Aggrecan Fragments Research Articles

Safety, Tolerability, and Pharmacodynamics of the ADAMTS-5 Nanobody M6495: Two Phase 1, Single-Center, Double-Blind, Randomized, Placebo-Controlled Studies in Healthy Subjects and Patients With Osteoarthritis.

To assess the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple injections of M6495, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) nanobody, in healthy volunteers and patients with osteoarthritis. Two randomized, placebo-controlled, double-blind studies were performed. Study 1 enrolled 54 healthy male volunteers who received one subcutaneous (s.c.) injection of M6495 (1-300 mg) or placebo (ratio 2:1), evaluating safety, PK, and PD as changes in the serum aggrecan fragment alanine-arginine-glycine-serine (ARGS). Study 2 enrolled 32 patients with osteoarthritis with Kellgren-Lawrence grades 2 to 4 and pain greater than or equal to 40 on the Western Ontario and McMaster Universities Arthritis Index pain subscale at screening and evaluated the safety, PK, and PD of three doses every two weeks (75-300 mg per dose) or six once-weekly M6495 s.c. doses (300 mg) or placebo (ratio 3:1) over 106 days' follow-up. M6495 in single and multiple doses of less than or equal to 300 mg s.c. weekly was well tolerated with no clinically significant changes in any safety parameter. Adverse events more frequently reported in the M6495 groups were mostly mild cases of injection site reactions, myalgia, and nausea, which resolved after treatment cessation. The elimination half-life of single s.c. doses of M6495 ranged from 79 to 267 hours. M6495 administration substantially reduced serum ARGS levels, indicative of target engagement and indicating disease-modifying potential of M6495. Treatment with M6495 in single and multiple doses up to and including 300 mg s.c. was found to be well tolerated and adequately safe for further clinical evaluation of potential disease-modifying effects.

Characterization and Advancement of an Evaluation Method for the Treatment of Spontaneous Osteoarthritis in STR/ort Mice: GRGDS Peptides as a Potential Treatment for Osteoarthritis

STR/ort mice spontaneously exhibit the typical osteoarthritis (OA) phenotype. However, studies describing the relationship between cartilage histology, epiphyseal trabecular bone, and age are lacking. We aimed to evaluate the typical OA markers and quantify the subchondral bone trabecular parameters in STR/ort male mice at different weeks of age. We then developed an evaluation model for OA treatment. We graded the knee cartilage damage using the Osteoarthritis Research Society International (OARSI) score in STR/ort male mice with or without GRGDS treatment. We measured the levels of typical OA markers, including aggrecan fragments, matrix metallopeptidase-13 (MMP-13), collagen type X alpha 1 chain (COL10A1), and SRY-box transcription factor 9 (Sox9), and quantified epiphyseal trabecular parameters. Compared to the young age group, elderly mice showed an increased OARSI score, decreased chondrocyte columns of the growth plate, elevated expression of OA markers (aggrecan fragments, MMP13, and COL10A1), and decreased expression of Sox9 at the articular cartilage region in elderly STR/ort mice. Aging also significantly enhanced the subchondral bone remodeling and microstructure change in the tibial plateau. Moreover, GRGDS treatment mitigated these subchondral abnormalities. Our study presents suitable evaluation methods to characterize and measure the efficacy of cartilage damage treatments in STR/ort mice with spontaneous OA.

Structural changes in collagen and aggrecan during extended aging may improve beef tenderness

The aim of this study was to characterize structural and property modifications of intramuscular connective tissue (IMCT) during extended aging. Longissimus lumborum (LL), Gluteus medius (GM), and Gastrocnemius (GT) muscles were collected from 10 USDA choice carcasses, fabricated and assigned to one of four aging periods: 3, 21, 42, or 63 days (n = 120). As expected, tenderness improved, and IMCT texture weakened after 21 days of postmortem aging (dpm; P < 0.05). In addition, transition temperature of collagen decreased (P < 0.01) after 42 dpm. It is interesting to note the collagen structure was also altered where relative % of γ chain decreased after 42 dpm (P < 0.05), and the α1 chain % increased at 63 days (P < 0.01). Finally, The LL and GT had a decrease in the 75 kDa aggrecan fragments from 3 to 21 to 42 dpm (P < 0.05). This study provided evidence that IMCT weakens during postmortem aging due to the modifications of IMCT components such as collagen and proteoglycan.

