High in vivo levels of adipsin lead to increased knee tissue degradation in osteoarthritis: data from humans and animal models.

Objective: This study explored the role of the adipokine adipsin in OA. Methods: Control and OA articular tissues, cells and serum were obtained from human individuals. Serum adipsin levels of human OA individuals were compared with cartilage volume loss as assessed by MRI at 48 months. Human adipsin expression was determined by PCR, its production in tissues by immunohistochemistry, and in SF and serum by a specific assay. OA was surgically induced in wild-type (Df+/+) and adipsin-deficient (Df-/-) mice, and synovial membrane and cartilage processed for histology and immunohistochemistry. Results: Adipsin levels were significantly increased in human OA serum, SF, synovial membrane and cartilage compared with controls, but the expression was similar in chondrocytes, synoviocytes and osteoblasts. Multivariate analysis demonstrated that human serum adipsin levels were significantly associated (P = 0.045) with cartilage volume loss in the lateral compartment of the knee. Destabilization of the medial meniscus-Df-/- mice showed a preservation of the OA synovial membrane and cartilage lesions (P ⩽ 0.026), the latter corroborated by the decreased production of cartilage degradation products and proteases (P ⩽ 0.047). The adipsin effect is likely due to a deficient alternative complement pathway (P ⩽ 0.036). Conclusion: In human OA, higher serum adipsin levels were associated with greater cartilage volume loss in the lateral compartment, and adipsin deficiency led to a preservation of knee structure. Importantly, we documented an association between adipsin and OA synovial membrane and cartilage degeneration through the activation of the complement pathway. This study highlights the clinical relevance of adipsin as a valuable biomarker and potential therapeutic target for OA.

Levels of serum biomarkers from a two-year multicentre trial are associated with treatment response on knee osteoarthritis cartilage loss as assessed by magnetic resonance imaging: an exploratory study.

BACKGROUND: There is an obvious need to identify biomarkers that could predict patient response to an osteoarthritis (OA) treatment. This post hoc study explored in a 2-year randomized controlled trial in patients with knee OA, the likelihood of some serum biomarkers to be associated with a better response to chondroitin sulfate in reducing cartilage volume loss. METHODS: Eight biomarkers were studied: hyaluronic acid (HA), C reactive protein (CRP), adipsin, leptin, N-terminal propeptide of collagen IIα (PIIANP), C-terminal crosslinked telopeptide of type I collagen (CTX-1), matrix metalloproteinase-1 (MMP-1), and MMP-3. Patients were treated with chondroitin sulfate (1200 mg/day; n = 57) or celecoxib (200 mg/day; n = 62). Serum biomarkers were measured at baseline. The cartilage volume at baseline and its loss at 2 years were assessed by quantitative magnetic resonance imaging (MRI). Statistical analysis included analysis of covariance. RESULTS: As data from the original MOSAIC trial showed no differences in cartilage volume and loss in the lateral compartment of the knee joint between the two treatment groups in any comparison, only the medial compartment and its subregions were studied. Stratification according to the median biomarker levels was used to discriminate treatment effect. In patients with levels of biomarkers of inflammation (HA, leptin and adipsin) lower than the median, those treated with chondroitin sulfate demonstrated less cartilage volume loss in the medial compartment, condyle, and plateau (p ≤ 0.047). In contrast, patients treated with chondroitin sulfate with higher levels of MMP-1 and MMP-3, biomarkers of cartilage catabolism, had less cartilage volume loss in the medial compartment, condyle, and plateau (p ≤ 0.050). Patients with higher levels of PIIANP and CTX-1, biomarkers related to collagen anabolism and bone catabolism, respectively, had reduced cartilage volume loss in the medial condyle (p ≤ 0.026) in the chondroitin sulfate group. CONCLUSION: This study is suggestive of a potentially greater response to chondroitin sulfate treatment on cartilage volume loss in patients with knee OA with low level of inflammation and/or greater level of cartilage catabolism. TRIAL REGISTRATION: This is a post hoc study. Original trial registration: ClinicalTrials.gov, NCT01354145 . Registered on 13 May 2011.

Influence of Tumor Necrosis Factor α, Parathyroid Hormone, and Vitamin D3 on Modulation of the RANKL2 Isoform: A Pilot Study.

RANKL exists as three isoforms: RANKL1, 2, and 3. RANKL1 and 3 were reported to be differently expressed upon treatment with some osteotropic factors, but RANKL2 expression could not be reliably determined. Here, we investigated through a mechanistic model, human 293 cells stably transfected with the RANKL2cDNA, the production and modulation of RANKL2 protein stability upon treatment with TNF-α, vitamin D3, and PTH. Data showed that TNF-a significantly increased (p<0.03) RANKL2 production and its half-life/stability (p<0.005). Vitamin D3 and PTH had no effect. This information will help to better define and differentiate the pathological mechanisms operating during osteolytic diseases.

