ABSTRACT
Neuropathic pain (NP) is a challenging condition to treat, as the need for new drugs to treat NP is an unmet goal. We investigated the analgesic potential of a new sulfated disaccharide compound, named BIS014. Oral administration (p.o.) of this compound induced ameliorative effects in formalin-induced nociception and capsaicin-induced secondary mechanical hypersensitivity in mice, but also after partial sciatic nerve transection (spared nerve injury), chemotherapy (paclitaxel)-induced NP, and diabetic neuropathy induced by streptozotocin. Importantly, BIS014, at doses active on neuropathic hypersensitivity (60 mg/kg/p.o.), did not alter exploratory activity or motor coordination (in the rotarod test), unlike a standard dose of gabapentin (40 mg/kg/p.o.) which although inducing antiallodynic effects on the NP models, it also markedly decreased exploration and motor coordination. In docking and molecular dynamic simulation studies, BIS014 interacted with TRPV1, a receptor involved in pain transmission where it behaved as a partial agonist. Additionally, similar to capsaicin, BIS014 increased cytosolic Ca2+ concentration ([Ca2+]c) in neuroblastoma cells expressing TRPV1 receptors; these elevations were blocked by ruthenium red. BIS014 did not block capsaicin-elicited [Ca2+]c transients, but inhibited the increase in the firing rate of action potentials in bradykinin-sensitized dorsal root ganglion neurons stimulated with capsaicin. Perspective: We report that the oral administration of a new sulfated disaccharide compound, named BIS014, decreases neuropathic pain from diverse etiology in mice. Unlike the comparator gabapentin, BIS014 does not induce sedation. Thus, BIS014 has the potential to become a new efficacious non-sedative oral medication for the treatment of neuropathic pain.
Subject(s)
Capsaicin , Neuralgia , Mice , Animals , Capsaicin/adverse effects , Hyaluronic Acid/pharmacology , Gabapentin , TRPV Cation Channels , Hyperalgesia/drug therapyABSTRACT
Chondroitin sulphate (CS) has long been used to treat osteoarthritis. Some investigations have also shown that the treatment with CS could reduce coronary events in patients with heart disease but no studies have identified the mechanistic role of these therapeutic effects. We aimed to investigate how the treatment with CS can interfere with the progress of atherosclerosis. The aortic arch, thoracic aorta and serum were obtained from apolipoprotein E (ApoE) knockout mice fed for 10 weeks with high-fat diet and then treated with CS (300 mg/kg, n = 15) or vehicle (n = 15) for 4 weeks. Atheromatous plaques were highlighted in aortas with Oil Red staining and analysed by microscopy. ApoE knockout mice treated with CS exhibited attenuated atheroma lesion size by 68% as compared with animals receiving vehicle. Serum lipids, glucose and C-reactive protein were not affected by treatment with CS. To investigate whether CS locally affects the inflamed endothelium or the formation of foam cells in plaques, human endothelial cells and monocytes were stimulated with tumour necrosis factor α or phorbol myristate acetate in the presence or absence of CS. CS reduced the expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 and ephrin-B2 and improved the migration of inflamed endothelial cells. CS inhibited foam cell formation in vivo and concomitantly CD36 and CD146 expression and oxidized low-density lipoprotein uptake and accumulation in cultured activated human monocytes and macrophages. Reported cardioprotective effects of CS may arise from modulation of pro-inflammatory activation of endothelium and monocytes and foam cell formation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Aorta, Thoracic/drug effects , Aortic Diseases/prevention & control , Atherosclerosis/prevention & control , Chondroitin Sulfates/pharmacology , Inflammation Mediators/metabolism , Inflammation/prevention & control , Animals , Aorta, Thoracic/metabolism , Aorta, Thoracic/pathology , Aortic Diseases/genetics , Aortic Diseases/metabolism , Aortic Diseases/pathology , Atherosclerosis/genetics , Atherosclerosis/metabolism , Atherosclerosis/pathology , Blood Glucose/metabolism , C-Reactive Protein/metabolism , Diet, High-Fat , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Foam Cells/drug effects , Foam Cells/metabolism , Foam Cells/pathology , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/blood , Lipids/blood , Lipoproteins, LDL/metabolism , Male , Mice, Knockout, ApoE , Monocytes/drug effects , Monocytes/metabolism , Monocytes/pathology , Plaque, Atherosclerotic , THP-1 CellsABSTRACT