Hypochlorous Acid and Chloramines Induce Specific Fragmentation and Cross-Linking of the G1-IGD-G2 Domains of Recombinant Human Aggrecan, and Inhibit ADAMTS1 Activity

Atherosclerosis is a chronic inflammatory disease and a leading cause of mortality. It is characterized by arterial wall plaques that contain high levels of cholesterol and other lipids and activated leukocytes covered by a fibrous cap of extracellular matrix (ECM). The ECM undergoes remodelling during atherogenesis, with increased expression of aggrecan, a proteoglycan that binds low-density-lipoproteins (LDL). Aggrecan levels are regulated by proteases, including a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1). Activated leukocytes release myeloperoxidase (MPO) extracellularly, where it binds to proteins and proteoglycans. Aggrecan may therefore mediate colocalization of MPO and LDL. MPO generates hypochlorous acid (HOCl) and chloramines (RNHCl species, from reaction of HOCl with amines on amino acids and proteins) that damage LDL and proteins, but effects on aggrecan have not been examined. The present study demonstrates that HOCl cleaves truncated (G1-IGD-G2) recombinant human aggrecan at specific sites within the IGD domain, with these being different from those induced by ADAMTS1 which also cleaves within this region. Irreversible protein cross-links are also formed dose-dependently. These effects are limited by the HOCl scavenger methionine. Chloramines including those formed on amino acids, proteins, and ECM materials induce similar damage. HOCl and taurine chloramines inactivate ADAMTS1 consistent with a switch from proteolytic to oxidative aggrecan fragmentation. Evidence is also presented for colocalization of aggrecan and HOCl-generated epitopes in advanced human atherosclerotic plaques. Overall, these data show that HOCl and chloramines can induce specific modifications on aggrecan, and that these effects are distinct from those of ADAMTS1.

Proteolysis: a key post-translational modification regulating proteoglycans.

Proteoglycans are composite molecules comprising a protein backbone, i.e., the core protein, with covalently attached glycosaminoglycan chains of distinct chemical types. Most proteoglycans are secreted or attached to the cell membrane. Their specialized structures, binding properties, and biophysical attributes underlie diverse biological roles, which include modulation of tissue mechanics, cell adhesion, and the sequestration and regulated release of morphogens, growth factors, and cytokines. As an irreversible post-translational modification, proteolysis has a profound impact on proteoglycan function, abundance, and localization. Proteolysis is required for molecular maturation of some proteoglycans, clearance of extracellular matrix proteoglycans during tissue remodeling, generation of bioactive fragments from proteoglycans, and ectodomain shedding of cell-surface proteoglycans. Genetic evidence shows that proteoglycan core protein proteolysis is essential for diverse morphogenetic events during embryonic development. In contrast, dysregulated proteoglycan proteolysis contributes to osteoarthritis, cardiovascular disorders, cancer, and inflammation. Proteolytic fragments of perlecan, versican, aggrecan, brevican, collagen XVIII, and other proteoglycans are associated with independent biological activities as so-called matrikines. Yet, proteoglycan proteolysis has been investigated to only a limited extent to date. Here, we review the actions of proteases on proteoglycans and illustrate their functional impact with several examples. We discuss the applications and limitations of strategies used to define cleavage sites in proteoglycans and explain how proteoglycanome-wide proteolytic mapping, which is desirable to fully understand the impact of proteolysis on proteoglycans, can be facilitated by integrating classical proteoglycan isolation methods with mass spectrometry-based proteomics.

