investigate the potential role of sortilin and senescence in osteoarthritis-dependent cartilage calcification. Osteoarthritic cartilage from human knee joints was collected after joint replacement, and samples were analyzed by immunohistochemistry and quantitative RT-PCR analysis. Human chondrocytes were treated with osteogenic medium for up to 21 days to induce calcification. Western blots for sortilin and ALP, as well as an ALP activity assay, were performed. Human chondrocytes were treated with mitomycin C to induce senescence, and sortilin expression was quantified at the protein and gene levels. Sections of knee joints from a murine model of osteoarthritis were stained for sortilin and p16 and analyzed by immunohistochemistry. Treatment of wild-type chondrocytes using an osteogenic medium similar to human chondrocytes was performed. Osteoarthritic cartilage from mouse and human knee joints showed an increased number of sortilin and p16-positive chondrocytes compared to healthy cartilage. This observation was corroborated by increased gene expression of sortilin and p16 in mild and moderate osteoarthritic cartilage samples. To investigate the mechanism of sortilin regulation, human chondrocytes were treated with osteogenic medium to induce calcification. Sortilin protein levels and expression were increased after 7 days of stimulation, whereas ALP levels and activity were upregulated after 21 days of stimulation. Similar observations were made in a murine osteoarthritis model. Mechanistically, senescent chondrocytes induced by mitomycin C showed an upregulation of sortilin and ALP gene expression compared to non-senescent chondrocytes. Our data indicate that sortilin and ALP are upregulated during cartilage calcification, which is associated with chondrocyte senescence and thus might contribute to the pathogenesis of osteoarthritis. Cellular senescence seems to induce sortilin expression.

Noncanonical Pyroptosis Triggered by Macrophage-Derived Extracellular Vesicles in Chondrocytes Leading to Cartilage Catabolism in Osteoarthritis.

The severity of osteoarthritis (OA) and cartilage degeneration is highly correlated with the development of synovitis, which is mediated by the activity of inflammatory macrophages. A better understanding of intercellular communication between inflammatory macrophages and chondrocytes should aid in the discovery of novel therapeutic targets. We undertook this study to explore the pathologic role of inflammatory macrophage extracellular vesicles (EVs) in cartilage degeneration. Macrophages were stimulated by treatment with bacterial lipopolysaccharides to mimic the state of inflammatory macrophages, and the resulting EVs were harvested for chondrocyte stimulation in vitro and for intraarticular injection in a mouse model. The stimulated chondrocytes were further subjected to RNA-sequencing analysis and other functional assays. The action of caspase 11 was disrupted in vitro using a specific small interfering RNA or wedelolactone, and in experimental murine OA models by intraarticular injection of wedelolactone. Stimulated chondrocytes exhibited a significant elevation in the expression of chondrocyte catabolic factors. Consistent with these results, RNA-sequencing analyses of stimulated chondrocytes indicated that up-regulated genes were mainly categorized into apoptotic process and tumor necrosis factor signaling pathways, which suggests the induction of apoptotic process. Moreover, these chondrocytes exhibited a significant elevation in the expression of pyroptosis-related molecules that were correlated with the expression of chondrocyte catabolic factors. The disruption of caspase 11 significantly alleviated pyroptotic and catabolic processes in stimulated chondrocytes and pathologic changes in collagenase-induced and joint instability-induced OA models. Our results provide new insight into the pathologic mechanisms of OA and suggest that noncanonical pyroptosis in chondrocytes represents an attractive therapeutic target for treatment.

Evaluation of expanded peripheral blood derived CD34+ cells for the treatment of moderate knee osteoarthritis.

Knee osteoarthritis (OA) is a degenerative joint disease of the knee that results from the progressive loss of articular cartilage. It is most common in the elderly and affects millions of people worldwide, leading to a continuous increase in the number of total knee replacement surgeries. These surgeries improve the patient's physical mobility, but can lead to late infection, loosening of the prosthesis, and persistent pain. We would like to investigate if cell-based therapies can avoid or delay such surgeries in patients with moderate OA by injecting expanded autologous peripheral blood derived CD34+ cells (ProtheraCytes®) into the articular joint. In this study we evaluated the survival of ProtheraCytes® when exposed to synovial fluid and their performance in vitro with a model consisting of their co-culture with human OA chondrocytes in separate layers of Transwells and in vivo with a murine model of OA. Here we show that ProtheraCytes® maintain high viability (>95%) when exposed for up to 96 hours to synovial fluid from OA patients. Additionally, when co-cultured with OA chondrocytes, ProtheraCytes® can modulate the expression of some chondrogenic (collagen II and Sox9) and inflammatory/degrading (IL1β, TNF, and MMP-13) markers at gene or protein levels. Finally, ProtheraCytes® survive after injection into the knee of a collagenase-induced osteoarthritis mouse model, engrafting mainly in the synovial membrane, probably due to the fact that ProtheraCytes® express CD44, a receptor of hyaluronic acid, which is abundantly present in the synovial membrane. This report provides preliminary evidence of the therapeutic potential of CD34+ cells on OA chondrocytes in vitro and their survival after in vivo implantation in the knee of mice and merits further investigation in future preclinical studies in OA models.