Monocyte chemoattractant protein-1 (MCP-1) overproduction from inflamed adipose tissue is a major contributor to obesity-related metabolic syndromes. 3T3-L1 embryonic fibroblasts were cultured and differentiated into adipocytes using an established protocol. Adipocytes were treated with lipopolysaccharide (LPS) to induce inflammation and thus MCP-1 release. At the same time, varying concentrations of chondroitin sulfate (CS) were added in a physiologically relevant range (10-200 µg/mL) to determine its impact on MCP-1 release. Chondroitin sulfate, a natural glycosaminoglycan of connective tissue including the cartilage extracellular matrix, was chosen on the basis of our previous studies demonstrating its anti-inflammatory effect on macrophages. Because the main action of MCP-1 is to induce monocyte migration, cultured THP-1 monocytes were used to test whether CS at the highest physiologically relevant concentration could inhibit cell migration induced by human recombinant MCP-1. Chondroitin sulfate (100-200 µg/mL) inhibited MCP-1 release from inflamed adipocytes in a dose-dependent manner (P < .01, 95% confidence interval [CI]: -5.89 to -3.858 at 100 µg/mL and P < .001, 95% CI: -6.028 to -3.996 at 200 µg/mL) but had no effect on MCP-1-driven chemotaxis of THP-1 monocytes. In summary, CS could be expected to reduce macrophage infiltration into adipose tissue by reduction in adipocyte expression and release of MCP-1 and as such might reduce adipose tissue inflammation in response to pro-inflammatory stimuli such as LPS, now increasingly recognized to be relevant in vivo.
ABSTRACT
CONTEXT: The efficacy of the combination chondroitin sulfate-glucosamine (CS-GlcN) in the treatment of knee osteoarthritis (OA) has been suggested in recent clinical studies. In vitro reports have also suggested anti-inflammatory and anti-resorptive effects of this combination. OBJECTIVE: The aim of this study was to characterize the effects of CS-GlcN on joint degradation in vivo including the assessment of inflammation and bone metabolism in a model of OA. MATERIALS AND METHODS: We have used the OA model induced by anterior cruciate ligament transection (ACLT) in ovariectomised rats. CS-GlcN was administered daily (oral gavage) from week 0 until week 12 after ovariectomy at the dose of 140 (CS)+175 (GlcN)(HCl) mg/kg. Histochemical analyses were performed, the levels of biomarkers and inflammatory mediators were measured by luminex or ELISA and bone microstructure was determined by µCT. RESULTS: CS-GlcN protected against cartilage degradation and reduced the levels of inflammatory mediators such as interleukin-1ß and tumor necrosis factor-α in the affected knee. In addition, serum biomarkers of inflammation and cartilage and bone degradation including matrix metalloproteinase-3, C-telopeptide of type II collagen and the ratio receptor activator of nuclear factor κB ligand/osteoprotegerin were significantly decreased by CS-GlcN. This treatment also tended to improve some bone microstructural parameters without reaching statistical significance. DISCUSSION AND CONCLUSIONS: These results demonstrate the chondroprotective effects of CS-GlcN in vivo, in the experimental model of ACLT in ovariectomised rats, and suggest that this combination may be useful to control the joint catabolic effects of inflammatory stress. These findings could have clinical relevance related to the prevention of joint degradation by CS-GlcN and support the potential development of OA treatments based on this combination.
Subject(s)
Anterior Cruciate Ligament Injuries/drug therapy , Anterior Cruciate Ligament/pathology , Cartilage, Articular/pathology , Chondroitin Sulfates/therapeutic use , Glucosamine/therapeutic use , Osteoarthritis, Knee/drug therapy , Protective Agents/therapeutic use , Animals , Anterior Cruciate Ligament/drug effects , Anterior Cruciate Ligament Injuries/pathology , Biomarkers/blood , Bone and Bones/drug effects , Bone and Bones/pathology , Cartilage, Articular/drug effects , Chondroitin Sulfates/pharmacology , Disease Models, Animal , Drug Therapy, Combination , Female , Glucosamine/pharmacology , Inflammation Mediators/metabolism , Joints/drug effects , Joints/pathology , Osteoarthritis, Knee/blood , Osteoarthritis, Knee/pathology , Ovariectomy , Protective Agents/pharmacology , Rats, Wistar , X-Ray MicrotomographyABSTRACT
BACKGROUND AND AIMS: Osteoarthritic patients treated with high doses of chondroitin sulfate (CS) have a lower incidence of coronary heart disease--but the mechanistic aspects of these beneficial effects of CS remain undefined. We examined how CS treatment affects the formation of atheroma via interaction with endothelial cells and monocytes. METHODS: We characterized arterial atheromatous plaques by multiphoton microscopy and serum pro-inflammatory cytokines by immunoenzymatic techniques in obese mice receiving CS (1 g/kg/day, i.p.) or vehicle for 6 days. Effects of CS on signaling pathways, cytokine secretion and macrophage migration were evaluated in cultures of human coronary endothelial cells and in a monocyte cell line stimulated with TNF-α by Western blot, immunoenzymatic techniques and transwell migration assays. RESULTS: Treatment of obese mice with CS reduced the extension of foam cell coverage in atheromatous plaques of arterial bifurcations by 62.5%, the serum concentration of IL1ß by 70%, TNF-α by 82% and selected chemokines by 25-35%. Cultures of coronary endothelial cells and monocytes stimulated with TNF-α secreted less pro-inflammatory cytokines in the presence of CS (P < 0.01). CS reduced the activation of the TNF-α signaling pathway in endothelial cells (pErk 36% of reduction, and NFκB 33% of reduction), and the migration of activated monocytes to inflamed endothelial cells in transwells (81 ± 6 vs. 13 ± 2, P < 0.001). CONCLUSIONS: CS interferes with the pro-inflammatory activation of monocytes and endothelial cells driven by TNF-α thus reducing the propagation of inflammation and preventing the formation of atherosclerotic plaques.