Glucosamine sulfate reduces prostaglandin E(2) production in osteoarthritic chondrocytes through inhibition of microsomal PGE synthase-1.

OBJECTIVE: Glucosamine sulfate (GS) has been inferred to have a potential antiinflammatory effect on osteoarthritis (OA). We investigated its effect on prostaglandin E(2) (PGE(2)) in human OA chondrocytes, and the level in the PGE(2) pathway at which its effect takes place. METHODS: We investigated the effect of GS treatment (0.05, 0.2, 1.0, and 2.0 mM) in OA chondrocytes in the absence or presence of interleukin 1ß (IL-1ß; 100 pg/ml). We determined the expression levels and protein production/activity of PGE(2), cyclooxygenase-1 (COX-1), COX-2, microsomal PGE synthase-1 (mPGES-1), glutathione, and peroxisome proliferator-activated receptor-γ (PPARγ), using specific primers, antibodies, and assays. RESULTS: GS treatment at 1 and 2 mM significantly inhibited (p ≤ 0.03) production of endogenous and IL-1ß-induced PGE(2). GS in both the absence and presence of IL-1ß did not significantly modulate COX-1 protein production, but GS at 1 and 2 mM demonstrated a decrease in COX-2 glycosylation in that it reduced the molecular mass of COX-2 synthesis. Under IL-1ß stimulation, GS significantly inhibited mPGES-1 messenger RNA expression and synthesis at 1 and 2 mM (p ≤ 0.02) as well as the activity of glutathione (p ≤ 0.05) at 2 mM. Finally, in both the absence and presence of IL-1ß, PPARγ was significantly induced by GS at 1 and 2 mM (p ≤ 0.03). CONCLUSION: Our data document the potential mode of action of GS in reducing the catabolism of OA cartilage. GS inhibits PGE(2) synthesis through reduction in the activity of COX-2 and the production and activity of mPGES-1. These findings may, in part, explain the mechanisms by which this drug exerts its positive effect on OA pathophysiology.

Strontium ranelate inhibits key factors affecting bone remodeling in human osteoarthritic subchondral bone osteoblasts.

INTRODUCTION: In osteoarthritis (OA) the progression of cartilage degeneration has been associated with remodeling of the subchondral bone. Human OA subchondral bone osteoblasts were shown to have an abnormal phenotype and altered metabolism leading to an abnormal resorptive process. Bone resorption is suggested to occur, at least in part, through the increased levels of two proteolytic enzymes, MMP-2 and MMP-9, and RANKL, which are mainly produced by osteoblasts. In this study, we investigated in human OA subchondral bone osteoblasts the modulatory effect of strontium ranelate on the above key factors. METHODS: Human subchondral bone osteoblasts were cultured in a medium containing 0.1, 1 and 2 mM of strontium ranelate for 18 h for mRNA and 72 h for protein determination. The effect of strontium ranelate was evaluated on the expression (qPCR) of MMP-2, MMP-9, OPG, RANKL (total), RANKL-1, and RANKL-3, on the production of OPG (ELISA), membranous RANKL (flow cytometry), and MT1-MMP, ADAM17, and ADAM19 (Western blot). After incubation of osteoblasts with pre-osteoclasts (i.e., differentiated human peripheral blood mononuclear cells), the resorbed surface was measured using a sub-micron synthetic calcium phosphate thin film. RESULTS: Firstly, the expression levels of MMP-2, MMP-9, OPG, and RANKL were determined in normal and OA subchondral bone osteoblasts. As expected, the gene expression of MMP-9 and RANKL were not detectable in normal cells, whereas MMP-2 was very low but detectable and OPG demonstrated high gene expression. Further experiments looking at the effect of strontium ranelate on expression levels, except for OPG, were performed only on the OA subchondral bone osteoblasts. In OA cells, the expression levels of MMP-2 and MMP-9 were significantly decreased by strontium ranelate at 1mM (p≤0.005, p≤0.02, respectively) and 2 mM (p≤0.003, p≤0.007), and for MMP-9 only at 0.1 mM (p≤0.05). In normal cells, the expression of OPG was increased with strontium ranelate at 2 mM, and in OA both the expression (p≤0.02) and synthesis (p≤0.002) of OPG were significantly increased with strontium ranelate at 1 and 2 mM. RANKL (total) as well as the isoforms RANKL-1 and RANKL-3 were significantly increased by strontium ranelate at 1 and 2 mM. Of note, it is known that the different RANKL isoforms differentially regulate RANKL membranous localization: RANKL-3, in contrast to RANKL-1, prevents such membranous localization. This is reflected by the significant (p≤0.02) reduction in the level of membranous RANKL by strontium ranelate at 2 mM. This latter finding was not likely to be related to a proteolytic cleavage of membranous RANKL, as the enzymes known to cleave it, MT1-MMP, ADAM17 and ADAM19, were unaffected by strontium ranelate. In addition, OA osteoblasts treated with strontium ranelate induced a significant (p≤0.002) decrease in resorbed surface at the three tested concentrations. CONCLUSION: This study provides new insights into the mode of action of strontium ranelate on the metabolism of human OA subchondral bone osteoblasts. These data suggest that strontium ranelate may exert a positive effect on OA pathophysiology by inhibiting, in these cells, the synthesis of key factors leading to bone resorption, a feature associated with the OA process.