Novel strategy of senescence elimination via toxicity-exempted kinome perturbations by nanoliposome-based thermosensitive hydrogel for osteoarthritis therapy

Cellular senescence and the senescence-associated secretory phenotype (SASP) have been implicated in osteoarthritis (OA). This study aims to determine whether multi-kinase inhibitor YKL-05-099 (Y099) has potential in senescence elimination and OA therapy and whether delivering Y099 by nanoliposmal hydrogel improves the performance of the kinase inhibitor. Y099 inhibited IL-1β-induced inflammation and catabolism and promoted anabolism of chondrocytes. To attenuate the inhibition of cell viability, nanoliposomal Y099-loaded thermosensitive hydrogel (Y099-Lip-Gel) was developed for sustained release and toxicity exemption. Notably, Y099-Lip-Gel exhibited a pronounced effect on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. Moreover, Y099-Lip-Gel remarkably increased the master regulator of chondrocyte phenotype Sox9 expression. After four intra-articular injections of Y099-Lip-Gel in the OA murine model, the histological lesions of cartilage were attenuated by Y099-Lip-Gel with subchondral bone loss and osteoclast formation inhibited. Transcriptomic analysis and experimental validations revealed that Y099-Lip-Gel suppressed cellular senescence by inhibiting the expression of senescence inducers and SASP factors. Furthermore, the phosphoproteomic analysis showed that Y099-Lip-Gel exerted a significant influence on kinome phosphorylation, inhibiting the MAPK and NF-κB signaling activations. The protective effects of Y099-Lip-Gel were also validated in cultured human OA cartilage explants. In conclusion, nanoliposomal Y099-loaded thermosensitive hydrogel has considerable potential in OA therapy. Nanoliposome-based hydrogel system has strength in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of protein kinase inhibitors. Senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology is a promising strategy for OA.Graphical

DLX5 promotes Col10a1 expression and chondrocyte hypertrophy and is involved in osteoarthritis progression

Osteoarthritis (OA) has been considered non-reversible as articular cartilage wears down with limited repair capacity. Enhanced chondrocyte hypertrophy and increased type X collagen gene (COL10A1) expression have been associated with OA. Therefore, regulators controlling collagen X expression and chondrocyte hypertrophy may play a role in OA intervention. Here, we investigated how Distal-less homeobox 5 (DLX5), the distal-less homeobox family member, controls murine Col10a1 gene expression and chondrocyte hypertrophy in chondrogenic cell models and its role in a murine OA model. Through qRT-PCR and Western blot analyses, we detected significantly increased levels of COL10A1 and DLX5 in hypertrophic MCT and ATDC5 cells compared to their proliferative stage. Forced expression of Dlx5 further increases, while knockdown of Dlx5 decreases COL10A1 expression in hypertrophic MCT cells. We have performed dual-luciferase reporter and ChIP assays and demonstrated that DLX5 promotes reporter activity through direct interaction with Col10a1 cis-enhancer. We established a murine OA model and detected markedly increased COL10A1 and DLX5 in the articular cartilage and subchondral bone of the OA mice compared with the controls. Notably, forced overexpression of DLX5 in hypertrophic MCT cells up-regulates RUNX2, and adjacent DLX5 and RUNX2 binding sites have previously been found within the Col10a1 cis-enhancer. Together, our data suggest that DLX5 may cooperate with RUNX2 to control cell-specific Col10a1 expression and chondrocyte hypertrophy and is involved in OA pathogenesis.

Silencing miR-146a-5p Protects against Injury-Induced Osteoarthritis in Mice.

