Proteoglycan Turnover Research Articles

Tibial bone versican content decreases with zoledronate treatment in adult mice

In bone remodeling, the expression and turnover of the proteoglycans versican and aggrecan are poorly understood. We report changes in adult mouse bone contents of versican and aggrecan associated with bothage and treatment with the drug zoledronate. The data may have implications for experimental animal models of osteoporosis and related conditions. Versican and aggrecan are large, aggregating proteoglycans involved in skeletal development, but little is known about their roles in bone remodeling. The purpose of this study was to investigate versican and aggrecan contents in adult mouse bones, and changes in their contents in response to the bisphosphonate zoledronate (ZOL). Mice (9weeks old) were treated with 125μg/kg ZOL or vehicle for 3 or 15weeks. Versican and aggrecan were isolated from tibial bones for Western blotting, automated integrated densitometry, and analysis (two-way ANOVA, α = 0.05). In ZOL-treated mouse bones, compared to vehicle, 340 and 60kDa versican content decreased significantly, and 100 and 60kDa aggrecan content decreased significantly (drug effect). In 24-week-old mouse bones, compared to 12weeks, statistically significant decreases were observed in 340, 80, 60, and 11kDa versican, and in 100, 70, and 40kDa aggrecan (age effect). There was a statistically significant ZOL-age interaction for 330kDa aggrecan. This is the first study to assess physiological versican and aggrecan adaptations in adult mammalian bone tissue, in the presence and absence of ZOL. We observed large decreases in some versican and aggrecan species from 12 to 24weeks. We also observed decreases in several versican and aggrecan species in the presence of ZOL. This indicates that bone proteoglycan expression and turnover may be important in bone remodeling.

Long non-coding rnas in osteoarthritis and chondrogenesis

Long non-coding rnas in osteoarthritis and chondrogenesis

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.

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.

FRI0314 Aetiology of experimentally induced osteoarthritis of the knee; biomechanical or biochemical factors?

BackgroundAt the moment, osteoarthritis (OA) is seen as a multifactorial disease, caused by a combination of i.a. genetics, overloading, obesity and trauma in the past. Disturbance of joint homeostasis, due...

Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study

To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

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.

Nonoperative management of ankle arthritis

Although hip and knee osteoarthritis are mostly of primary origin, ankle osteoarthritis is of posttraumatic origin. In sports injuries, the ankle is the second most commonly injured body site after the knee. In addition, compared to the cartilage in the knee joint, ankle cartilage has a higher content of proteoglycans and water, and an increased rate of proteoglycan turnover and synthesis, all of which are responsible for its increased incidence of posttraumatic origin osteoarthritis. Nonoperative management of ankle arthritis typically starts with weight reduction, activity modification, and oral non-steroidal anti-inflammatory drugs, physical therapy, and can progress to gait-aids including a cane, shoe-wear modification, patellar tendon weight-bearing ankle-foot orthosis, and intra-articular injections with corticosteroids or hyaluronic acid.

Coagulation aggravates blood‐induced joint damage in dogs

Joint bleeding due to trauma, major joint surgery, or hemophilia leads to joint damage. It is unclear if there are differences between coagulating blood and anticoagulated blood with respect to joint degeneration, especially in vivo. Therefore, we undertook this study to evaluate in a canine in vivo model whether intraarticular exposure to coagulating blood is more destructive than exposure to anticoagulated blood, and whether inflammation plays a role in the cartilage- damaging process. In 7 dogs the left knees were injected with coagulating blood 4 times a week during weeks 1 and 4, and the right knees were injected with saline. In 7 other dogs, anticoagulated heparinized blood was injected, and heparinized saline was used as control. Ten weeks after the last injection, cartilage matrix turnover and synovial inflammation were analyzed. To study inflammation-independent cartilage damage, explants of cartilage from at least 6 human donors per group were exposed in vitro to coagulating and anticoagulated blood, plasma, and serum for 4 days. Cartilage matrix turnover was determined after a recovery period of 12 days. Canine knees injected with coagulating blood showed more disturbed proteoglycan turnover than knees injected with anticoagulated blood. Synovial inflammation was present only after intraarticular injections with coagulating blood. In in vitro experiments, exposure of human cartilage explants to coagulating blood resulted in more damage than did exposure to anticoagulated blood, while exposure to plasma and serum did not alter cartilage matrix turnover. This study shows that coagulating blood causes more long-lasting in vivo joint damage than anticoagulated blood, thereby suggesting that along with joint bleeding in hemophilia, exposure to intraarticular blood should also be avoided during surgery and trauma to prevent joint damage.