Subject(s)
Atherosclerosis/drug therapy , Chondroitin Sulfates/therapeutic use , Inflammation/drug therapy , Obesity/drug therapy , Animals , Atherosclerosis/etiology , Atherosclerosis/metabolism , Cell Line , Disease Models, Animal , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Inflammation/complications , Inflammation/metabolism , Male , Mice , Mice, Obese , Obesity/complications , Obesity/pathologyABSTRACT
It is currently known that in CNS the extracellular matrix is involved in synaptic stabilization and limitation of synaptic plasticity. However, it has been reported that the treatment with chondroitinase following injury allows the formation of new synapses and increased plasticity and functional recovery. So, we hypothesize that some components of extracellular matrix may modulate synaptic transmission. To test this hypothesis we evaluated the effects of chondroitin sulphate (CS) on excitatory synaptic transmission, cellular excitability, and neuronal plasticity using extracellular recordings in the CA1 area of rat hippocampal slices. CS caused a reversible depression of evoked field excitatory postsynaptic potentials in a concentration-dependent manner. CS also reduced the population spike amplitude evoked after orthodromic stimulation but not when the population spikes were antidromically evoked; in this last case a potentiation was observed. CS also enhanced paired-pulse facilitation and long-term potentiation. Our study provides evidence that CS, a major component of the brain perineuronal net and extracellular matrix, has a function beyond the structural one, namely, the modulation of synaptic transmission and neuronal plasticity in the hippocampus.
Subject(s)
CA1 Region, Hippocampal/physiology , Chondroitin Sulfates/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Long-Term Potentiation/drug effects , Animals , CA1 Region, Hippocampal/drug effects , Chondroitin ABC Lyase/pharmacology , Male , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Osteoarthritis (OA) is the most frequent articular disease and a leading cause of disability. There is a need for effective treatments able to slow the progression of disease. Some of the available treatments are dietary supplements providing natural components. Recent studies have shown that estrogen deficiency contributes to the pathophysiological events of OA progression. METHODS: We have used the anterior cruciate ligament transection model of OA in ovariectomised rats to study the effects of BIS076, a new formulation of a natural porcine cartilage extract associated with hydroxyapatite (as a source of calcium) and vitamin D3. Cartilage degradation, proteoglycan depletion and synovitis were followed by histochemistry. Effects on bone microstructure were determined by µCT. The levels of biomarkers in serum and inflammatory mediators in knee homogenates were measured by luminex or ELISA. RESULTS: Oral administration of BIS076 reduced articular cartilage damage and serum levels of cartilage degradation markers C-telopeptide of type II collagen and cartilage oligomeric matrix protein, as well as matrix metalloproteinase-3. The local inflammatory response was down-regulated by BIS076 with lower production of pro-inflammatory cytokines and prostaglandin E2 in joint tissues. In addition, BIS076 was effective on metaphyseal bone alterations as this formulation increased volumetric bone mineral density and improved bone micro-architecture. These effects were related to the modification of bone metabolism reflected by changes in bone biomarkers with reductions in the ratio receptor activator of nuclear factor κB ligand/osteoprotegerin and the levels of tartrate-resistant acid phosphatase-5b, suggesting an inhibitory activity of BIS076 on trabecular bone resorption. CONCLUSIONS: We have demonstrated the protective properties of a new formulation (BIS076) on joint lesion and bone alterations in an experimental model of OA in ovariectomised rats. This study supports the interest of BIS076 in OA treatments.
