Dietary fatty acids and arthritis.

Musculoskeletal complaints are the second most frequent reason for medical treatments. Within these diseases rheumatoid arthritis (RA) and, especially, osteoarthritis (OA) are common. Although the causes of arthritis are multifactorial and not fully understood, clinical trials have generally shown benefit from dietary n-3 polyunsaturated fatty acids. This has usually been attributed to their anti-inflammatory properties. Recently we have used in vitro model systems to study the molecular mechanism(s) by which n-3 PUFAs may act to alleviate the symptoms of arthritis. These experiments showed that n-3 PUFAs reduce expression of cartilage-degrading proteinases, cyclooxygenase-2 and inflammatory cytokines. Eicosapentaenoic acid (EPA) was more effective than docosahexaenoic acid (DHA) or alpha-linolenic acid. The data provide a scientific rationale for the consumption of n-3 fatty acids as part of a healthy diet and perhaps in treating arthritis.

Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis.

OBJECTIVE: To assess the relative efficacy of three different omega-3 (n-3) polyunsaturated fatty acids (PUFAs) in suppressing the mRNA levels for important proteins involved in the etiology of osteoarthritis (OA). METHODS: A model cell culture system (bovine chondrocytes) was used. Inflammatory factors and enzymes involved in OA were induced by exposure of the chondrocyte cultures to interleukin-1alpha (IL-1alpha). The effect of pre-incubating cultures with various amounts of exogenous fatty acids on subsequent levels of mRNAs was assessed by reverse transcription-polymerase chain reactions (RT-PCR). RESULTS: Exposure of cultures to IL-1alpha induced expression of the cartilage proteinases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)-4 and ADAMTS-5, cyclooxygenase (COX)-2, the matrix metalloproteinase (MMP)-3 and the inflammatory cytokines IL-1alpha, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). n-3 PUFAs were able to reduce the levels of mRNA for ADAMTS-4, ADAMTS-5, MMP-3, MMP-13, COX-2 (but not COX-1), IL-1alpha, IL-1beta and TNF-alpha. Eicosapentaenoic acid (EPA) was the most effective, followed by docosahexaenoic (DHA) and then alpha-linolenic (ALA) acid. The n-6 PUFA, arachidonic acid (AA) had no effect. CONCLUSION: These results show that omega-3 (n-3) PUFAs cause a reduction in the mRNA levels for various proteins known to be important in the pathology of OA. They provide a molecular explanation, at least in part, for beneficial effects of dietary omega-3 PUFAs for the amelioration of symptoms of the disease. The relative efficacy of EPA suggests that this omega-3 PUFA may be especially useful for dietary supplementation in patients with OA.

Oxidative stress induces expression of osteoarthritis markers procollagen IIA and 3B3(-) in adult bovine articular cartilage.

OBJECTIVE: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype. METHODS: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants. RESULTS: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants. CONCLUSIONS: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis.

n-3 fatty acids specifically modulate catabolic factors involved in articular cartilage degradation.

This study describes specific molecular mechanisms by which supplementation with n-3 fatty acids (i.e. those present in fish oils) can modulate the expression and activity of degradative and inflammatory factors that cause cartilage destruction during arthritis. Our data show that incorporation of n-3 fatty acids (but not other polyunsaturated or saturated fatty acids) into articular cartilage chondrocyte membranes results in a dose-dependent reduction in: (i) the expression and activity of proteoglycan degrading enzymes (aggrecanases) and (ii) the expression of inflammation-inducible cytokines (interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha) and cyclooxygenase (COX-2), but not the constitutively expressed cyclooxygenase COX-1. These findings provide evidence that n-3 fatty acid supplementation can specifically affect regulatory mechanisms involved in chondrocyte gene transcription and thus further advocate a beneficial role for dietary fish oil supplementation in alleviation of several of the physiological parameters that cause and propogate arthritic disease.

Effects of culture conditions and exposure to catabolic stimulators (IL-1 and retinoic acid) on the expression of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) by articular cartilage chondrocytes.

The chondrocytes of articular cartilage synthesize a number of proteinases which are capable of degrading the component molecules of this specialized extracellular matrix. The use of class-specific proteinase inhibitors indicates that major activities responsible for catabolism of proteoglycan (aggrecan) and collagen are attributable to zinc-dependent metalloproteinases. In this study, we have compared the mRNA expression profiles of two matrix metalloproteinases (MMP-3 and MMP-13) and five disintegrin-metalloproteinases (ADAM-10, ADAM-9, ADAM-15, TNF-alpha-converting enzyme and decysin) by chondrocytes (human, porcine and bovine) from fresh cartilage and in cartilage explant cultures and isolated cells cultured in monolayer or in agarose gels. Such cultures were maintained in the presence or absence of interleukin-1 (IL-1) or all-trans-retinoic acid, two agents which promote cartilage matrix degradation in vitro. Whereas transcripts for all metalloproteinases examined were detected in chondrocytes from human osteoarthritic cartilage in monolayer cultures, mRNAs for ADAM-15 and decysin were not present in fresh osteoarthritic human cartilage or explant cultures. Similarly, expression of porcine and bovine metalloproteinase mRNAs varied with different culture conditions. Novel cDNA sequences obtained for porcine and bovine MMP-3 and MMP-13, porcine ADAM-10, porcine and bovine ADAM-9 and porcine TACE confirmed expression of mRNAs for these molecules by articular chondrocytes. Quantitative RT-PCR analysis was used to determine the effects of IL-1 and retinoic acid on metalloproteinase mRNA levels in human chondrocytes cultured in monolayer and in porcine chondrocytes cultured in agarose. For the MMPs, IL-1 treatment resulted in an approximately two to threefold increase in human and porcine MMP-3 and MMP-13 mRNAs, while retinoic acid treatment caused a statistically significant increase in human MMP-3 mRNA levels, but no significant change in transcript levels for porcine MMP-3 nor human or porcine MMP-13. The mRNA levels for ADAM-15 were elevated in human monolayer chondrocytes exposed to IL-1 or retinoic acid, while transcripts levels for TNF-alpha converting enzyme were increased in response to retinoic acid. In contrast, ADAM-9 mRNA levels were decreased in human monolayer chondrocytes exposed to IL-1 or retinoic acid. The results demonstrate that chondrocyte metalloproteinase expression can vary dependent on cell environment in situ and in vitro, and information on chondrocyte MMP and ADAM gene expression following cytokine (IL-1) or retinoid stimulation.

Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions.

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.