Serum CTXii Correlates With Articular Cartilage Degeneration After Anterior Cruciate Ligament Transection or Arthrotomy Followed by Standardized Exercise

Background:Anterior cruciate ligament injury increases risk for accelerated development of osteoarthritis. The effect of exercise on articular cartilage following joint injury is not well understood. Biochemical biomarkers of collagen degradation and proteoglycan turnover are potential indicators for early articular cartilage degeneration.Hypothesis:This study tests the hypothesis that serum concentrations of CS846 and CTXii correlate with structural changes to articular cartilage following joint injury in exercised animals.Study Design:Controlled laboratory study.Methods:Twenty-four Sprague-Dawley rats underwent either arthrotomy alone (sham surgery) or anterior cruciate ligament transection (ACLT). Animals were recovered for 3 weeks and then exercised on a treadmill at 18 m per minute, 1 hour per day, 5 days per week, until sacrifice either 6 or 12 weeks later. Articular cartilage was assessed grossly, and histology was graded using modified Mankin, toluidine blue, and modified David-Vaudey scales. Serum collected preoperatively and at sacrifice was assayed by ELISA for CTXii and CS846.Results:At 6 weeks, gross grades (P < 0.01), modified Mankin scores (P < 0.03), and toluidine blue scores (P < 0.04) were higher, reflecting increased degeneration in ACLT animals compared with sham surgery animals. Serum CS846 increased after 6 weeks in ACLT animals (P < 0.05). Serum CTXii levels strongly correlated with Mankin degenerative scores (coefficient = 0.81, P < 0.01) and David-Vaudey histology grades (coefficient = 0.73, P < 0.01) at 6 weeks. While gross grades remained higher at 12 weeks in ACLT animals (P < 0.04), no differences were seen in serum CS846 and CTXii. Histology scores also showed no differences between ACLT and sham due to increasing degeneration in the sham surgery group.Conclusion:The strong correlation between serum CTXii and microstructural changes to articular cartilage following joint injury demonstrates potential use of serum biomarkers for early detection of cartilage degeneration. Increasing cartilage degeneration in exercised sham-surgery animals suggests that early loading may have negative effects on articular cartilage due to either mechanical injury or hemarthrosis after arthrotomy.Clinical Relevance:Patients with anterior cruciate ligament injury are at increased risk for development of posttraumatic osteoarthritis. CTXii may be useful for early detection of joint degeneration. Further study on the effects of exercise after injury is important to postinjury and postoperative rehabilitation.

Novel Role of ADAMTS-5 Protein in Proteoglycan Turnover and Lipoprotein Retention in Atherosclerosis

Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE(-/-)) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE(441)) and aggrecan ((374)ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE(441) versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.

Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells

Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. Therapeutic aggrecanase inhibition for treatment of arthritic disease may thus not impede regenerative self-healing pathways based on chondrogenesis of local progenitor cells in the joint.

Large Proteoglycan Complexes and Disturbed Collagen Architecture in the Corneal Extracellular Matrix of Mucopolysaccharidosis Type VII (Sly Syndrome)

Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.

