Effect of Resveratrol on Serum Levels of Type II Collagen and Aggrecan in Patients with Knee Osteoarthritis: A Pilot Clinical Study.

Treatment of knee osteoarthritis (OA) remains a challenging concern. Preclinical studies provided accumulating evidence on resveratrol efficacy in ameliorating degenerative articular damage. The present study was conducted to evaluate the effects of resveratrol as monotherapy on the serum level of type II collagen (Coll 2-1) and aggrecan in patients with knee osteoarthritis. The study was an open-labeled noncontrolled clinical trial. Resveratrol 500 mg/day in a single oral dose was given to the patients with knee osteoarthritis for 90 days. The serum levels of Coll-2-1, aggrecan, and biomarkers of inflammation were measured pre- and posttreatment. Hematological profiles and both hepatic and renal function markers were investigated at the baseline and at the end of the treatment for evaluating the tolerability and safety of resveratrol. Visual Analog Scale (VAS) for pain and Knee injury and Osteoarthritis Outcome Score (KOOS) for disease activity were clinically assessed monthly. Administration of 500 mg resveratrol for three months led to a nonsignificant decrease in the serum level of Coll 2-1 while a significant increase in aggrecan serum level. Resveratrol significantly improves pain score measured by VAS and KOOS after 30 days. Improvements in patients' activity and functional status were also evident at day 30 and kept on for three months which was reflected by KOOS subscale scores and with a significant improvement in all KOOS areas. In conclusion, oral administration of resveratrol as a monotherapy provides a remarkable improvement in the clinical status of the patients but has no significant effect on serum levels of Coll 2-1.

Aggrecan: a new biomarker for acute type A aortic dissection.

Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac enzymes like creatine kinase MB and cardiac troponin T showed no correlation with ACAN levels in ATAAD patients. Receiver-operator characteristics (ROC) curve analysis for ATAAD patients versus control subjects an optimum discrimination limit of ACAN plasma levels at 14.3 ng/mL with a corresponding sensitivity of 97% and specificity of 81%. According to our findings ACAN is a reliable potential biomarker in plasma samples to detect ATAAD with high sensitivity and specificity.

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.

The influence of a sustained 10-day hypoxic bed rest on cartilage biomarkers and subchondral bone in females: The FemHab study.

This study assessed the influence of a 10-day hypoxic bed rest on cartilage biomarkers and subchondral bone density across the patellofemoral joint (PFJ). Within clinical settings hypoxic tissue may arise in several types of disorders. Furthermore, a hypoxic environment is being considered for space flight habitats in the near future. Female participants (N = 12) participated in this study comprising three 10-day interventions: hypoxic ambulation (HAMB), normoxic bed rest (NBR), and hypoxic bed rest (HBR). Venous samples were collected prior to (day -2: Pre) and during the intervention (days 2 and 5), immediately before reambulation (D11) and 24 hr post intervention (R1). Blood samples were analyzed for: aggrecan, hyaluronan, Type IIA procollagen amino terminal propeptide (PIIANP), and cartilage oligomeric matrix protein (COMP). Total bone mineral density (BMD) in eight regions (2 mm × 10 mm) across the PFJ was determined. The three interventions (HAMB, HBR, and NBR) did not induce any significant changes in the cartilage biomarkers of hyaluronan or PIIANP. Aggrecan increased during the HAMB trial to 2.02 fold the Pre value. COMP decreased significantly in both NBR & HBR compared to HAMB on D5. There were significant differences in BMD measured across the PFJ from cortical patellar bone (735 to 800 mg/cm3 ) to femur trabecular (195 to 226 mg/cm3 ). However, there were no significant changes in BMD from Pre to Post bed rest. These results indicate that there were no significant detectable effects of inactivity/unloading on subchondral bone density. The biomarker of cartilage, COMP, decreased on D5, whereas the addition of hypoxia to bed rest had no effect, it appears that hypoxia in combination with ambulation counteracted this decrease.

Time between anterior cruciate ligament injury and reconstruction and cartilage metabolism six-months following reconstruction.