Subject(s)
Anterior Cruciate Ligament Injuries , Collagen Type II/therapeutic use , Glycosaminoglycans/therapeutic use , Osteoarthritis, Knee/drug therapy , Osteoarthritis, Knee/etiology , Ovariectomy/adverse effects , Tissue Extracts/therapeutic use , Animals , Biomarkers/blood , Cartilage Oligomeric Matrix Protein/blood , Collagen Type II/blood , Cytokines/blood , Dinoprostone/blood , Disease Models, Animal , Durapatite/therapeutic use , Female , Matrix Metalloproteinase 3/blood , Osteoarthritis, Knee/blood , Peptide Fragments/blood , Rats , Rats, Wistar , Swine , Treatment Outcome , Vitamin D/therapeutic useABSTRACT
Osteoarthritis is a common, progressive joint disease, and treatments generally aim for symptomatic improvement. However, SYmptomatic Slow-Acting Drugs in Osteoarthritis (SYSADOAs) not only reduce joint pain, but slow structural disease progression. One such agent is chondroitin sulfate-a complex, heterogeneous polysaccharide. It is extracted from various animal cartilages, thus has a wide range of molecular weights and different amounts and patterns of sulfation. Chondroitin sulfate has an excellent safety profile, and although various meta-analyses have concluded that it has a beneficial effect on symptoms and structure, others have concluded little or no benefit. This may be due, at least partly, to variations in the quality of the chondroitin sulfate used for a particular study. Chondroitin sulfate is available as pharmaceutical- and nutraceutical-grade products, and the latter have great variations in preparation, composition, purity and effects. Moreover, some products contain a negligible amount of chondroitin sulfate and among samples with reasonable amounts, in vitro testing showed widely varying effects. Of importance, although some showed anti-inflammatory effects, others demonstrated weak effects, and some instances were even pro-inflammatory. This could be related to contaminants, which depend on the origin, production and purification process. It is therefore vitally important that only pharmaceutical-grade chondroitin sulfate be used for treating osteoarthritis patients.
Subject(s)
Chemistry, Pharmaceutical/methods , Chondroitin Sulfates/chemistry , Dietary Supplements/standards , Quality Control , Animals , Chondroitin Sulfates/therapeutic use , Osteoarthritis/drug therapyABSTRACT
OBJECTIVE: The aim of this study was to develop a genetic prognostic tool to predict radiographic progression towards severe disease in primary knee OA (KOA) patients. METHODS: This investigation was a cross-sectional, retrospective, multicentric association study in 595 Spanish KOA patients. Caucasian patients aged ≥40 years at the time of diagnosis of primary KOA of Kellgren-Lawrence grade 2 or 3 were included. Patients who progressed to Kellgren-Lawrence score 4 or who were referred for total knee replacement within 8 years after diagnosis were classified as progressors to severe disease. Clinical variables of the initial stages of the disease (gender, BMI, age at diagnosis, OA in the contralateral knee, and OA in other joints) were registered as potential predictors. Single nucleotide polymorphisms and clinical variables with an association of P < 0.05 were included in the multivariate analysis using forward logistic regression. RESULTS: A total of 23 single nucleotide polymorphisms and the time of primary KOA diagnosis were significantly associated with KOA severe progression in the exploratory cohort (n = 220; P < 0.05). The predictive accuracy of the clinical variables was limited: area under the curve (AUC) = 0.66. When genetic variables were added to the clinical model (full model), the prediction of KOA progression was significantly improved (AUC = 0.82). Combining only genetic variables (rs2073508, rs10845493, rs2206593, rs10519263, rs874692, rs7342880, rs780094 and rs12009), a predictive model with good accuracy was also obtained (AUC = 0.78). The predictive ability for KOA progression of the full model was confirmed on the replication cohort (two-sample Z-test; n = 62; P = 0.190). CONCLUSION: An accurate prognostic tool to predict primary KOA progression has been developed based on genetic and clinical information from OA patients.