Assessment of synovial fluid biomarkers in healthy foals and in foals with tarsocrural osteochondrosis

Although alterations in biomarkers of cartilage turnover in synovial fluid (SF) have been demonstrated in horses with osteochondrosis (OC), there have been few investigations of such alterations in animals <1 year old. In this study tarsocrural SF samples from foals aged 18, 22 and 52 weeks of age were assessed for: (1) 'turnover' biomarkers of type II collagen (CPII and C2C) and proteoglycan (CS846 and glycosaminoglycans [GAG]); (2) matrix metalloproteinase (MMP) activity; (3) insulin-like growth factor (IGF)-1; (4) transforming growth factor (TGF)-β1; (5) prostaglandin (PG) E(2); and (6) leukotriene B(4). Using a linear mixed model, the concentration of biomarkers was compared between animals that developed or did not develop radiographic evidence of OC at 24 or 48 weeks of age. The CPII:C2C ratio tended to be higher in OC-affected joints compared to controls at all ages, and this difference was statistically significant at 22 weeks of age. The concentrations of CS846 and IGF-1, and the CS846:GAG ratio were reduced in OC-affected joints relative to controls at 18 weeks of age only. At 52 weeks of age, the PGE(2) concentration was lower in joints with OC. Overall, there appears to be a consistent anabolic shift in type II collagen turnover in juvenile joints affected by OC. Aberrant proteoglycan turnover is not a hallmark of the late repair of this lesion but reduced concentrations of IGF-1 in SF may be associated with early-stage lesions.

Response to: cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection

We welcome the interest of Rutgers and colleagues [1] in providing additional information on the mode of action of autologous conditioned serum (ACS). Studies such as the one they published in a recent issue of Arthritis Research & Th erapy are important because the Orthokine ACS treatment (Orthogen, Dusseldorf, Germany) has been shown to be safe and eff ective in a number of clinical studies and is used widely in Europe. Randomized controlled trials have confi rmed the effi cacy of this treatment in osteoarthritis of the knee and lumbar radiculopathy. Additional human studies show promise in treating muscle injury and tunnel widening after anterior cruciate ligament (ACL) reconstruction. All clinical and preclinical studies confi rm an excellent riskto-benefi t ratio. We would like to off er some comments concerning the paper of Rutgers and colleagues. Th e method of producing ACS has been carefully optimized to enrich for anti-infl ammatory cytokines, such as interleukin (IL)-1Ra, IL-10, and IL-13, while keeping low the concentration of infl ammatory cyto kines, such as IL-1β and tumor necrosis factor-alpha (TNF-α). Th e ACS preparation of Rutgers and colleagues had increased concen trations of IL-1β and TNF-α, and this raises questions about the composition of the product that was tested. Nevertheless, despite the elevated concentrations of these two cytokines, there was no adverse eff ect of ACS on proteoglycan turnover in the cartilage explant cultures. Th is suggests that anti-infl ammatory and possibly chondro protective ingredients within ACS pre dominate. Further studies using additional controls and autologous instead of heterologous serum would be interesting. Th e third component of the study by Rutgers and colleagues measured cytokine levels in synovial fl uids before and after treatment with ACS. No changes were found, but it should be noted that Orthokine should be used only when knee eff usion has been removed eff ectively. Th e described possible aspiration of synovial fl uid after treatment with ACS raises questions about the selection of these patients or the composition of the injected ACS. In this regard, it is worth noting that another study [2,3] involving the injection of Orthokine ACS into knees after ACL plasty showed evidence of reduced IL-1β content in synovial fl uid. Th is reduction corresponded with im proved pain and function and with reduced tunnel widening. Clearly, the mechanisms through which ACS brings about clinical improvement are incompletely understood and should be the subject of additional research.

Cartilage integrity and proteoglycan turnover are comparable in canine experimentally induced and human joint degeneration

The value of experimental models of osteoarthritis (OA) largely depends on the ability to translate observations to human OA. Surprisingly, direct comparison of characteristics of human and experimental OA is scarce. In the present study, cartilage integrity and matrix turnover in a canine model of joint degeneration were compared to human clinical OA. In 23 Beagle dogs, joint degeneration was induced in one knee, the contra-lateral knee served as a control. For comparison, human osteoarthritic and healthy knee cartilage were obtained at arthroplasty (n=14) and post-mortem (n=13). Cartilage was analyzed by histology and biochemistry. Values for cartilage integrity and proteoglycan (PG) synthesis showed species specific differences; GAG content of healthy cartilage was 2-fold higher in canine cartilage and PG synthesis even 8-fold. However, the relative decrease in PG content between healthy and OA cartilage was similar for humans and canines (-17% vs. -15%, respectively), as was the histological damage (+7.0 vs. +6.1, respectively) and the increase of PG synthesis (+100% vs. +70%, respectively). Remarkably, the percentage release of total and of newly formed PGs in human and canine controls was similar, as was the increase due to degeneration (+65% vs. +81% and +91% vs. +52%, respectively). Despite differences in control conditions, the observed changes in characteristics of cartilage integrity and matrix turnover are similar in a canine model of joint degeneration and human clinical OA. The canine Groove model shows that its characteristics reflect those of human OA which makes the model appropriate for studying human OA.