Dynamic compression inhibits cytokine-mediated type II collagen degradation

ObjectiveTo examine the effect of dynamic compressive loading applied intermittently on bovine cartilage explants stimulated with proinflammatory cytokines over 21 days. DesignCartilage explants were cultured for 21 days with Oncostatin M and TNFα (O ​+ ​T) [10/5 ​ng/mL] or in culture medium alone (w/o). The explants were either left free-swelling or subjected to dynamic compressive loading of 20 ​min, at 1 ​Hz, with loads ranging between 0.1 and 1 ​MPa, 5 times weekly. Metabolic activity was measured once weekly using Alamar Blue and cartilage turnover was assessed with biomarkers targeting degradation fragments of aggrecan (AGNx1) and type II collagen (C2M). Glycosaminoglycan degradation was quantified was the DMMB assay. Furthermore, explant weight and histological analysis was used to assess the cartilage degradation. ResultsDynamic compression of cartilage explants attenuated the O ​+ ​T-mediated C2M release on day 21 with 40% (p ​= ​0.0068) compared to the unloaded explants. Additionally, the change in explant weight from day −1 to day 21 showed that O ​+ ​T stimulation alone mediated a cartilage loss of 11%, whereas O ​+ ​T-stimulated explants subjected to compressive loading demonstrated a decreased cartilage weight loss of 6%, which was supported by the histological analysis. However, loading had no effect on aggrecan degradation. ConclusionIn cartilage explants cultured in a proinflammatory milieu, dynamic compressive loading confers anti-catabolic effects, inhibiting type II collagen degradation and reducing explant cartilage loss. These results demonstrate that compressive loading alters cartilage tissue turnover and enforces the need to include mechanical loading in a translation ex vivo cartilage model.

Immunoblot Investigation of Aggrecan Protein in Post-mortem Brain Tissue

The MS tissue banks estimated that more than fifty millions patient in the world, each patient cost more than one millions for treatment, has social and economic effects. The breakdown of blood brain barrier considered as autoimmune disease and early events in MS parthenogenesis. This study include investigation the modification of aggrecan glycoprotein in MS (30 samples) postmortem compared to NAWM (20 samples) via using western blotting. Fifty blocks of postmortem brain tissue were extracted their proteins for protein electrophoresis. Results indicated that human brain tissue express aggrecan proteins and its fragments were increased in MS active lesion compared to the normal control. The presence of aggrecan fragments considered as evidence of activation of different matrix mattaloproeinase enzymes to help neuron outgrowth axons.

Diabetes Mellitus and the Development of Lumbar Canal Stenosis: Is There Any Relevance?

Study DesignRetrospective study.PurposeTo assess the relationship between the severity of lumbar canal stenosis (LCS) and type-II diabetes mellitus (DM).Overview of LiteratureDM is a multiorgan disorder that has an effect on all types of connective tissues. LCS is a narrowing of the spinal canal with nerve root impingement that causes neurological claudication and radiculopathy. Identification of the risk factors of LCS is key in the prevention of its onset or progression.MethodsLCS patients were divided into three groups as per DM status: group A without DM (n=150); group B patients with well-controlled DM; and group C patients with uncontrolled DM. Groups B and C were subdivided into group B1: patients with DM with a duration of ≤10 years (n=76), group B2: DM with duration of >10 years (n=68), group-C1 DM duration ≤10 years (n=56), and group C2 DM duration >10 years (n=48). The severity of LCS was evaluated using the Swiss Spinal Stenosis Scale (SSSS) and Modified Oswestry Disability score (MODS). Operated patients ligamentum flavum sent for histological staining and quantitative immunofluorescence analysis.ResultsThe demographic data of groups did not show any difference except in age. There was no difference between the mean SSSS and MODS of groups A and B1. Groups B2, C1, and C2 had higher average SSSS and MODS than group A (p<0.05). Groups B2 and C2 had higher SSSS and MODS than groups B1 and C1. Groups C1 and C2 had higher scores than groups B1 and B2 (p<0.05). The severity of LCS was significantly related to the duration of DM in groups B and C (p<0.05). Uncontrolled and longer duration of DM had significant elastin fibers loss and also higher rate of disk apoptosis, high matrix aggrecan fragmentation, and high disk glycosaminoglycan content.ConclusionsLonger duration and uncontrolled diabetes were risk factors for LCS and directly correlate with the severity of LCS.

