Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis

Introduction

Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods

Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results

Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion

These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.     

Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model

Introduction

Cell therapy is a rapidly growing area of research for the treatment of osteoarthritis (OA). This work is aimed to investigate the efficacy of intra-articular adipose-derived stromal cell (ASC) injection in the healing process on cartilage, synovial membrane and menisci in an experimental rabbit model.

Methods

The induction of OA was performed surgically through bilateral anterior cruciate ligament transection (ACLT) to achieve eight weeks from ACLT a mild grade of OA. A total of 2 × 10⁶ and 6 × 10⁶ autologous ASCs isolated from inguinal fat, expanded in vitro and suspended in 4% rabbit serum albumin (RSA) were delivered in the hind limbs; 4% RSA was used as the control. Local bio-distribution of the cells was verified by injecting chloro-methyl-benzamido-1,1''-dioctadecyl-3,3,3''3''-tetra-methyl-indo-carbocyanine per-chlorate (CM-Dil) labeled ASCs in the hind limbs. Cartilage and synovial histological sections were scored by Laverty''s scoring system to assess the severity of the pathology. Protein expression of some extracellular matrix molecules (collagen I and II), catabolic (metalloproteinase-1 and -3) and inflammatory (tumor necrosis factor- α) markers were detected by immunohistochemistry. Assessments were carried out at 16 and 24 weeks.

Results

Labeled-ASCs were detected unexpectedly in the synovial membrane and medial meniscus but not in cartilage tissue at 3 and 20 days from ASC-treatment. Intra-articular ASC administration decreases OA progression and exerts a healing contribution in the treated animals in comparison to OA and 4% RSA groups.

Conclusions

Our data reveal a healing capacity of ASCs in promoting cartilage and menisci repair and attenuating inflammatory events in synovial membrane inhibiting OA progression. On the basis of the local bio-distribution findings, the benefits obtained by ASC treatment could be due to a trophic mechanism of action by the release of growth factors and cytokines.     

Coenzyme Q10 Ameliorates Pain and Cartilage Degradation in a Rat Model of Osteoarthritis by Regulating Nitric Oxide and Inflammatory Cytokines

Objective

To investigate the effect of CoenzymeQ10 (CoQ10) on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA).

Materials and Methods

OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA) to the knee. Oral administration of CoQ10 was initiated on day 4 after MIA injection. Pain severity was assessed by measuring secondary tactile allodynia using the von Frey assessment test. The degree of cartilage degradation was determined by measuring cartilage thickness and the amount of proteoglycan. The mankin scoring system was also used. Expressions of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), IL-6, IL-15, inducible nitric oxide synthase (iNOS), nitrotyrosine and receptor for advanced glycation end products (RAGE) were analyzed using immunohistochemistry.

Results

Treatment with CoQ10 demonstrated an antinociceptive effect in the OA animal model. The reduction in secondary tactile allodynia was shown by an increased pain withdrawal latency and pain withdrawal threshold. CoQ10 also attenuated cartilage degeneration in the osteoarthritic joints. MMP-13, IL-1β, IL-6, IL-15, iNOS, nitrotyrosine and RAGE expressions were upregulated in OA joints and significantly reduced with CoQ10 treatment.

Conclusion

CoQ10 exerts a therapeutic effect on OA via pain suppression and cartilage degeneration by inhibiting inflammatory mediators, which play a vital role in OA pathogenesis.     

CCN4 induces IL-6 production through αvβ5 receptor,PI3K,Akt, and NF-κB singling pathway in human synovial fibroblasts

Introduction

Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.

Methods

CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.

Conclusions

Our results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.     

Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis

Background

Osteoarthritis (OA) is a common joint disease that causes disabilities in elderly. However, few agents with high efficacy and low side effects have been developed to treat OA. In this study, we evaluated the effects of the alginate extract named CTX in OA cell and rabbit models.