Abnormal post‐translational and extracellular processing of brevican in plaque‐bearing mice over‐expressing APPsw

Aggregation of amyloid-beta (Abeta) in the forebrain of Alzheimer's disease (AD) subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are major components of this extracellular environment. To test the hypothesis that Abeta, or another amyloid precursor protein (APP) dependent mechanism modifies the accumulation and/or turnover of extracellular proteoglycans, we examined whether the expression and processing of brevican, an abundant extracellular, chondroitin sulfate (CS)-bearing proteoglycan, were altered in brains of Abeta-depositing transgenic mice (APPsw - APP gene bearing the Swedish mutation) as a model of AD. The molecular size of CS chains attached to brevican was smaller in hippocampal tissue from APPsw mice bearing Abeta deposits compared to non-transgenic mice, likely because of changes in the CS chains. Also, the abundance of the major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice, yet these immunoreactive fragments appeared to accumulate adjacent to the plaque edge. These results suggest that Abeta or APP exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. As proteoglycans stabilize synaptic structure and inhibit molecular plasticity, defective brevican processing observed in Abeta-bearing mice and potentially end-stage human AD, may contribute to deficient neural plasticity.

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

Metabolism of proteoglycans in tendon

Tendons are dense, fibrous connective tissues that are responsible for transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects function) is very common, both within the sporting arena and in the workplace. Importantly, proteoglycans are essential components of the tendon extracellular matrix (ECM) and changes in their expression and metabolism/turnover have been associated with tendinopathy. Within tendons, the small leucine-rich proteoglycans (SLPRs), decorin, fibromodulin, lumican and keratocan predominate within tensional regions, while in tendon fibrocartilage, increased concentrations of proteoglycans common to the articular cartilage phenotype are present, including aggrecan, biglycan and proteoglycan 4. However, the rate of proteoglycan turnover within tendon is markedly higher than that of cartilage, mediated via the "aggrecanases," which are constitutively active in the tendon matrix. Data suggest that this increased proteoglycan turnover is likely to be required to maintain normal tendon homeostasis, with perturbations in proteoglycan metabolism contributing to tissue dysfunction. Thus, future studies aimed at furthering our fundamental knowledge of tendon proteoglycan metabolism in health and disease are important in the development of improved treatments for tendon disorders.

Interleukin‐10 protects against blood‐induced joint damage

Despite prophylactic treatment, haemophilia patients suffer from spontaneous joint bleeds, which lead to severe joint damage. Also after joint trauma, an intra-articular haemorrhage can add to joint damage over time. This study evaluated interleukin 10 (IL-10) in the search for possible interventions to prevent or limit the damaging effects of joint bleeds. Human articular cartilage tissue explants were cultured in the presence or absence of 50% v/v blood (or its cellular components) for 4 d (the expected blood load in vivo after a joint haemorrhage), followed by a recovery period of 12 d. Pharmacological dosages of IL-10 reached during treatment (1 or 10 ng/ml) were added. Additionally, cartilage and synovial tissue obtained from joints with end-stage haemophilic arthropathy (HA) were cultured in the presence of IL-10 (10 ng/ml). IL-10 protected cartilage from the damaging effects of blood exposure, measured by its effects on proteoglycan turnover. In addition, IL-10 beneficially influenced cartilage from patients with HA and reduced the production of the inflammatory cytokines IL-1beta and tumour necrosis factor-alpha by haemophilic synovial tissue. Taken together, although effects were obtained in vitro, IL-10 protects against blood-induced joint damage and might be further evaluated as candidate in treatment of tissue damaging effects of joint haemorrhages.