Proteoglycan Release Research Articles

Interleukin-1B IS essential for blood-induced cartilage damage in vitro

Purpose: Exposure of joint cartilage to blood can occur after joint trauma, during or after major joint surgery, or due to hemophilia. This ultimately leads to joint damage, both by direct effects of blood on cartilage and via synovial inflammation. Cartilage destruction is considered to result from both cartilage matrix-degrading proteases as well as cartilage-destructive pro-inflammatory cytokines such as IL1β. To unravel the role of IL1β in the pathogenesis of blood-induced cartilage damage, we investigated whether direct blocking of IL1β specifically prevents blood-induced cartilage damage in vitro. Methods: Full thickness healthy human articular cartilage explants, obtained post-mortem, were cultured for 4 days in presence or absence of 50% v/v whole blood. A recombinant human IL1β monoclonal antibody (IL1β mAb) was only added during blood exposure in a concentration of 0, 1, 2, 10, 30, or 100 ng/mL. Cartilage matrix proteoglycan turnover was determined 12 days later to analyse long-term effects. Moreover, to investigate the direct effects of IL1β mAb on cartilage, explants were cultured for 4 days in the presence of 10 ng/mL IL1β mAb in the absence of blood. Results: Exposure to blood decreased the proteoglycan-synthesis rate (-62%) and proteoglycan content (-19%), and increased the proteoglycan release (+191%, all p < 0.05). Adding IL1βmAb resulted in a dose-dependent increase of the proteoglycan synthesis rate leading to normalisation at higher concentrations (see figure A). Moreover, proteoglycan release was statistically significantly decreased from a concentration of 3ng/mL and above (see figure B). Similar, the proteoglycan content increased statistically significantly upon addition of the IL1βmAb (3 ng/mL and above; all p < 0.05). In the absence of blood, IL1βmAb did not have direct effects on cartilage proteoglycan turnover (p = 0.51). Conclusions: This study demonstrates that IL1β is a crucial factor in the development of blood-induced cartilage damage in vitro. Blocking this pro-inflammatory cytokine with a monoclonal antibody protects cartilage from the damaging effects of blood exposure. Further research is warranted to investigate the in vivo capacity of IL1β mAb in prevention and its position as a treatment of hemophilic arthropathy.

Lack of sclerostin promotes osteoarthritis by activating canonical and non-canonical WNT pathways

Lack of sclerostin promotes osteoarthritis by activating canonical and non-canonical WNT pathways

A fusion protein of IL4 and IL10 (IL4-10 synerkine), is very effective in protecting cartilage from blood-induced damage

Purpose: Interleukin 4 (IL4), IL10 or the combination of both protects cartilage from blood-induced damage in vitro. Because IL4 and IL10 use different signaling pathways they are able to exert different, but also potentially additive effects. It has been postulated that they can act in synergy enhancing their beneficial effects and controlling their individual adverse effects. Therefore a combination of IL4 and IL10 might be the best option to administer in vivo. To overcome the low bioavailability of IL4 and IL10 in vivo, a fusion protein has been developed: IL4-10 synerkine. It has been demonstrated that this novel drug inhibits numerous pro-inflammatory cytokines secreted by monocytes and T-cells. This study investigates whether this IL4-10 synerkine protects against blood-induced cartilage damage similarly as the combination of the individual components. Methods: Human cartilage explants were exposed to 50% v/v whole blood for 4 days and simultaneously to a broad concentration range (0-100 ng/mL) of the IL4-10 synerkine. Effects of 10 ng/mL IL4-10 synerkine were compared to the same concentrations of the individual cytokines and the combination. Cartilage matrix proteoglycan turnover was assessed after a recovery period of 12 days. Moreover, the influence of IL4-10 synerkine (10 ng/mL) and its individual components on levels of IL1β and IL6 were investigated in a 4 days 50% v/v whole blood culture. Results: Exposure to 50% v/v whole blood resulted in a clear statistical significant decrease of proteoglycan (PG) synthesis rate (-74%; p = 0.012) and an increase of PG release (+92%, p = 0.017) compared to control. Adding of IL4-10 synerkine, resulted in a clear dose-response curve, leading to full normalisation of PG synthesis rate and -release at higher concentrations (>10 ng/mL). These results were similar to the effect of the two individual cytokines combined (see figure). The individual cytokines had a more distinct profile where IL4 was superior over IL10 (see figure). Moreover, addition of IL4-10 synerkine reduced IL1β and IL6 production in whole blood cultures to control levels (both p < 0.05), similar to the combination of IL4 plus IL10 (p = 0.180 and p = 0.173, respectively). Conclusions: IL4-10 synerkine strongly protects against blood-induced cartilage damage in vitro, presumably at least in part by reduction of pro-inflammatory cytokines including IL1β and IL6. Considering better bioavailability and more easy application of this synerkine compared to the individual cytokines, testing the IL4-10 synerkine in an in vivo model of blood-induced cartilage damage is warranted.

