Rose K. Davidson

Superoxide dismutase downregulation in osteoarthritis progression and end-stage disease

Background Oxidative stress is proposed as an important factor in osteoarthritis (OA). Objective To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. Methods SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin–Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. Results All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin–Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. Conclusion This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

OBJECTIVE Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). METHODS Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. RESULTS Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. CONCLUSION Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

Class I Histone Deacetylase Inhibition Modulates Metalloproteinase Expression and Blocks Cytokine‐Induced Cartilage Degradation

OBJECTIVE To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro. METHODS A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro. RESULTS Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression. CONCLUSION Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.

Objective Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis. Methods Gene expression, histone acetylation, and signaling of the transcription factors NF-E2–related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels. Results SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB–dependent genes. Compared with cytokines alone, SFN (10 μM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 μmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes. Conclusion SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.

The potential for dietary factors to prevent or treat osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease-modifying drugs. It is a leading cause of disability in the UK. Increasing age and obesity are both major risk factors for OA and the health and economic burden of this disease will increase in the future. Focusing on compounds from the habitual diet that may prevent the onset or slow the progression of OA is a strategy that has been under-investigated to date. An approach that relies on dietary modification is clearly attractive in terms of risk/benefit and more likely to be implementable at the population level. However, before undertaking a full clinical trial to examine potential efficacy, detailed molecular studies are required in order to optimise the design. This review focuses on potential dietary factors that may reduce the risk or progression of OA, including micronutrients, fatty acids, flavonoids and other phytochemicals. It therefore ignores data coming from classical inflammatory arthritides and nutraceuticals such as glucosamine and chondroitin. In conclusion, diet offers a route by which the health of the joint can be protected and OA incidence or progression decreased. In a chronic disease, with risk factors increasing in the population and with no pharmaceutical cure, an understanding of this will be crucial.

Dickkopf-3 is upregulated in osteoarthritis and has a chondroprotective role.

Summary Objective Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance. Methods Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1β (IL1β) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFβ and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays. Results Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1β and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFβ signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited. Conclusions We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.

Isothiocyanates are detected in human synovial fluid following broccoli consumption and can affect the tissues of the knee joint

Osteoarthritis is a major cause of disability and there is no current pharmaceutical treatment which can prevent the disease or slow its progression. Dietary advice or supplementation is clearly an attractive option since it has low toxicity and ease of implementation on a population level. We have previously demonstrated that sulforaphane, a dietary isothiocyanate derived from its glucosinolate precursor which is found in broccoli, can prevent cartilage destruction in cells, in in vitro and in vivo models of osteoarthritis. As the next phase of this research, we enrolled 40 patients with knee osteoarthritis undergoing total knee replacement into a proof-of-principle trial. Patients were randomised to either a low or high glucosinolate diet for 14 days prior to surgery. We detected ITCs in the synovial fluid of the high glucosinolate group, but not the low glucosinolate group. This was mirrored by an increase in ITCs and specifically sulforaphane in the plasma. Proteomic analysis of synovial fluid showed significantly distinct profiles between groups with 125 differentially expressed proteins. The functional consequence of this diet will now be tested in a clinical trial.