Variable effects of 3 different chondroitin sulfate compounds on human osteoarthritic cartilage/chondrocytes: relevance of purity and production process.

OBJECTIVE: During osteoarthritis (OA), the altered metabolism of cartilage involves proinflammatory factors and matrix metalloprotease (MMP) activity. Studies showed that chondroitin sulfate (CS) may exert a positive effect on the cartilage. Because of differences in CS in terms of purity and the production/purification process, we compared the effects of 3 different types of CS on human OA cartilage. METHODS: Three types of CS were tested: CS1 (porcine, purity 90.4%), CS2 (bovine, purity 96.2%), and CS3 (bovine, purity 99.9%). Treatment with CS at 200 and 1000 microg/ml was performed on human OA cartilage explants in the presence/absence of interleukin 1ss (IL-1ss), and the protein modulations of factors including prostaglandin E(2) (PGE(2)), IL-6, and MMP-1 measured by ELISA. The CS effect on the expression of collagen type II was also investigated on OA chondrocytes using quantitative polymerase chain reaction. RESULTS: In the presence of IL-1ss, CS2 at 1000 microg/ml significantly inhibited IL-6 and PGE(2) production, and CS3 at 200 microg/ml markedly reduced the level of IL-6. CS1 was much less efficient at reducing the catabolic markers and in the absence of IL-1ss, it significantly increased IL-6 and MMP-1. IL-1ss significantly inhibited the gene expression level of collagen type II; only CS3 was able to limit this inhibition. CS1, in the presence or absence of IL-1ss, further markedly decreased collagen type II expression. CONCLUSION: Our data indicate that among the 3 tested CS, CS1 increased production of some catabolic pathways and inhibited the gene expression level of collagen type II. Our study provides new information in the context of prescribing CS for alleviating OA symptoms, as the purity and/or production/purification of the CS compound could orient the current OA disease process toward increased catabolic pathways.

The peroxisome proliferator-activated receptor gamma agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: in vivo protective effects mediated through the inhibition of key signaling and catabolic pathways.

OBJECTIVE: Emerging evidence indicates that peroxisome proliferator-activated receptor gamma (PPARgamma) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARgamma agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes. METHODS: OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP-1), ADAMTS-5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK-1/2, p38, JNK, and NF-kappaB p65. RESULTS: Pioglitazone reduced the development of cartilage lesions in a dose-dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP-1, ADAMTS-5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK-1/2, p38, and NF-kappaB. CONCLUSION: These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARgamma activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site.

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.

The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases.

This study sought to evaluate the levels of mRNA expression and protein synthesis of MMP-13, cathepsin K, aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5) and 5-lipoxygenase (5-LOX) in cartilage in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA), and to examine the effects of treatment with licofelone, a 5-lipoxygenase (LOX)/cyclooxygenase (COX) inhibitor, on the levels of these catabolic factors. Sectioning of the ACL of the right knee was performed in three experimental groups: group 1 received no active treatment (placebo group); and groups 2 and 3 received therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day orally, respectively) for 8 weeks, beginning the day following surgery. A fourth group consisted of untreated dogs that were used as normal controls. Specimens of cartilage were selected from lesional areas of OA femoral condyles and tibial plateaus, and were processed for real-time quantitative PCR and immunohistochemical analyses. The levels of MMP-13, cathepsin K, ADAMTS-4, ADAMTS-5 and 5-LOX were found to be significantly increased in OA cartilage. Licofelone treatment decreased the levels of both mRNA expression and protein synthesis of the factors studied. Of note was the marked reduction in the level of 5-LOX gene expression. The effects of the drug were about the same at both tested dosages. In vivo treatment with therapeutic dosages of licofelone has been found to reduce the degradation of OA cartilage in experimental OA. This, coupled with the results of the present study, indicates that the effects of licofelone are mediated by the inhibition of the major cartilage catabolic pathways involved in the destruction of cartilage matrix macromolecules. Moreover, our findings also indicate the possible auto-regulation of 5-LOX gene expression by licofelone in OA cartilage.

Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids.