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes.

IntroductionOsteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.MethodsHuman synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.ResultsExosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.ConclusionsExosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage

Objective: Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation. Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E 2 (PGE 2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days. Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death ( p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG ( p<0.05) and PGE 2 release ( p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release ( p<0.01). Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.

Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage

Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation. Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E 2 (PGE 2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days. Curcumin induced chondrocyte death in primary cultures (50μM p<0.001and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death ( p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG ( p<0.05) and PGE 2 release ( p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release ( p<0.01). Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.

Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo

Cartilage destruction is a crucial process in arthritis and is characterized by the degradation of cartilage proteins, proteoglycans, and type II collagen (CII), which are embedded within the extracellular matrix. While proteoglycan loss can be reversed, the degradation of CII is irreversible and has been correlated with an over-expression and over-activation of matrix metalloproteinases (MMPs). Among the various MMPs, the collagenase MMP-13 possesses the greatest catalytic activity for CII degradation. Here we show that the pomegranate-derived polyphenols, punicalagin (PA) and ellagic acid (EA), inhibit MMP-13-mediated degradation of CII in vitro. Surface plasmon resonance studies and molecular docking simulations suggested multiple binding interactions of PA and EA with CII. The effects of PA on bovine cartilage degradation (stimulated with IL-1β) were investigated by assaying proteoglycan and CII release into cartilage culture media. PA inhibited the degradation of both proteins in a concentration-dependent manner. Finally, the anti-inflammatory effects of PA (daily IP delivery at 10 and 50mg/kg for 14days) were tested in an adjuvant-induced arthritis rat model. Disease development was assessed by daily measurements of body weight and paw volume (using the water displacement method). PA had no effect on disease development at the lower dose but inhibited paw volume (P<0.05) at the higher dose.

THU0035 Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

BackgroundOsteoarthritis (OA) and Rheumatoid Arthrisis (RA) are characterized by degradation of the cartilage. Proteases of the coagulation cascade and the fibrinolytic system, such as thrombin and plasmin, are elevated in...

AB0134 Blood coagulation aggravates joint damage after an experimental haemorrhage in the canine knee

BackgroundJoint bleeding due to trauma, major joint surgery, or haemophilia leads to joint damage. However, it is unclear if there are differences between coagulating blood and anticoagulated blood with respect...

A short time window to profit from protection of blood-induced cartilage damage by IL-4 plus IL-10

IL-4 plus IL-10 prevents blood-induced cartilage damage. The aim of the present study was to evaluate whether cartilage damage can still be averted by addition of IL-4 plus IL-10 when added after the onset of a bleed and whether aspiration of blood prior to addition of IL-4 plus IL-10 is of additive protective value. Healthy canine hip and human shoulder cartilage was exposed to whole blood for 4 days. IL-4 plus IL-10 was administered directly or after a delay of several hours up to 2 days. Furthermore, blood was aspirated after 1 or 2 days and subsequently IL-4 plus IL-10 was added. IL-1β concentration and cartilage matrix proteoglycan turnover were determined. Exposure of canine and human cartilage to blood decreased the proteoglycan synthesis rate and content and increased proteoglycan release. IL-4 plus IL-10 only prevented blood-induced damage of canine cartilage when added directly, not after 4 h or later. For human cartilage, IL-4 plus IL-10 limited blood-induced damage as well as IL-1β production when administered within 4-8 h after the onset of a bleed, but not thereafter. Aspiration of blood within 24 h fully prevented cartilage damage. Subsequent addition of IL-4 plus IL-10 was not of additive value. For humans, there is a short time window after onset of a joint bleed in which IL-4 plus IL-10 can limit blood-induced cartilage damage. Furthermore, aspiration of a joint to shorten blood exposure fully prevents cartilage damage. Both options can be considered in the treatment of a joint haemorrhage.

Histamine contributes to increased RANKL to osteoprotegerin ratio through altered nuclear receptor 4A activity in human chondrocytes

To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage. Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA. Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression. Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.