Subject(s)
Disease Progression , Osteoarthritis, Knee/diagnosis , Osteoarthritis, Knee/genetics , Polymorphism, Single Nucleotide/genetics , Severity of Illness Index , Aged , Cross-Sectional Studies , Female , Humans , Knee Joint/diagnostic imaging , Logistic Models , Longitudinal Studies , Male , Middle Aged , Multivariate Analysis , Osteoarthritis, Knee/diagnostic imaging , Predictive Value of Tests , Prognosis , Radiography , Retrospective Studies , SpainABSTRACT
BACKGROUND: Chondroitin Sulphate (CS), a natural glycosaminoglycan of the extracellular matrix, has clinical benefit in symptomatic osteoarthritis but has never been tested in gout. In vitro, CS has anti-inflammatory and positive effects on osteoarthritic chondrocytes, synoviocytes and subchondral bone osteoblasts, but its effect on macrophages is unknown. The purpose of our study was to evaluate the in vitro effects of CS on monosodium urate (MSU)-stimulated cytokine production by macrophages. METHODS: THP-1 monocytes were differentiated into mature macrophages using a phorbol ester, pretreated for 4 hours with CS in a physiologically achievable range of concentrations (10-200 µg/ml) followed by MSU crystal stimulation for 24 hours. Cell culture media were analyzed by immunoassay for factors known to be upregulated during gouty inflammation including IL-1ß, IL-8 and TNFα. The specificity of inflammasome activation by MSU crystals was tested with a caspase-1 inhibitor (0.01 µM-10 µM). RESULTS: MSU crystals ≥10 mg/dl increased macrophage production of IL-1ß, IL-8 and TNFα a mean 7-, 3- and 4-fold respectively. Induction of IL-1ß by MSU was fully inhibited by a caspase-1 inhibitor confirming inflammasome activation as the mechanism for generating this cytokine. In a dose-dependent manner, CS significantly inhibited IL-1ß (p = 0.003), and TNFα (p = 0.02) production from macrophages in response to MSU. A similar trend was observed for IL-8 but was not statistically significant (p = 0.41). CONCLUSIONS: CS attenuated MSU crystal induced macrophage inflammation, suggesting a possible role for CS in gout prophylaxis.
Subject(s)
Chondroitin Sulfates/pharmacology , Gout , Macrophages/drug effects , Uric Acid/toxicity , Cell Line , Chondroitin Sulfates/therapeutic use , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Gout/pathology , Gout/prevention & control , Humans , Inflammation/pathology , Inflammation/prevention & control , Macrophages/pathology , Monocytes/drug effects , Monocytes/pathologyABSTRACT
Osteoarthritis (OA) is the most common age-related rheumatic disease. Chondrocytes play a primary role in mediating cartilage destruction and extracellular matrix (ECM) breakdown, which are main features of the OA joint. Quantitative proteomics technologies are demonstrating a very interesting power for studying the molecular effects of some drugs currently used to treat OA patients, such as chondroitin sulfate (CS) and glucosamine (GlcN). In this work, we employed the iTRAQ (isobaric tags for relative and absolute quantitation) technique to assess the effect of CS and GlcN, both alone and in combination, in modifying cartilage ECM metabolism by the analysis of OA chondrocytes secretome. 186 different proteins secreted by the treated OA chondrocytes were identified. 36 of them presented statistically significant differences (p ≤ 0.05) between untreated and treated samples: 32 were increased and 4 decreased. The synergistic chondroprotective effect of CS and GlcN, firstly reported by our group at the intracellular level, is now demonstrated also at the extracellular level.
Subject(s)
Chondroitin Sulfates/pharmacology , Glucosamine/pharmacology , Aged , Aged, 80 and over , Cartilage, Articular/drug effects , Cells, Cultured , Chondrocytes/drug effects , Drug Synergism , Humans , Osteoarthritis/drug therapyABSTRACT
OBJECTIVE: To compare the gene expression patterns of synovial cells from inflamed or normal/reactive areas of synovial membrane obtained from the same patient with osteoarthritis (OA). METHODS: At the time of total knee replacement, synovial tissues were obtained from 12 patients with knee OA. The inflammation status of the synovial membrane was characterized according to macroscopic criteria and classified as normal/reactive or inflamed. Biopsy samples were cultured separately for 7 days. Microarray gene expression profiling was performed on normal/reactive and inflamed areas. Western blot and immunohistochemistry were used to confirm the identified genes that were differentially expressed. RESULTS: We identified 896 genes that were differentially expressed between normal/reactive and inflamed areas. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling, and angiogenesis. In the inflammation network, the genes TREM1 and S100A9 were strongly up-regulated. The genes MMP3, MMP9, CTSH (cathepsin H), and CTSS (cathepsin S) were significantly up-regulated in the cartilage catabolism pathway, while the most up-regulated anabolism enzyme gene was HAS1. In the Wnt signaling pathway, the genes for Wnt-5a and low-density lipoprotein receptor-related protein 5 were up-regulated, while the gene FZD2 and the gene for Dkk-3 were down-regulated. Finally, STC1, which codes for a protein involved in angiogenesis, was identified as the most up-regulated gene in inflamed compared with normal/reactive areas. CONCLUSION: This study is the first to identify different expression patterns between 2 areas of the synovial membrane from the same patient. These differences concern several key pathways involved in OA pathogenesis. This analysis also provides information regarding new genes and proteins as potential targets of treatment.
