E. Montell

Chondroitin and glucosamine sulfate in combination decrease the pro-resorptive properties of human osteoarthritis subchondral bone osteoblasts: a basic science study.

Early in the pathological process of osteoarthritis (OA), subchondral bone remodelling, which is related to altered osteoblast metabolism, takes place. In the present study, we explored in human OA subchondral bone whether chondroitin sulfate (CS), glucosamine sulfate (GS), or both together affect the major bone biomarkers, osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and the pro-resorptive activity of OA osteoblasts. The effect of CS (200 μg/mL), GS (50 and 200 μg/mL), or both together on human OA subchondral bone osteoblasts, in the presence or absence of 1,25(OH)2D3 (vitamin D3) (50 nM), was determined on the bone biomarkers alkaline phosphatase and osteocalcin, on the expression (mRNA) and production (enzyme-linked immunosorbent assay) of bone remodelling factors OPG and RANKL, and on the pro-resorptive activity of these cells. For the latter experiments, human OA osteoblasts were incubated with differentiated peripheral blood mononuclear cells on a sub-micron synthetic calcium phosphate thin film. Data showed that CS and GS affected neither basal nor vitamin D3-induced alkaline phosphatase or osteocalcin release. Interestingly, OPG expression and production under basal conditions or vitamin D3 treatment were upregulated by CS and by both CS and GS incubated together. Under basal conditions, RANKL expression was significantly reduced by CS and by both drugs incubated together. Under vitamin D3, these drugs also showed a decrease in RANKL level, which, however, did not reach statistical significance. Importantly, under basal conditions, CS and both compounds combined significantly upregulated the expression ratio of OPG/RANKL. Vitamin D3 decreased this ratio, and GS further decreased it. Both drugs reduced the resorption activity, and statistical significance was reached for GS and when CS and GS were incubated together. Our data indicate that CS and GS do not overly affect cell integrity or bone biomarkers. Yet CS and both compounds together increase the expression ratio of OPG/RANKL, suggesting a positive effect on OA subchondral bone structural changes. This was confirmed by the decreased resorptive activity for the combination of CS and GS. These data are of major significance and may help to explain how these two drugs exert a positive effect on OA pathophysiology.

Immunomodulatory and anti-inflammatory effects of chondroitin sulphate.

•  Biochemistry of chondrotin sulphate •  Mechanism of action of chondroitin sulphate ‐  Effect of chondroitin sulphate on the chondrocyte ‐  Effect of chondroitin sulphate on the synovial membrane ‐  Effect of chondroitin sulphate on subchondral bone •  Human use of chondroitin sulphate ‐  Chondrotin sulphate in osteoarthritis ‐  Chondrotin sulphate in psoriasis ‐  Chondrotin sulphate in atherosclerosis ‐  Chondroitin sulphate in IBD ‐  Chondroitin sulphate in degenerative diseases of the central nervous system (CNS) ‐  Other autoimmune diseases that may benefit from chondroitin sulphate •  Conclusions

Chondroitin Sulfate Protects SH-SY5Y Cells from Oxidative Stress by Inducing Heme Oxygenase-1 via Phosphatidylinositol 3-Kinase/Akt

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 μM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 μM. Cell death caused by a combination of 10 μM rotenone plus 1 μM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 μM; in this toxicity model, maximum protection was achieved at 3 μM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.

Gene expression pattern of cells from inflamed and normal areas of osteoarthritis synovial membrane

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).

Gene expression pattern of synovial cells from inflammatory and normal areas of osteoarthritis synovial membrane.

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).

Pharmacoproteomic study of the effects of chondroitin and glucosamine sulfate on human articular chondrocytes

IntroductionChondroitin 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.MethodsChondrocytes obtained from three healthy donors were treated with GS 10 mM and/or CS 200 μg/mL, and then stimulated with interleukin-1β (IL-1β) 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.ResultsA 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-1β in chondrocytes.ConclusionsCS 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 IL1β-stimulated human articular chondrocytes.