Osteoarthritis (OA), the most prevalent joint disease and the leading cause of disability, remains an incurable disease largely because the etiology and pathogenesis underlying this degenerative process are poorly understood. Low-grade inflammation within joints is a well-established factor that disturbs joint homeostasis and leads to an imbalance between anabolic and catabolic processes in articular cartilage; however, the complexity of the network between inflammatory factors that often involves positive and negative feedback loops makes current anti-cytokine therapy ineffective. MicroRNAs (miRNAs) have emerged as key regulators to control inflammation, and aberrant miRNAs expression has recently been linked to OA pathophysiology. In the present study, we characterized transcriptomic profiles of miRNAs in primary murine articular chondrocytes in response to a proinflammatory cytokine, IL-1β, and identified miR-146a-5p as the most responsive miRNA to IL-1β. miR-146a-5p was also found to be upregulated in human OA cartilage. We further demonstrated that knockdown of miR-146a-5p antagonized IL-1β-mediated inflammatory responses and IL-1β-induced catabolism in vitro, and silencing of miR-146a in chondrocytes ameliorated articular cartilage destruction and reduced OA-evoked pain in an injury-induced murine OA model. Moreover, parallel RNA sequencing revealed that differentially expressed genes in response to IL-1β were enriched in pathways related to inflammatory processes, cartilage matrix homeostasis, and cell metabolism. Bioinformatic analyses of putative miR-146a-5p gene targets and following prediction of protein-protein interactions suggest a functional role of miR-146a-5p in mediating inflammatory processes and regulation of cartilage homeostasis. Our genetic and transcriptomic data define a crucial role of miR-146a-5p in OA pathogenesis and implicate modulation of miR-146a-5p in articular chondrocytes as a potential therapeutic strategy to alleviate OA.

Targeting Vascular Endothelial Growth Factor Receptors as a Therapeutic Strategy for Osteoarthritis and Associated Pain.

Pain is the major reason that patients suffering from osteoarthritis (OA) seek medical care. We found that vascular endothelial growth factors (VEGFs) mediate signaling in OA pain pathways. To determine the specific contributions of VEGFs and their receptors (VEGFRs) to joint pathology and pain transmission during OA progression, we studied intra-articular (IA) injections of VEGF ligands into murine knee joints. Only VEGF ligands specific for the activation of VEGFR1, but not VEGFR2, induced allodynia within 30 min. Interventions in OA by inhibitors of VEGFRs were done in vivo using a preclinical murine OA model by IA injections of selective inhibitors of VEGFR1/VEGFR2 kinase (pazopanib) or VEGFR2 kinase (vandetanib). OA phenotypes were evaluated using pain-associated murine behavioral tests and histopathologic analyses. Alterations in VEGF/VEGFR signaling by drugs were determined in knee joints, dorsal root ganglia, and spinal cord by immunofluorescence microscopy. Pazopanib immediately relieved OA pain by interfering with pain transmission pathways. Pain reduction by vandetanib was mainly due to the inhibition of cartilage degeneration by suppressing VEGFR2 expression. In conclusion, IA administration of pazopanib, which simultaneously inhibits VEGFR1 and VEGFR2, can be developed as an ideal OA disease-modifying drug that rapidly reduces joint pain and simultaneously inhibits cartilage degeneration.

Moderate exercise protects against joint disease in a murine model of osteoarthritis.

Exercise is recommended as a non-pharmacological therapy for osteoarthritis (OA). Various exercise regimes, with differing intensities and duration, have been used in a range of OA rodent models. These studies show gentle or moderate exercise reduces the severity of OA parameters while high intensity load bearing exercise is detrimental. However, these studies were largely conducted in rats or in mouse models induced by severe injury, age or obesity, whilst destabilization of the medial meniscus (DMM) in mice has become a widely accepted model due to its lower variability, moderate progression and timescale. The present study was undertaken to provide insight into the effect of moderate exercise on early joint pathology in the DMM mouse model. Exercise was induced a week after induction by forced wheel walking for three or 7weeks. Joints were analyzed by microcomputed tomography and histology. Assessment of skeletal parameters revealed that exercise offered protection against cartilage damage after 7weeks of exercise, and a temporary protection against osteosclerosis was displayed after 3weeks of exercise. Furthermore, exercise modified the metaphyseal trabecular microarchitecture of the osteoarthritic leg in both time points examined. Collectively, our findings corroborate previous studies showing that exercise has an important effect on bone in OA, which subsequently, at 8weeks post-induction, translates into less cartilage damage. Thus, providing an exercise protocol in a surgical mouse model of OA, which can be used in the future to further dissect the mechanisms by which moderate exercise ameliorates OA.

A Pilot Analysis of Genome-Wide DNA Methylation Patterns in Mouse Cartilage Reveals Overlapping Epigenetic Signatures of Aging and Osteoarthritis.