Thrombospondin‐1 signaling via CD47 regulates T lymphocyte glycosaminoglycan biosynthesis

T cells express two cell surface proteoglycans that bind thrombospondin‐1: CD47 and APLP2. CD47 mediates inhibition of T cell signaling by thrombospondin‐1, which requires glycosaminoglycan modification of CD47 at Ser64. This prompted us to examine how T cell activation modulates CD47 glycosylation. T cell activation increased both heparan and chondroitin sulfate biosynthesis by globally inducing mRNA levels of the respective glycosyltransferases and sulfotransferases. Activation resulted in larger glycosaminoglycan chains with a decreased density of sulfation, a net shift towards increased chondroitin sulfate, and increased release of proteoglycan into the medium. These changes resulted in altered modification of CD47. T cell activation in the presence of thrombospondin‐1 inhibited these changes, but not in cells lacking CD47. Remarkably, CD47 null T cells exhibited delayed induction of mRNAs for glycosaminoglycan biosynthetic enzymes upon anti‐CD3 stimulation. Re‐expression of CD47 in CD47 mutant cells restored activation‐dependent mRNA induction and sensitivity to thrombospondin‐1. These data reveal a negative feedback loop by which thrombospondin‐1 controls the level of an essential post‐translational modification of its own signaling receptor in T cells.

Dkk3 Can prevent cartilage degradation and modulate TGFbeta and Wnt signalling

Purpose: We have previously shown that Dkk3 expression is increased in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The factors regulating Dkk3 expression in cartilage and the effect of Dkk3 on chondrocyte function are poorly ascribed. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFb signalling in other cell systems. Methods: Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1 (IL1) and oncostatin-M (OSM). The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/-Wnt3a, TGFb and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/-TGFb or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFb signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/Dkk3 and proteoglycan output assessed using alcian blue staining. b-catenin was silenced in primary HAC prior to TGFb and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results: Dkk3 expression was decreased in primary HAC following IL1/ OSM treatment. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonized Wnt signalling, decreasing Wnt3a-induced AXIN2 expression and luciferase expression from the TOPFlash reporter. Dkk3 also antagonized Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFb signalling, increasing TGFb-induced TIMP3 and ADAM12 expression and TGFb-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonized Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. b-catenin knockdown did not significantly alter TGFbor Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Knockdown of Dkk3 in primary HAC resulted in altered expression of a number of genes, with decreases in DICER and BMPR2, and increases in CTGF detected. Conclusions: OA pathogenesis is likely regulated by a multitude of factors relating to cell signalling including the balance of cytokines in the articular joint. Dkk3 expression is increased in OA but can be regulated IL1 and OSM. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA

Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

Inhibition of Wnt/B-catenin pathway by sclerostin promotes cartilage maintenance

Purpose: Cartilage homeostasis is regulated by several mechanisms that influence both anabolic and catabolic balance. Among them, the local activation of Wnt pathways plays a major role in chondrocyte activity which might be regulated by Wnt inhibitors. Sclerostin, a Wnt inhibitor mainly produced by osteocytes and hypertrophic chondrocytes, might participate to the crosstalk between bone and cartilage. Therefore, the aim of this study is to determine the effect of Sclerostin in chondrocyte function. Methods: Primary murine chondrocytes, isolated from long bone epiphysis of 6 day-old mice, were cultured with or without Wnt3a conditioned media (Wnt3aCM) and in the presence or absence of mouse recombinant Sclerostin. Proteoglycan release induced by Wnt was quantified in the supernatant of chondrocytes by a colorimetric assay. Activation and inhibition of Wnt pathway was shown by the translocation of β-catenin (IF, TOP-GAL activity). BrdU and Tunel assays were performed to test the effect of Wnt and Sclerostin on cell proliferation and apoptosis. Chondrocyte differentiation induced by Sclerostin was investigated by RT-qPCR through the relative expression of type II and X collagen, Sox9, aggrecan, MMP-3, MMP-13 ADAMTS-4 & 5 genes. Results: Wnt3aCM reduced proteoglycan release in a dose-dependent manner and was rescued by Sclerostin (20ng/ml). This dose was chosen for the following experiments. Wnt inhibited the gene expression of collagen type II (X18-fold), Sox9 (X140 fold) and Aggrecan (X90-fold) and increases the gene expression of metalloproteinases such as Adamts-4 (X5-fold), Adamts-5 (X5.5-fold), MMP3 (X7-fold) and MMP13 (X6.6-fold). Sclerostin was able to inhibit the nuclear translocation of β-catenin induced by Wnt, but failed to exert any effect in the proliferation or apoptosis in primary chondrocytes regardless of Wnt. However, Wnt3a increased the gene expression of collagen type X (X3-fold) which was abolished by Sclerostin (X1.6-fold). Sclerostin significantly inhibited the Wnt-induced increase of catabolic genes (X1.7-fold Adamts-4, X1.6-fold Adamts-5, X3.4-fold MMP3 and X4-fold MMP13) and rescues partially the expression of anabolic genes (X5- fold collagen type II, X6-fold Sox-9 and X11-fold Aggrecan). In addition, Wnt3a induced the expression of Sost mRNA suggesting that Sclerostin might be regulated by Wnt. We further observed an enhanced expression of Sclerostin in osteoarthritic cartilage compared to cartilage of control mice. Conclusions: We here show that Wnt3a increases the catabolic activity and inhibits the anabolic activity in murine chondrocytes. Sclerostin alleviates the expression of catabolic genes induced by Wnt and rescued proteoglycan production. Moreover, Sclerostin promotes the cartilage maintenance through the inhibition of chondrocyte hypertrophy. Finally, the expression of Sclerostin expression in osteoarthritic mice suggests that Sclerostin might be involved in the pathophysiology in cartilage damage and might constitute a target for the prevention of osteoarthritis.