Antioxidant, antiinflammatory and neuroprotective actions of chondroitin sulfate and proteoglycans

The antiinflammatory and antiapoptotic effects of chondroitin sulfate (CS) are being used to treat osteoarthritis. Recent evidence has revealed that those peripheral effects of CS may also have therapeutic interest in diseases of the central nervous system (CNS). We review here such evidence. Perineuronal nets (PNNs) formed by chondroitin sulfate proteoglycans (CSPGs) may have a neuroprotective action against oxidative stress potentially involved in neurodegeneration. On the other hand, in human neuroblastoma SH-SY5Y cells CS has antioxidant and neuroprotective effects by activating the signaling pathway PKC/PI3K/Akt and inducing the antioxidant enzyme hemoxygenase-1. Consistent with this is the observation that protein kinase C (PKC) blockade overcomes inhibition of neurite outgrowth elicited by CSPGs. In addition, CS protects cortical neurons against excytotoxic death by phosphorylation of intracellular signals and the suppression of caspase-3 activation. Of interest is the finding that a disaccharide derived from CSPG degradation (CSGP-DS) protects neurons against toxicity both in vitro and in vivo. Furthermore, CSGP-DS efficiently protects against neuronal loss in experimental autoimmune encephalomyelitis and uveitis, decreases secretion of tumor necrosis factor-alpha (TNF-alpha) and block necrosis factor kappa B (NF-kappaB) translocation. In conclusion, CS may have neuroprotective properties linked to its antioxidant and antiinflammatory effects.

Pharmacoproteomic Study of Three Different Chondroitin Sulfate Compounds on Intracellular and Extracellular Human Chondrocyte Proteomes

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.

Characterization of synovial angiogenesis in osteoarthritis patients and its modulation by chondroitin sulfate

IntroductionThis 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)-1β and chondroitin sulfate (CS) on the expression of pro- and anti-angiogenic factors by synovial fibroblasts cells (SFC) were also studied.MethodsBiopsies 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-1β (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.ResultsImmunohistochemistry 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-1β. A time dependent regulation of the expression of anti-angiogenic factor genes was observed. IL-1β stimulated the expression of anti-angiogenic factor genes but inhibited it after 24 h. CS reversed the inhibitory effect of IL-1β on anti-angiogenic factors, VEGI and TSP-1.ConclusionsWe demonstrated that synovial biopsies from I areas expressed a pro-angiogenic phenotype. IL-1β induced an imbalance between pro- and anti-angiogenic factors in SFC and CS tended to normalize this IL-1β-induced imbalance, providing a new possible mechanism of action of this drug.

Effectiveness of chondroitin sulphate in patients with concomitant knee osteoarthritis and psoriasis: a randomized, double-blind, placebo-controlled study

OBJECTIVE The aim of the trial was to assess the efficacy of chondroitin sulphate (CS) on symptomatic knee osteoarthritis (OA) associated to psoriasis. METHODS In this randomized, double-blind, placebo (PBO)-controlled clinical trial 129 patients with symptomatic knee OA and concomitant psoriasis were randomized into two groups receiving 800 mg daily of CS or PBO for 3 months. The primary efficacy outcome for knee OA was the Huskissons visual analogue scale (VAS) and for psoriasis was the Psoriasis Area and Severity Index (PASI). Additionally, other secondary efficacy criteria for both conditions were assessed. RESULTS After 3 months of treatment, CS was more effective than PBO, relieving pain VAS (CS -26.9+/-24.8 vs PBO -14.23+/-20.8mm, P<0.01), decreasing the Lequesne index (CS -4.8+/-3.4 vs PBO -3.3+/-3.5, P<0.05) and reducing the number of patients using acetaminophen as rescue medication (CS 43% vs PBO 64%, P<0.05). Regarding PASI, Overall Lesion Severity Scale and Physicians Global Assessment of Change no statistically significant changes were detected in front of PBO. However, CS improved plantar psoriasis compared to PBO (CS 87% vs PBO 27%, P<0.05). Quality of life improved significantly in CS-treated patients according to the Short Form-36 health survey and the Dermatology Life Quality Index (DLQI). CS tolerability was excellent. Adverse events were infrequent and evenly distributed among groups. The incidence of psoriatic flares did not increase after treatments. CONCLUSIONS This study confirms the efficacy and safety of CS as a symptomatic slow-acting drug in patients with knee OA and shows that CS improves plantar psoriasis. The use of CS could represent a special benefit in patients with both pathologies since non-steroidal anti-inflammatory drugs have been reported to induce or exacerbate psoriasis.