Rapamycin Ameliorates Age‐Associated Intervertebral Disc Degeneration in Male Marmosets

Introduction Age-associated intervertebral disc degeneration (IDD) is a major contributor to low back pain and physical disability in the US. Rapamycin has been shown in several animal models, including mammalian, to increase lifespan and decrease aging and age-associated pathologies, including cancer, cardiac and neurodegenerative diseases. Rapamycin is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR) complex 1, which is a master regulator of cell growth and proliferation signals. It has not yet been determined if rapamycin treatment influences age-associated IDD. Hence, the goal of this study was to determine the effect of a one-year oral rapamycin treatment on IDD progression in old marmosets. Methods Old male marmosets were given rapamycin using a delivery system that microencapsulates rapamycin in a eudagrit coating and is mixed into yogurt for feeding via a syringe to accurately account for each daily dose. As control, old animals were given empty eudagrit capsules delivered the same way, for one year. Spine specimens were collected from young (<5 years old), old (> 10 years old) + vehicle, and old + rapamycin marmosets. Treatment was given for one year prior to sacrifice. Cellularity and histological structure were assessed using H&E staining. Disc matrix anabolism was assessed by aggrecan immunohistochemistry (IHC) and DMMB assay to quantify sulfated glycosaminoglycans. Disc matrix catabolism was assessed via WB for aggrecan fragments. Autophagy was measured via WB for established autophagy protein markers. Student's t-test was used for significance, n=3-4. Results Rapamycin treatment significantly decreased histological scoring for IDD characteristics compared to control old marmosets (Figure 1a-c.) Rapamycin treatment significantly increased older marmosets GAG levels and increased aggrecan IHC staining compared to control (Figure 2a, b). Similarly, rapamycin treatment decreased ADAMTS-mediated and MMP-mediated aggrecan fragmentation (Figure 2c, d). Rapamycin treatment did not significantly affect the autophagy markers LC3-II, Atg7, beclin-1, or Atg12-Atg5, but it did significantly decrease p62 expression. Discussion Overall, rapamycin treatment significantly improved disc catabolism, increasing aggrecan production, and histological score in old marmosets' discs. These results suggest that one-year daily rapamycin treatment delayed IDD progression in older male marmosets. Interestingly, autophagy related proteins were not significantly increased in older male marmosets treated with rapamycin, although rapamycin treatment has been shown to increase autophagy in other tissues, as it is a known inhibitor of mTOR. This implies either rapamycin given orally cannot diffuse into the disc, and systemic effects of rapamycin were observed in other assays, or autophagy is not regulated by rapamycin in the disc. This unique finding warrants further study. Significance This is the first study to investigate the in vivo effects of rapamycin treatment on age-associated IDD in a nonhuman primate model and suggests rapamycin might be a viable treatment to improve IDD characteristics in patients.

Development of a highly sensitive chemiluminescence immunoassay for quantification of aggrecanase-generated ARGS aggrecan fragments in serum