BACKGROUND: To determine the association between time from injury to ACL reconstruction (TimeInjury-ACLR) and biochemical markers of cartilage metabolism and inflammation six months following ACL reconstruction (ACLR). METHODS: Individuals with a unilateral ACL injury were enrolled at initial presentation in the orthopedic clinic; blood was collected six months following ACLR. Enzyme-linked immunosorbent assays were used to analyze the ratio of serum concentrations of type-II collagen breakdown (C2C) to synthesis (CPII), plasma matrix metalloproteinase-3 (MMP-3), interleukin-6 (IL-6), and serum aggrecan neoepitope (ARGS). We used separate linear regressions to assess associations between biochemical markers and TimeInjury-ACLR. RESULTS: Twenty-two participants (50% females, mean [SD], age 21.9 [4.5] years old; BMI 23.8 [2.6] kg/m2) completed the study. TimeInjury-ACLR ranged from nine to 67days (31.0 [14.4days]). Greater TimeInjury-ACLR predicted greater serum C2C:CPII ratios six months following ACLR (C2C:CPII=0.15 [0.02], R2=0.213, P=0.030). Males (R2=0.733, P=0.001) but not females (R2=0.030, P=0.609) demonstrated a significant association between greater C2C:CPII and TimeInjury-ACLR at the six-month follow-up exam. TimeInjury-ACLR did not associate with IL-6, MMP-3, or ARGS at six months. CONCLUSIONS: Greater time between injury and ACL reconstruction was associated with greater serum C2C:CPII six months following ACLR in males but not females, and IL-6, MMP-3, and ARGS levels were not associated with TimeInjury-ACLR in males or females. The time between ACL injury and ACLR may affect collagen metabolism in males and should be further investigated in a larger study along with other patient-relevant outcomes.

The role of serum ADAMTS-1 and aggrecan on polycystic ovary syndrome in adolescents and younger-aged females.

PURPOSE: The aim of this study was to analyze serum a disintegrin-like and metalloproteinase with thrombospondin-type motifs-1 (ADAMTS-1) and aggrecan levels in adolescents and younger-aged females with polycystic ovary syndrome (PCOS) compared with ovulatory controls to determine whether these are potential markers for the prediction of PCOS diagnosis. We also aimed to determine whether they could predict the development of clinical implications associated with PCOS. METHOD: PCOS (n = 49) and ovulatory age-matched controls (n = 41) (mean age, 18.6 ± 2.5) were recruited. Anthropometric measurements were recorded and biochemical parameters were analyzed. Serum ADAMTS-1 and aggrecan levels were determined with enzyme-linked immunosorbent assay. The predictive effects of ADAMTS-1 and aggrecan on the diagnosis of PCOS and for the development of cardiovascular disease (CVD) risk and insulin resistance (IR) were evaluated. The correlation between investigated markers and anthropometric, biochemical, and hormonal parameters were also investigated. RESULTS: Mean serum ADAMTS-1 level was increased in adolescents and younger-aged females with PCOS compared to ovulatory controls. An elevated ADAMTS-1 level was positive predictive of the diagnosis of PCOS with the best cut-off value of 2.5 ng/ml (sensitivity 69% and specificity 78%). A positive predictive role of ADAMTS-1 on the development of CVD risk and IR was found among all patients. Serum ADAMTS-1 and aggrecan levels were significantly and positively correlated with each other. CONCLUSION: Increased levels of ADAMTS-1 could be a potential marker for the etiopathogenesis of PCOS in adolescents and younger-aged females and predict the development of CVD risk and IR among all patients with the same age.

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA. METHODS: CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry. FINDINGS: Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage. CONCLUSIONS: CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Walking Speed As a Potential Indicator of Cartilage Breakdown Following Anterior Cruciate Ligament Reconstruction.

OBJECTIVE: To determine whether or not self-selected walking speed associates with serum biomarkers of cartilage (collagen and proteoglycan) breakdown in anterior cruciate ligament reconstructed (ACLR) individuals. METHODS: Twenty individuals with a history of a primary unilateral ACLR participated in this cross-sectional study. Resting blood was collected from each participant prior to completing 5 walking gait trials at a self-selected comfortable speed. Walking speed was evaluated in a 3-dimensional motion capture laboratory and determined from the velocity of the pelvic center of mass. Sera were assessed for collagen type II cleavage product (C2C) and proteoglycan (aggrecan) concentrations using commercially available specific enzyme-linked immunosorbent assays. Pearson's product-moment (r) and Spearman's (ρ) correlations were used to evaluate associations between walking speed and biomarkers of cartilage breakdown metabolism. Partial correlations were used to determine whether covariates influenced associations between walking speed and biomarkers of cartilage breakdown. RESULTS: ACLR individuals with a slower walking speed demonstrated higher concentrations of serum C2C (r = -0.52, P = 0.02), while there was no significant association between walking speed and aggrecan concentrations (ρ = -0.29, P = 0.31). After accounting for the variance associated with stance phase duration, ACLR individuals with a slower walking speed still demonstrated greater serum C2C concentrations (partial r = -0.53, P = 0.02). CONCLUSION: ACLR individuals who habitually walk slower may experience a greater degree of collagen breakdown, suggesting that walking speed may be a future useful clinical indicator for identifying individuals with higher levels of cartilage breakdown and preradiographic osteoarthritic joint changes.