Cartilage epigenetic changes are strongly associated with human osteoarthritis (OA). However, the influence of individual environmental OA risk factors on these epigenetic patterns has not been determined; herein we characterize cartilage DNA methylation patterns associated with aging and OA in a mouse model. Murine knee cartilage DNA was extracted from healthy young (16-week, n=6), old (82-week, n=6), and young 4-week post-destabilization of the medial meniscus (DMM) OA (n=6) C57BL6/J mice. Genome-wide DNA methylation patterns were determined via Illumina BeadChip. Gene set enrichment analysis was performed by Ingenuity Pathway Analysis. The top seven most differentially methylated positions (DMPs) were confirmed by pyrosequencing in an independent animal set. Results were compared to previously published human OA methylation data. Aging was associated with 20,940 DMPs, whereas OA was associated with 761 DMPs. Merging these two conditions revealed 279 shared DMPs. All demonstrated similar directionality and magnitude of change (Δβ 1.0% ± 0.2%, mean methylation change ± SEM). Shared DMPs were enriched in OA-associated pathways, including RhoA signaling (P=1.57 × 10-4 ), protein kinase A signaling (P=3.38 × 10-4 ), and NFAT signaling (P=6.14 × 10-4 ). Upstream regulators, including TET3 (P=6.15 × 10-4 ), immunoglobulin (P=6.14 × 10-4 ), and TLR7 (P=7.53 × 10-4 ), were also enriched. Pyrosequencing confirmed six of the seven top DMPs in an independent cohort. Aging and early OA following DMM surgery induce similar DNA methylation changes within a murine OA model, suggesting that aging may induce pro-OA epigenetic "poising" within articular cartilage. Future research should focus on confirming and expanding these findings to other environmental OA risk factors, including obesity, as well as determining late OA changes in mice.

Oxymatrine Protects Chondrocytes against IL-1β-triggered Apoptosis in Vitro and Inhibits Osteoarthritis in Mice Model.

Background Osteoarthritis (OA) is a multifactorial disease with various risk factors, resulting in the degeneration of articular cartilage and whole joints. However, to date, no effective disease-modifying therapy for OA has been developed. Oxymatrine (OMT) is associated with many pharmacological effects, including anti-inflammatory, antiapoptotic, and antioxidative properties. However, the role of OMT in OA remains unclear. Materials and Methods An IL-1β-induced chondrocyte model and anterior cruciate ligament transection (ACLT)-induced murine model of OA were constructed. The effect of OMT on chondrocyte viability was assessed using the CCK-8 assay. The protein level was assessed by Western blot analysis, and the apoptosis rate was assessed by flow cytometry in vitro and TUNEL staining in OA model mice. The effect of OMT on the degradation of articular cartilage in ACLT-induced OA mice was assessed by histological analysis. Results OMT at 0–2 mg/mL showed no conspicuous cytotoxicity on chondrocytes after 24 hours of incubation. OMT at 0.5, 1, and 2 mg/mL inhibited IL-1β-triggered apoptosis, upregulated MMP13, MMP9, and Col X, and upregulated Col II in chondrocytes in vitro. OMT represses the NF-κB signaling cascade in IL-1β-triggered chondrocytes in vitro. In an in vivo study, OMT decreased the apoptosis rate of chondrocytes and exerted a protective effect against the degradation of articular cartilage in ACLT-triggered OA mice. Conclusion OMT plays a protective role against chondrocyte injury induced by IL-1β in vitro or ACLT in vivo. OMT may play a role in chondrocytes during OA by inhibiting NF-κB signaling by decreasing the phosphorylation of p65 and IκB. OMT treatment may be a promising chondroprotective approach to delay OA cartilage progression.

TRPV1 as an anti-ferroptotic target in osteoarthritis.

TRPV1 as an anti-ferroptotic target in osteoarthritis.

Interplay between cellular changes in the knee joint, circulating lipids and pain behaviours in a slowly progressing murine model of osteoarthritis.

Synovial inflammation has known contributions to chronic osteoarthritis (OA) pain, but the potential role in transitions from early to late stages of OA pain is unclear. The slowly progressing surgical destabilization of the medial meniscus (DMM) murine OA model and sham control, was used in male C57BL/6J mice to investigate the interplay between knee inflammation, plasma pro- and anti-inflammatory oxylipins and pain responses during OA progression. Changes in joint histology, macrophage infiltration, chemokine receptor CX3CR1 expression, weight bearing asymmetry, and paw withdrawal thresholds were quantified 4, 8 and 16 weeks after surgery. Plasma levels of multiple bioactive lipid mediators were quantified using liquid chromatography with tandem mass-spectrometry (LC-MS/MS). Structural joint damage was evident at 8 weeks post-DMM surgery onwards. At 16 weeks post-DMM surgery, synovial scores, numbers of CD68 and CD206 positive macrophages and pain responses were significantly increased. Plasma levels of oxylipins were negatively correlated with joint damage and synovitis scores at 4 and 8 weeks post-DMM surgery. Higher circulating levels of the pro-resolving oxylipin pre-cursor 17-HDHA were associated with lower weight bearing asymmetry at week 16. The transition to chronic OA pathology and pain is likely influenced by both joint inflammation and plasma oxylipin mediators of inflammation and levels of pro-resolution molecules. Using a slow progressing surgical model of osteoarthritis we show how the changing balance between local and systemic inflammation may be of importance in the progression of pain behaviours during the transition to chronic osteoarthritis pain.