ObjectiveCartilage degradation is a hallmark of osteoarthritis (OA). Aggrecan, a major proteoglycan of articular cartilage extracellular matrix (ECM), is degraded by ADAMTS-5 resulting in the release of ARGS-G2 fragments to synovial fluid and circulation. The aim was to quantify ARGS-G2 in the serum of OA patients using the huARGS immunoassay. MethodsThe immunoassay was produced under GMP conditions and the technical performance was assessed. The biological relevance of the immunoassay was assessed in the conditioned media from a bovine full-depth cartilage explant (BEX) model. The diurnal and inter-day variations of ARGS levels were evaluated in OA patients’ serum. Post-hoc analysis of huARGS was conducted in a sub-cohort of a phase III OA trial testing the safety and efficacy of oral salmon calcitonin. ResultsTechnical performance: huARGS demonstrated good technical performance. Biological relevance: ARGS release was induced by inflamatory facotrs stimulation compared to the vehicle group, reaching a peak at day 3 and gradually decreasing to base level at day 12. The ARGS release was suppressed by the addition of the ADAMTS-4/-5 activation inhibitor. Biological variation: No significant diurnal or inter-day effect was found. Phase III clinical trial: The participants in the lowest group (Q1) of baseline huARGS levels were more likely to progress radiographically than the highest group (Q4): OR 3.38[0.81-14.02]. ConclusionsThe huARGS shows good technical performance and low biological variation. It has the potential to aid drug development in various stages, both as a PD biomarker and identifying progressors who might be likely to respond to an OA drug.

Degeneration of the articular disc in the human triangular fibrocartilage complex.

Traumatic injuries of the triangular fibrocartilage complex (TFCC) are frequent reasons for ulnar wrist pain. The assessment of the extent of articular disc (AD) degeneration is important for the differentiation of acute injuries versus chronic lesions. The AD of the TFCC of eleven human cadaver wrists was dissected. Degeneration was analyzed according to the grading of Krenn et al. Hematoxylin-eosin was used to determine the tissue morphology. Degeneration was evaluated using the staining intensity of alcian blue, the immunohistochemistry of the proteoglycan versican and the immunoreactivity of NITEGE, an aggrecan fragment. The staining homogeneity of HE decreased with higher degeneration of the AD and basophilic tissue areas were more frequently seen. Two specimens were characterized as degeneration grade 1, five specimens as grade 2, and four specimens as grade 3, respectively. Staining intensity of alcian blue increased with higher degeneration grade of the specimens. Immunoreactivity for NITEGE was detected around tissue fissures and perforations as well as matrix splits. Immunoreactivity for versican was found concentrated in the tissue around matrix fissures and lesions as well as loose connective tissue at the ulnar border of the AD. Specimens with degeneration grade 2 had the strongest immunoreactivity of NITEGE and versican. Cell clusters were observed in specimens with degeneration grade 2 and 3, which were stained by alcian blue and immunoreactive for NITEGE and versican. Increasing age of the cadaver wrists correlated with a higher degree of degeneration (p < 0.0001, r = 0.68). The fibrocartilage of degenerated ADs contains NITEGE and versican. The amount of the immunoreactivity of these markers allows the differentiation of degenerative changes into three grades. The degeneration of the AD increases with age and emphasizes its important mechanical function.

Higher aggrecan 1-F21 epitope concentration in synovial fluid early after anterior cruciate ligament injury is associated with worse knee cartilage quality assessed by gadolinium enhanced magnetic resonance imaging 20\u2009years later

BackgroundTo investigate if cartilage related biomarkers in synovial fluid are associated with knee cartilage status 20 years after an anterior cruciate ligament (ACL) injury.MethodsWe studied 25 patients with a complete ACL rupture without subsequent ACL reconstruction or radiographic knee OA. All had a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) 20 years after the ACL injury, using the T1 transverse relaxation time in the presence of gadolinium (T1Gd) which estimates the concentration of glycosaminoglycans in hyaline cartilage. Synovial fluid samples were aspirated acutely (between 0 and 18 days) and during 1 to 5 follow up visits between 0.5 and 7.5 years after injury. We quantified synovial fluid concentrations of aggrecan (epitopes 1-F21 and ARGS), cartilage oligomeric matrix protein, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by immunoassays, and sulfated glycosaminoglycans by Alcian blue precipitation. Western blot was used for qualitative analyses of aggrecan fragments in synovial fluid and cartilage samples.ResultsWestern blot indicated that the 1-F21 epitope was located within the chondroitin sulfate 2 region of aggrecan. Linear regression analyses (adjusted for age, sex, body mass index and time between injury and sampling) showed that acute higher synovial fluid 1-F21-aggrecan concentrations were associated with shorter T1Gd values 20 years after injury, i.e. inferior cartilage quality (standardized effects between − 0.67 and − 1.0). No other statistically significant association was found between molecular biomarkers and T1Gd values.ConclusionHigher acute synovial fluid 1-F21-aggrecan concentrations in ACL injured patients, who managed to cope without ACL reconstruction and were without radiographic knee OA, were associated with inferior knee cartilage quality assessed by dGEMRIC 20 years after injury.