Intra-articular blood coagulation aggravates joint damage after a bleed in a canine in vivo model

Intra-articular blood coagulation aggravates joint damage after a bleed in a canine in vivo model

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism.

ObjectiveSubchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.MethodsOA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.ResultsBone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.ConclusionThe inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.

Elevation of cartilage AGEs does not accelerate initiation of canine experimental osteoarthritis upon mild surgical damage

Osteoarthritis is a highly prevalent disease, age being the main risk factor. The age-related accumulation of advanced-glycation-endproducts (AGEs) adversely affects the mechanical and biochemical properties of cartilage. The hypothesis that accumulation of cartilage AGEs in combination with surgically induced damage predisposes to the development of osteoarthritis was tested in vivo in a canine model. To artificially increase cartilage AGEs, right knee joints of eight dogs were repeatedly injected with ribose/threose (AGEd-joints). Left joints with vehicle alone served as control. Subsequently, minimal surgically applied cartilage damage was induced and loading restrained as much as possible. Thirty weeks after surgery, joint tissues of all dogs were analyzed for biochemical and histological features of OA. Cartilage pentosidine levels were ∼5-fold enhanced (p = 0.001 vs. control-joints). On average, no statistically significant differences in joint degeneration were found between AGEd and control-joints. Enhanced cartilage pentosidine levels did correlate with less cartilage proteoglycan release (R = -0.762 and R = -0.810 for total and newly-formed proteoglycans, respectively; p = 0.028 and 0.015 for both). The current data support the diminished cartilage turnover, but only a tendency towards enhanced cartilage damage in AGEd articular cartilage was observed. As such, elevated AGEs do not unambiguously accelerate the development of early canine OA upon minimal surgical damage.

Anti-inflammatory Effects of Polyphenolic-Enriched Red Raspberry Extract in an Antigen-Induced Arthritis Rat Model

The red raspberry ( Rubus idaeus ) fruit contains bioactive polyphenols including anthocyanins and ellagitannins with reported anti-inflammatory properties. This study sought to investigate the cartilage-protecting and anti-inflammatory effects of a polyphenolic-enriched red raspberry extract (RRE; standardized to total polyphenol, anthocyanin, and ellagitannin contents) using (1) an in vitro bovine nasal explant cell culture model and (2) an in vivo adjuvant-induced arthritis rat model. RRE contained 20% total polyphenols (as gallic acid equivalents), 5% anthocyanins (as cyanidin-3-glucoside equivalents), and 9.25% ellagitannins (as ellagic acid equivalents). In the in vitro studies, bovine nasal explants were stimulated with 10 ng/mL IL-1β to induce the release of proteoglycan and type II collagen. On treatment with RRE (50 μg/mL), there was a decrease in the rate of degradation of both proteoglycan and type II collagen. In the in vivo antigen-induced arthritis rat model, animals were gavaged daily with RRE (at doses of 30 and 120 mg/kg, respectively) for 30 days after adjuvant injection (750 μg of Mycobacterium tuberculosis suspension in squalene). At the higher dose, animals treated with RRE had a lower incidence and severity of arthritis compared to control animals. Also, histological analyses revealed significant inhibition of inflammation, pannus formation, cartilage damage, and bone resorption by RRE. This study suggests that red raspberry polyphenols may afford cartilage protection and/or modulate the onset and severity of arthritis.