Subject(s)
Gene Expression , Knee Joint/metabolism , Osteoarthritis, Knee/genetics , Synovial Membrane/metabolism , Aged , Aged, 80 and over , Chondrocytes/metabolism , Chondrocytes/pathology , Female , Gene Expression Profiling , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Knee Joint/pathology , Male , Middle Aged , Osteoarthritis, Knee/metabolism , Osteoarthritis, Knee/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Synovial Membrane/pathologyABSTRACT
The anterior cruciate ligament transection (ACLT) model of osteoarthritis (OA) in young rats is widely used to study the pathogenesis of OA and possible treatment approaches. As aging is a key factor in the progression of this condition, it is hypothesized that animals may vary in their responses to ACLT according to their age. The histopathological features of young (2month-old) and middle-aged (12month-old) rats in the presence or absence of ACLT were compared. The results indicated that moderate degradative changes can be detected in the knee joints of sham-operated middle-aged rats compared with young animals. After ACLT, cartilage degradation was significantly higher in middle-aged rats in relation to young animals. An increase in interleukin(IL)-1ß and IL-17 suggests the presence of a local inflammatory response represented by synovitis in ACLT rats which is not dependent on age. Our study indicates that age is an important factor affecting the pathogenesis of OA changes after ACLT and it should be considered in studies using this experimental model.
Subject(s)
Aging/pathology , Anterior Cruciate Ligament Injuries , Arthritis, Experimental/etiology , Osteoarthritis/etiology , Aging/metabolism , Animals , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Cartilage, Articular/pathology , Collagen Type II/metabolism , Cytokines/metabolism , Disease Progression , Inflammation Mediators/metabolism , Male , Osteoarthritis/metabolism , Osteoarthritis/pathology , Proteoglycans/metabolism , Rats , Rats, Wistar , Synovitis/etiology , Synovitis/metabolism , Synovitis/pathologyABSTRACT
Chondroitin sulfate (CS) proteoglycans (CSPGs) are the most abundant PGs of the brain extracellular matrix (ECM). Free CS could be released during ECM degradation and exert physiological functions; thus, we aimed to investigate the effects of CS on voltage- and current-clamped rat embryo hippocampal neurons in primary cultures. We found that CS elicited a whole-cell Na(+)-dependent inward current (ICS) that produced drastic cell depolarization, and a cytosolic calcium transient ([Ca(2+)]c). Those effects were similar to those elicited by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, were completely blocked by NBQX and CNQX, were partially blocked by GYKI, and were unaffected by MK801 and D-APV. Furthermore, ICS and AMPA currents were similarly potentiated by cyclothiazide, a positive allosteric modulator of AMPA receptors. Because CSPGs have been attributed Ca(2) (+) -dependent roles, such as neural network development, axon pathfinding, plasticity and regeneration after CNS injury, CS action after ECM degradation could be contributing to the mediation of these effects through its interaction with AMPA and kainate receptors.
Subject(s)
Action Potentials/physiology , Chondroitin Sulfates/metabolism , Neurons/metabolism , Receptors, AMPA/metabolism , Receptors, Kainic Acid/metabolism , Animals , Cells, Cultured , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Hippocampus/metabolism , Patch-Clamp Techniques , Rats , Rats, Sprague-DawleyABSTRACT
INTRODUCTION: Chondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1ß) is regulated by CS, employing a novel quantitative proteomic approach. METHODS: Human articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1ß 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results. RESULTS: We could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1ß, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1. CONCLUSION: We have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1ß. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.