Diabetes Mellitus-A Risk Factor for the Development of Lumbar Disc Degeneration: A Retrospective Study of an Indian Population.

Study Design:A retrospective study.Objectives:To determine the association between type-2 diabetes mellitus (T2DM) and the severity of lumbar disc degeneration disease (LDDD).Methods:We included 199 patients with low back pain (LBP) who visited our hospital from 2016 to 2018. All patients were divided into 3 groups as per inclusion criteria. Group A, patients without DM (n = 75); group B, patients with controlled DM (n = 72); and group C, patients with uncontrolled DM (n = 52). The patients were further subdivided into group B1, DM duration ≤10 years (n = 38); group B2, DM duration >10 years (n = 34); group C1 DM duration ≤10 years (n = 28); and group C2, DM duration >10 years (n = 24). Sex, age, body mass index, occupation, smoking history, alcohol use, and duration of T2DM were recorded. The severity of LDDD was evaluated using the 5-level Pfirrmann grading system. Operated patients’ disc materials were sent for histological examination.Results:Demographic data showed no difference among groups (P > 0.5), except age. Patients with DM showed more severe disc degeneration compared with patients without DM. The average Pfirrmann scores between groups A and B1 had no difference; groups B2, C1, and C2 showed higher average Pfirrmann scores than group A (P < 0.05). Groups B2 and C2 showed higher average Pfirrmann scores than groups B1 and C1 (P < 0.05). Groups C1 and C2 showed higher average Pfirrmann scores than groups B1 and B2 (P < 0.05). The severity of LDDD was significantly related to DM duration in both groups B and C (P < 0.05). DM groups showed increased disc apoptosis and matrix aggrecan fragmentation, disc glycosaminoglycan content and histological analysis were significantly different; the results are similar to Pfirrmann score results.Conclusions:DM duration >10 years and uncontrolled DM were risk factors for LDDD.

Hyperglycemia and its influence on development of lumbar degenerative disc disease

Study DesignA Retrospective observational study. ObjectivesTo determine the influence of hyperglycemia on severity of lumbar degenerative disc disease (LDDD). MethodsWe retrospectively included 199 patients with low back pain (LBP) who visited our tertiary care hospital from June 2016 to December 2018. All patients divided into three groups as per inclusion and exclusion criteria. Group-A had patients without DM (n = 75). Group B had well-controlled DM patients (n = 72) and Group-C had uncontrolled DM patients (n = 52). Group B and C subdivided according to dutation of DM. Group-B1 DM duration was ≤ 10 years (n = 38), Group-B2 DM duration was >10 years (n = 34), Group-C1 DM duration ≤10 years (n = 28), Group-C2 DM duration >10 years (n = 24). Sex, age, BMI, occupation, smoking history, alcohol use and duration of type-II DM were recorded. The severity of LDDD was evaluated using the five-level Pfirrmann grading system. Operated patient's disc material sent for histological examination. ResultsPatients with DM showed more severe disc degeneration compared to patients without DM. The average Pfirrmann scores between Groups A and B1 had no difference; Groups B2, C1, and C2 showed higher average Pfirrmann-scores than Group-A (p > 0.05). Group-B2 and Group-C2 showed higher average Pfirrmann-scores than Group-B1 and Group-C1 (p > 0.05). Group-C1 and Group-C2 showed higher average Pfirrmann-scores than Group-B1 and B2 (p > 0.05). The severity of LDDD was significantly related to DM duration both in groups B & C (p > 0.05). DM groups showed increased disc apoptosis and matrix aggrecan fragmentation, Disc glycosaminoglycan content and histological significantly different, the results are similar to Pfirrmann-score results. ConclusionsThere is a positive relationship between diabetes and LDDD. A longer the duration and poor control of hyperglycemia could aggravate disc degeneration.