Results

CTX was formulated by hydrolyzing sodium alginate polymers with alginate lyase and then mixing with pectin. HPLC was used to analyze the CTX content. Human chondrosarcoma SW1353 cells treated with interleukin-1β were used as OA model cells to investigate the effects of CTX on chondrocyte inflammation and anabolism. CTX at concentrations up to 1000 μg/ml exerted low cytotoxicity. It inhibited the gene expression of proinflammatory matrix metalloproteinases (MMPs) including MMP1, MMP3 and MMP13 in a dose-dependent manner and increased the mRNA level of aggrecan, the major proteoglycan in articular cartilage, at 1000 μg/ml. Thirteen-week-old New Zealand White rabbits underwent a surgical anterior cruciate ligament transection and were orally treated with normal saline, glucosamine or CTX for up to 7 weeks. Examinations of the rabbit femur and tibia samples demonstrated that the rabbits taking oral CTX at a dosage of 30 mg/kg/day suffered lesser degrees of articular stiffness and histological cartilage damage than the control rabbits.

Conclusions

The gene expression profiles in the cell and the examinations done on the rabbit cartilage suggest that the alginate extract CTX is a pharmaco-therapeutic agent applicable for OA therapy.     

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

Introduction

Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.

Methods

sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.

Results

All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.

Conclusions

Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.     

Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis

Introduction

We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRβ). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRβ for treating rat antigen-induced arthritis.

Methods

A monoclonal antibody (mAb) to rat FRβ was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRβ gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRβ mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRβ mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund''s adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRβ-expressing macrophages and cathepsin K-positive cells on day 21.

Results

Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRβ-expressing macrophages and cathepsin K-positive cells.

Conclusions

Intra-articular administration of an immunotoxin to FRβ is effective for improving rat antigen-induced arthritis.     

Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR,mass spectrometry,histochemistry with plant/human lectins and lectin localization with a glycoprotein

Introduction

This study aimed to characterize the glycophenotype of osteoarthritic cartilage and human chondrocytes.

Methods

Articular knee cartilage was obtained from nine osteoarthritis (OA) patients. mRNA levels for 27 glycosyltransferases were analyzed in OA chondrocytes using RT-qPCR. Additionally, N- and O-glycans were quantified using mass-spectrometry. Histologically, two cartilage areas with Mankin scores (MS) either ≤4 or ≥9 were selected from each patient representing areas of mild and severe OA, respectively. Tissue sections were stained with (1) a selected panel of plant lectins for probing into the OA glycophenotype, (2) the human lectins galectins-1 and -3, and (3) the glycoprotein asialofetuin (ASF) for visualizing β-galactoside-specific endogenous lectins.

Results

We found that OA chondrocytes expressed oligomannosidic structures as well as non-, mono- and disialylated complex-type N-glycans, and core 2 O-glycans. Reflecting B4GALNT3 mRNA presence in OA chondrocytes, LacdiNAc-terminated structures were detected. Staining profiles for plant and human lectins were dependent on the grade of cartilage degeneration, and ASF-positive cells were observed in significantly higher rates in areas of severe degeneration.

Conclusions

In summary, distinct aspects of the glycome in OA cartilage are altered with progressing degeneration. In particular, the alterations measured by galectin-3 and the pan-galectin sensor ASF encourage detailed studies of galectin functionality in OA.     

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Magnetic Resonance Imaging of Osteophytic,Chondral, and Subchondral Structures in a Surgically-Induced Osteoarthritis Rabbit Model

Objective

This study aimed to assess changes in osteophytic, chondral, and subchondral structures in a surgically-induced osteoarthritis (OA) rabbit model in order to correlate MRI findings with the macroscopic progress of OA and to define the timepoint for disease status in this OA model.

Methods

The OA model was constructed by surgery in thirty rabbits with ten normal rabbits serving as controls (baseline). High-resolution three-dimensional MRI using a 1.5-T coil was performed at baseline, two, four, and eight weeks post-surgery. MRIs of cartilage lesions, subchondral bone lesions, and osteophyte formations were independently assessed by two blinded radiologists. Ten rabbits were sacrificed at baseline, two, four, and eight weeks post-surgery, and macroscopic evaluation was independently performed by two blinded orthopedic surgeons.