Subject(s)
Anabolic Agents/metabolism , Angiogenesis Inhibitors/metabolism , Anti-Inflammatory Agents/metabolism , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondroitin Sulfates/pharmacology , Cell Adhesion Molecules/metabolism , Cells, Cultured , Complement C1r/metabolism , Complement C1s/metabolism , Humans , Interleukin-1beta/pharmacology , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/metabolism , Thrombospondin 1/metabolismABSTRACT
This study aimed to assess whether chronic administration of chondroitin sulfate (CS) affects baseline expression of cytochrome P450 isoforms and impedes the decrease in expression and activity of CYP1A2 and CYP3A6 in rabbits with a turpentine-induced inflammatory reaction (TIIR). Seven groups of 5 rabbits, 3 control groups and 4 receiving 20 mg/kg/day of CS for 20 and 30 days, were used. The rabbits of 1 control group and 2 groups receiving CS had a TIIR; finally, the rabbits of one of the control groups remained in the animal facilities for 30 days to assess the effect of time and environment on cytochrome P450. In control rabbits, intake of CS for 20 and 30 days did not affect CYP3A6, CYP1A2 and NADPH cytochrome P450 reductase (CPR) mRNA, protein expression and activity. Compared with control rabbits, the TIIR not only reduced mRNA, protein expression and activity of CYP3A6 and CYP1A2 but also that of CPR. In rabbits with TIIR, CS prevented the decrease of CYP3A6 expression but not the reduction in activity. CS did not impede TIIR-induced down-regulation of CYP1A2. Hepatic NO() concentrations and NF-κB nuclear translocation were increased by the TIIR, effect reversed by CS. In vitro, in hepatocytes, CS did not alter the expression and activity of CYP3A6, CYP1A2, and CPR. In conclusion, oral CS elicits a systemic effect but does not affect CYP1A2, CYP3A6, and CPR in control rabbits, although in rabbits with TIIR, CS prevents CYP3A6 protein down-regulation but not that of CYP1A2.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Chondroitin Sulfates/pharmacology , Cytochrome P-450 CYP1A2/metabolism , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Turpentine/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Chondroitin Sulfates/chemistry , Cytochrome P-450 CYP1A2/biosynthesis , Cytochrome P-450 CYP1A2/genetics , Enzyme Inhibitors/chemistry , Inflammation/chemically induced , Inflammation/drug therapy , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Structure-Activity RelationshipABSTRACT
INTRODUCTION: This work aimed at comparing the production of inflammatory and pro- and anti-angiogenic factors by normal/reactive (N/R) or inflammatory (I) areas of the osteoarthritic synovial membrane. The effects of interleukin (IL)-1ß and chondroitin sulfate (CS) on the expression of pro- and anti-angiogenic factors by synovial fibroblasts cells (SFC) were also studied. METHODS: Biopsies from N/R or from I areas of osteoarthritic synovial membrane were collected at the time of surgery. The inflammatory status of the synovial membrane was characterized by the surgeon according to macroscopic criteria, including the synovial vascularization, the villi formation and the hypertrophic aspect of the tissue. We assessed the expression of CD45, von Willebrand factor and vascular endothelial growth factor (VEGF) antigen by immunohistochemistry in both N/R and I biopsies. The production of IL-6, -8, VEGF and thrombospondin (TSP)-1 by N/R or I synovial cells was quantified by ELISA. SFC were cultured in the absence or in the presence of IL-1ß (1 ng/ml) and with or without CS (10, 50, 200 µg/ml). Gene expression of pro-angiogenic factors (VEGF, basic fibroblast growth factor (bFGF), nerve growth factor (NGF), matrix metalloproteinase (MMP)-2 and angiopoietin (ang)-1) and anti-angiogenic factors (vascular endothelial growth inhibitor (VEGI), TSP-1 and -2) were determined by real time RT-PCR. Production of VEGI and TSP-1 was also estimated by ELISA. RESULTS: Immunohistochemistry showed the increase of lymphocyte infiltration, vascular density and VEGF expression in I compared to N/R synovial biopsies. Synovial cells from I areas produced more IL-6, IL-8 and VEGF but less TSP-1 than cells isolated from N/R synovial biopsies. The expression of pro-angiogenic factors by SFC was stimulated by IL-1ß. A time dependent regulation of the expression of anti-angiogenic factor genes was observed. IL-1ß stimulated the expression of anti-angiogenic factor genes but inhibited it after 24 h. CS reversed the inhibitory effect of IL-1ß on anti-angiogenic factors, VEGI and TSP-1. CONCLUSIONS: We demonstrated that synovial biopsies from I areas expressed a pro-angiogenic phenotype. IL-1ß induced an imbalance between pro- and anti-angiogenic factors in SFC and CS tended to normalize this IL-1ß-induced imbalance, providing a new possible mechanism of action of this drug.