Influences of circulatory factors on intervertebral disc aging phenotype.

Whether disc aging is influenced by factors beyond its local environment is an important unresolved question. Here we performed heterochronic parabiosis in mice to study the effects of circulating factors in young and old blood on age-associated intervertebral disc degeneration. Compared to young isochronic pairs (Y-Y), young mice paired with old mice (Y-O) showed significant increases in levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic tissue degeneration, but negligible changes in cellular senescence markers (p16INK4a, p21Cip1). Compared to old isochronic pairs (O-O), old mice paired with young mice (O-Y) exhibited a significant decrease in expression of cellular senescence markers (p16, p21, p53), but only marginal decreases in the levels of disc MMP-13 and ADAMTS4, aggrecan fragmentation, and histologic degeneration. Thus, exposing old mice to young blood circulation greatly suppressed disc cellular senescence, but only slightly decreased disc matrix imbalance and degeneration. Conversely, exposing young mice to old blood accelerated their disc matrix imbalance and tissue degeneration, with little effects on disc cellular senescence. Thus, non-cell autonomous effects of circulating factors on disc cellular senescence and matrix homeostasis are complex and suggest that disc matrix homeostasis is modulated by systemic factors and not solely through local disc cellular senescence.

Aggrecan Turnover in Women with Rheumatoid Arthritis Treated with TNF-α Inhibitors.

This study was performed to evaluate the effects of 15-month anti-tumor necrosis factor α (anti-TNF-α) therapy on the aggrecan turnover of female rheumatoid arthritis (RA) patients. Serum was obtained from healthy subjects and female RA patients treated with TNF-α inhibitors (TNFαI) in combination with methotrexate. We measured serum levels of aggrecan chondroitin sulfate 846 epitope (CS846), aggrecan fragments (AGC), disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and 5 (ADAMTS-5), as well as their natural inhibitor, known as tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), using immunoassay methods. Serum levels of CS846, AGC, ADAMTS-4, ADAMTS-5 and TIMP-3 were higher in female patients with RA before the treatment in comparison to healthy subjects. Ratio of ADAMTS-5 to TIMP-3 was significantly higher in RA women than in controls, whereas ADAMTS-4/TIMP-3 ratio did not differ from that in controls. During the anti-TNF-α therapy, the serum levels of 846 epitope increased, whereas levels of AGC decreased in female RA patients. Furthermore, 15 months of treatment with TNFαI downregulated serum levels of both ADAMTS, without any effect on TIMP-3 levels. These changes were accompanied by significantly reduced ratios of ADAMTS to TIMP-3. According to our results, anti-TNF-α therapy has a beneficial impact on aggrecan remodeling during RA.