Results

The signal intensities and morphologies of chondral and subchondral structures by MRI accurately reflected the degree of OA. Cartilage defects progressed from a grade of 0.05–0.15 to 1.15–1.30 to 1.90–1.97 to 3.00–3.35 at each successive time point, respectively (p<0.05). Subchondral bone lesions progressed from a grade of 0.00 to 0.78–0.90 to 1.27–1.58 to 1.95–2.23 at each successive time point, respectively (p = 0.000). Osteophytes progressed from a size (mm) of 0.00 to 0.87–1.06 to 1.24–1.87 to 2.21–3.21 at each successive time point, respectively (p = 0.000).

Conclusions

Serial observations revealed that MRI can accurately detect the progression of cartilage lesions and subchondral bone edema over an eight-week period but may not be accurate in detecting osteophyte sizes. Week four post-surgery was considered the timepoint between OA-negative and OA-positive status in this OA model. The combination of this OA model with MRI evaluation should provide a promising tool for the pre-clinical evaluation of new disease-modifying osteoarthritis drugs.     

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Background

Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.

Results

Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.

Conclusions

miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

Introduction

Although IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.

Methods

Cartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.

Results

SOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.

Conclusions

Our results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.     

Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2

Introduction

Rheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA₂V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.

Methods

Human synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA₂V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA₂V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.

Results

EPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA₂V co-localized with EPCR on RASFs. Suppression of sPLA₂V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA₂V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.

Conclusions

Our results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA₂V.     

Kinematic and dynamic gait compensations resulting from knee instability in a rat model of osteoarthritis

Introduction

Osteoarthritis (OA) results in pain and disability; however, preclinical OA models often focus on joint-level changes. Gait analysis is one method used to evaluate both preclinical OA models and OA patients. The objective of this study is to describe spatiotemporal and ground reaction force changes in a rat medial meniscus transection (MMT) model of knee OA and to compare these gait measures with assays of weight bearing and tactile allodynia.

Methods

Sixteen rats were used in the study. The medial collateral ligament (MCL) was transected in twelve Lewis rats (male, 200 to 250 g); in six rats, the medial meniscus was transected, and the remaining six rats served as sham controls. The remaining four rats served as naïve controls. Gait, weight-bearing as measured by an incapacitance meter, and tactile allodynia were assessed on postoperative days 9 to 24. On day 28, knee joints were collected for histology. Cytokine concentrations in the serum were assessed with a 10-plex cytokine panel.

Results

Weight bearing was not affected by sham or MMT surgery; however, the MMT group had decreased mechanical paw-withdrawal thresholds in the operated limb relative to the contralateral limb (P = 0.017). The gait of the MMT group became increasingly asymmetric from postoperative days 9 to 24 (P = 0.020); moreover, MMT animals tended to spend more time on their contralateral limb than their operated limb while walking (P < 0.1). Ground reaction forces confirmed temporal shifts in symmetry and stance time, as the MMT group had lower vertical and propulsive ground reaction forces in their operated limb relative to the contralateral limb, naïve, and sham controls (P < 0.05). Levels of interleukin 6 in the MMT group tended to be higher than naïve controls (P = 0.072). Histology confirmed increased cartilage damage in the MMT group, consistent with OA initiation. Post hoc analysis revealed that gait symmetry, stance time imbalance, peak propulsive force, and serum interleukin 6 concentrations had significant correlations to the severity of cartilage lesion formation.

Conclusion

These data indicate significant gait compensations were present in the MMT group relative to medial collateral ligament (MCL) injury (sham) alone and naïve controls. Moreover, these data suggest that gait compensations are likely driven by meniscal instability and/or cartilage damage, and not by MCL injury alone.     

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

Introduction

Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.

Methods

Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.

Results

Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.

Conclusions

Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.