Subject(s)
Chondroitin Sulfates/therapeutic use , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Osteoarthritis/drug therapy , Osteoarthritis/pathology , Synovial Membrane/pathology , Aged , Aged, 80 and over , Cells, Cultured , Female , Humans , Male , Middle Aged , Neovascularization, Pathologic/epidemiology , Osteoarthritis/epidemiology , Synovial Membrane/drug effectsABSTRACT
Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 µg/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies.
Subject(s)
Chondrocytes/drug effects , Chondrocytes/metabolism , Chondroitin Sulfates/pharmacology , Proteome/metabolism , Amino Acid Sequence , Cell Extracts/chemistry , Cells, Cultured , Extracellular Matrix Proteins/metabolism , Humans , Intracellular Fluid/metabolism , Molecular Sequence Data , Osteoarthritis/metabolism , Osteoarthritis/pathology , Peptide Fragments/chemistry , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Two-Dimensional Difference Gel ElectrophoresisABSTRACT
The glycosaminoglycan chondroitin sulfate (CS) is a major constituent of the extracellular matrix of the central nervous system where it can constitute part of the perineuronal nets. Constituents of the perineuronal nets are gaining interest because they have modulatory actions on their neighbouring neurons. In this study we have investigated if CS could afford protection in an acute in vitro ischemia/reoxygenation model by using isolated hippocampal slices subjected to 60min oxygen and glucose deprivation (OGD) followed by 120min reoxygenation (OGD/Reox). In this toxicity model, CS afforded protection of rat hippocampal slices measured as a reduction of lactate dehydrogenase (LDH) release; maximum protection (70% reduction of LDH) was obtained at the concentration of 3mM. To evaluate the intracellular signaling pathways implicated in the protective effect of CS, we first analysed the participation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 by western blot. OGD/Reox induced the phosphorylation of p38 and dephosphorylation of ERK1/2; however, CS only inhibited p38 but had no effect on ERK1/2. Furthermore, OGD/Reox-induced translocation of p65 to the nucleus was prevented in CS treated hippocampal slices. Finally, CS inhibited iNOS induction caused by OGD/Reox and thereby nitric oxide (NO) production measured as a reduction in DAF-2 DA fluorescence. In conclusion, the protective effect of CS in hippocampal slices subjected to OGD/Reox can be related to a modulatory action of the local immune response by a mechanism that implies inhibition of p38, NFκB, iNOS and the production of NO.
Subject(s)
Cell Death/drug effects , Chondroitin Sulfates/pharmacology , Glucose/metabolism , Hippocampus/drug effects , NF-kappa B/antagonists & inhibitors , Nitric Oxide Synthase Type II/antagonists & inhibitors , Oxygen/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Hippocampus/cytology , Hippocampus/metabolism , In Vitro Techniques , Male , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Phosphorylation , Rats , Rats, Sprague-Dawley , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
INTRODUCTION: Chondroitin sulfate (CS) and glucosamine sulfate (GS) are symptomatic slow-acting drugs for osteoarthritis (OA) widely used in clinic. Despite their widespread use, knowledge of the specific molecular mechanisms of their action is limited. The aim of this work is to explore the utility of a pharmacoproteomic approach for the identification of specific molecules involved in the pharmacological effect of GS and CS. METHODS: Chondrocytes obtained from three healthy donors were treated with GS 10 mM and/or CS 200 µg/mL, and then stimulated with interleukin-1ß (IL-1ß) 10 ng/mL. Whole cell proteins were isolated 24 hours later and resolved by two-dimensional electrophoresis. The gels were stained with SYPRORuby. Modulated proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry. Real-time PCR and Western blot analyses were performed to validate our results. RESULTS: A total of 31 different proteins were altered by GS or/and CS treatment when compared to control. Regarding their predicted biological function, 35% of the proteins modulated by GS are involved in signal transduction pathways, 15% in redox and stress response, and 25% in protein synthesis and folding processes. Interestingly, CS affects mainly energy production (31%) and metabolic pathways (13%), decreasing the expression levels of ten proteins. The chaperone GRP78 was found to be remarkably increased by GS alone and in combination with CS, a fact that unveils a putative mechanism for the reported anti-inflammatory effect of GS in OA. On the other hand, the antioxidant enzyme superoxide dismutase 2 (SOD2) was significantly decreased by both drugs and synergistically by their combination, thus suggesting a drug-induced decrease of the oxidative stress caused by IL-1ß in chondrocytes. CONCLUSIONS: CS and GS differentially modulate the proteomic profile of human chondrocytes. This pharmacoproteomic approach unravels the complex intracellular mechanisms that are modulated by these drugs on IL1ß-stimulated human articular chondrocytes.