OBJECTIVE: To explore the modulation of 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX) expression in human osteoarthritic (OA) chondrocytes, their relative implications in leukotriene B(4) (LTB(4)) production, the effect of different factors on this system, and the influence of increased LTB(4) production on the synthesis of catabolic factors of cartilage. METHODS: FLAP and 5-LOX expression and LTB(4) production were monitored following treatment with transforming growth factor beta1 (TGFbeta1; 5 ng/ml) and 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3); 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 mug/ml), NS-398 (10 muM), or FR122047 (5 muM), or a dual inhibitor of COX/5-LOX activity, licofelone (2.6 muM). LTB(4), prostaglandin E(2) (PGE(2)), and matrix metalloprotease 1 (MMP-1) production were measured by specific enzyme-linked immunosorbent assays, nitric oxide by the Griess reaction, and FLAP and 5-LOX expression by quantitative polymerase chain reaction. RESULTS: Human OA chondrocytes expressed both FLAP and 5-LOX. TGFbeta1 and/or 1,25(OH)(2)D(3) induced a rapid and marked enhancement ( approximately 4-13-fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB(4) production and PGE(2) synthesis. Treatment with COX inhibitors in the absence or presence of TGFbeta1 and 1,25(OH)(2)D(3) induced a rapid increase in LTB(4) production; this response was mediated by the sustained and significant (P < 0.01) up-regulation ( approximately 1.5-fold) of 5-LOX mRNA levels. Conversely, treatment with licofelone showed no effect on 5-LOX but significantly reduced FLAP expression levels. Coincubation of licofelone with TGFbeta1 plus 1,25(OH)(2)D(3) did not affect FLAP or 5-LOX levels. In the presence of TGFbeta1 plus 1,25(OH)(2)D(3), naproxen, but not licofelone, induced MMP-1 production and both drugs decreased nitric oxide levels. CONCLUSION: Both the eicosanoids PGE(2) and LTB(4) are important cofactors in regulating FLAP/5-LOX expression; the inhibition of PGE(2) up-regulates 5-LOX while down-regulating FLAP gene expression, and LTB(4) appears to be an up-regulating factor on the 5-LOX gene. Importantly, nonsteroidal antiinflammatory drugs up-regulate the synthesis of LTB(4), supporting the shunt hypothesis from COX to 5-LOX. We also demonstrated that LTB(4) likely contributes to the up-regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.

Nitric oxide induced cell death in human osteoarthritic synoviocytes is mediated by tyrosine kinase activation and hydrogen peroxide and/or superoxide formation.

OBJECTIVE: To investigate the regulation of osteoarthritis (OA) synovial fibroblast nitric oxide (NO) induced cell death. METHODS: Cultured synovial fibroblasts from human OA synovium were incubated with NO donor sodium nitroprusside (SNP) in the absence or presence of specific inhibitors of different protein kinases, cyclooxygenase-2 (COX-2), caspase-3 and caspase-9, inducible NO synthase, and in the absence or presence of prostaglandin E2 (PGE2). Experiments were also performed using scavengers of NO (carboxy-PXTO), peroxynitrite (uric acid), and superoxide (taxifolin). The level of cell death was measured by MTT and DNA fragmentation. RESULTS: Human OA synovial fibroblasts incubated with SNP decreased cell viability and increased DNA fragmentation in a dose dependent manner. This was associated with increased levels of both COX-2 and PGE2 production. Selective inhibition of COX-2 by NS-398 significantly inhibited SNP induced cell death, even in the presence of exogenously added PGE2. Experiments revealed that SNP treated cells expressed increased levels of active caspase-3 and caspase-9, while Bcl-2 was downregulated. Incubation of these treated cells with inhibitors of caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) protected viability of SNP treated OA synovial fibroblasts, indicating that NO mediated cell death was mainly related to apoptosis. This was also confirmed by measuring the DNA fragmentation (TUNEL method) and the level of active caspase-3 (immunocytochemistry) in these cells. Data also showed that SNP induces the activation of kinases MEK 1/2, p38, and tyrosine kinases. Specific inhibition of tyrosine kinases completely abrogated the SNP induced cell death. In turn, this cell death protection was associated with a marked inhibition of caspase-3 and caspase-9 activities, as well as COX-2/PGE2 production. Moreover, data showed that the NO donor SNP induced cell death was not solely related to the production of NO or peroxynitrite, but to the generation of reactive oxygen species (ROS) such as hydrogen peroxide and/or superoxide. CONCLUSION: Our results provided strong evidence of the role of tyrosine kinase and mitogen activated protein kinase activation, by upregulation of COX-2 expression, in NO induced OA synovial fibroblast death. The generation of ROS such as hydrogen peroxide and superoxide appeared to be a major factor in the death of these cells.