Pregnancy Associated Plasma Protein A (PappA) Knockout Mice: In Vivo Disc Study

INTRODUCTIONPast research in disc biology has highlighted the beneficial effects of growth factors, such as BMPs and IGF‐1, in vitro. Based on this research, it was proposed that exogenous IGF‐1 be used as a therapy for intervertebral disc degeneration (IDD). In the aging research field, suppressing the IGF‐1‐mediated signaling pathway has consistently decreased the incidence of age‐related disorders and increased lifespan of mammals and rodents. One model using PappA knockout mice has shown decreasing IGF‐1 signaling leads to positive effects on aging and longevity. Active PappA protease cleaves IGF binding protein 4 (IGFBP4), releasing active IGF‐1 which can bind to its receptor and exert downstream signaling effects. When you genetically remove PappA, IGF‐1 stays bound to IGFBP4. Here, we examined the discs of aged PAPPA−/− mice versus wild type (WT) to determine whether there was an effect on age‐associated IDD.METHODSThis study was conducted according to IACUC approved protocols at University of Pittsburgh. Genetic deletion of PAPP‐A protease in mice with a mixed C57BL/6‐129SV/E background was achieved by deletion of both alleles of PAPP‐A. Spine segments were harvested from WT and PAPPA−/− mice at 23 months. Disc proteoglycan content was assessed by safranin‐O‐histology and DMMB assay was used to quantify sulfated glycosaminoglycans (GAG) in lumbar NP tissue. Western blotting was performed to measure the disc aggrecan proteolytic fragments, and the catabolic markers MMP‐3 and ADAMTS‐4. Levels of disc cellular senescence (loss of lamin B1expression) was assessed by immunohistochemistry. Student’s t‐test was used to test significance between groups (p&lt;0.05). Average values (n=4 mice) were shown with SEM.RESULTSCompared to WT, PAPPA−/− mice had decreased proteoglycan content and two times less disc GAG content (Figure 1). PAPPA−/− mice also had significantly decreased aggrecan fragmentation and decreased protein levels of MMP‐3 and ADAMTS‐4 in their discs compared to WT mice (Figure 2). Additionally, greater level of disc lamin B1 was seen in PAPPA−/− compared to WT mice, suggesting reduced disc cellular senescence in PAPPA−/− mice.DISCUSSIONThe role of IGF‐1 in disc health and aging is currently unresolved. PAPPA−/− mice have reduced matrix content but the aggrecan matrix present after aging appears to be less fragmented based on our findings. Lowered disc fragmentation corresponded to a decrease in disc catabolic factors MMP‐3 and ADAMTS‐4 protein in PAPPA−/− mice. Lamin B1 staining suggests there are less disc senescent cells in old PAPPA−/− mice compared to normally aged mice. Further studies are needed to determine the mechanism for the decreased disc catabolism and more full length aggrecan in aged PAPPA−/− mice compared to aged WT mice. Additionally, further studies should confirm whether young PAPPA−/− mice also have decreased GAG content or this loss occurs during the aging process.SIGNIFICANCEThe results of this study suggest the net effect of IGF‐1 regarding both anabolic and catabolic effects must be examined to fully understand if IGF‐1 is beneficial for disc homeostasis during age‐associated IDD.Support or Funding InformationR01‐AG044376‐01 &amp; Ferguson Foundation GrantQualitative and quantitative results show decreased disc proteoglycan content, including aggrecan, in aged PAPPA −/− mice.Figure 1Despite less proteoglycan content, aged PAPPA −/− mice had less fragmented aggrecan and lower levels of catabolic markers, MMP‐3 and ADAMTS‐4 compared to aged WT mice.Figure 2

Serum aggrecan fragment ARGS neoepitope is associated with age and structural severity in osteoarthritis

Purpose: ARGS neoepitope (ARGS) originates from aggrecan, a proteoglycan abundant in cartilage of high importance for structure and function. ARGS is a product of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 and 5 (ADAMTS-4, -5) enzymatic degradation of aggrecan. ARGS has mainly been investigated in synovial fluid (SF) of knee trauma subjects, where it has been observed to increase following injury. Furthermore, studies have indicated that ARGS in SF may also be increased in knees affected by OA.

Development of a highly sensitive chemiluminescence immunoassay for detection of aggrecanase-generated args aggrecan fragments in serum

Purpose: Cartilage metabolism and turnover is a hallmark of Osteoarthritis (OA), and can be assessed by blood- or urine-based markers of extracellular matrix protein (ECMP) degradation. Levels of such markers may reflect disease activity and as such be applied in drug development as pharmacodynamic markers. Aggrecan, a major ECMP of articular cartilage, is degraded by ADAMTS-5, which is upregulated and activated in OA. One of the well-described ADAMTS-5 cleavage sites is at 373E-374A in the G1-G2 inter-global domain, resulting in the release of ARGS-G2 fragment to synovial fluid and circulation.

Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors

Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM+TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 invitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor invitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.