Circulating keratan sulfate as a marker of metabolic changes of cartilage proteoglycan in juvenile idiopathic arthritis; influence of growth factors as well as proteolytic and prooxidative agents on aggrecan alterations.

BACKGROUND: The aim of this study was to evaluate the plasma keratan sulfate (KS) level as a potential marker of joint damage in children with juvenile idiopathic arthritis (JIA). The influence of growth factors as well as proteolytic and prooxidative agents on aggrecan alterations were evaluated in this study. METHODS: Plasma levels of KS, transforming growth factor ß1 (TGF-ß1), platelet-derived growth factor BB (PDGF-BB), a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4 and ADAMTS-5), and thiol groups (TG) were quantified in samples obtained from 30 healthy subjects and 30 patients with JIA before and after treatment. RESULTS: Increased (p<0.01) plasma KS was observed in JIA patients before treatment. Therapy resulted in a decrease in KS level. However, plasma KS level remained higher (p<0.05) than in controls. Increased levels of TGF-ß1 (p<0.01) and PDGF-BB (p<0.05) in untreated JIA patients were recorded. Clinical improvement was accompanied by significant decrease in TGF-ß1 and PDGF-BB, compared with a pretreatment condition and a control group. The concentrations of proteinases were characterized by different trends of alterations. When the ADAMTS-4 level increased (p<0.01) in the blood of untreated patients, the concentration of ADAMTS-5 was found to be reduced (p<0.0001), compared with controls. JIA treatment resulted in the normalization of ADAMTS-4 level. Plasma TG concentration was decreased only in untreated patients (p<0.05). We have revealed a significant correlation between plasma KS level and ADAMTS-4, TGF-ß1, TG, C-reactive protein, and erythrocyte sedimentation rate levels. CONCLUSIONS: Plasma KS level in JIA patients, reflecting the aggrecan structure, indicates that treatment that modifies inflammation simultaneously does not contribute to total regeneration of articular matrix components and signalizes the need for further treatment.

An ARGS-aggrecan assay for analysis in blood and synovial fluid.

OBJECTIVE: To validate a modified ligand-binding assay for the detection of aggrecanase generated aggrecan fragments with the ARGS neoepitope in synovial fluid (SF) and blood, and to verify the identity of aggrecan fragments found in blood. DESIGN: An enzyme-linked immunosorbent assay (ELISA) on the Meso Scale Discovery (MSD) platform for detection of ARGS-aggrecan was validated, using a standard made from recombinant human aggrecan. Matched samples of SF, serum, plasma, and urine were obtained from 36 subjects at different time points after knee injury, and analysed for ARGS-aggrecan content. Aggrecan was purified from serum and plasma pools and analysed by Western blot. RESULTS: The limits of quantification for the ARGS-aggrecan assay was between 0.2 and 0.025 pmol ARGS/ml, and the sensitivity of the assay was improved two-fold compared to when using a standard purified from human donors. The ARGS concentrations were highest in SF (mean, range; 3.02, 0.36-30.22 pmol/ml), 20 times lower in the blood samples (0.14, 0.055-0.28 pmol/ml serum and 0.13, 0.053-0.28 pmol/ml plasma), and 80 times lower in urine (0.036, below detection - 0.087 pmol/ml). Serum-ARGS and plasma-ARGS concentrations were similar, and correlated (r(S) = 0.773, P < 0.001). SF concentration correlated with serum concentrations (r(S) = 0.420, P = 0.011). In blood, we identified 129-138 kDa aggrecan fragments containing the ARGS neoepitope. CONCLUSIONS: This novel ARGS-aggrecan assay is highly sensitive and suited for analysis of SF and blood samples. Both SF and blood contains ARGS-aggrecan, and ARGS concentrations in SF and serum are correlated.

Association between cartilage and bone biomarkers and incidence of radiographic knee osteoarthritis (RKOA) in UK females: a prospective study.

OBJECTIVE: There is a need to find biochemical markers that would identify people with increased risk of developing radiographic knee osteoarthritis (RKOA). The aim of this study was to evaluate the ability of cartilage and bone biomarkers (cartilage oligomeric matrix protein (COMP), aggrecan, cellular inhibitor of apoptosis protein (cIAP), N-telopeptide-to-helix (NTx)) to predict RKOA incidence in a 10-year follow-up of UK females from the Chingford community study. METHOD: Joint space narrowing (JSN), osteophytes (OSP) and Kellgren-Lawrence (K/L) grades were scored from radiographs of both knees at study baseline and 10 years later in 1,003 women aged 45-64. Circulating levels of biomarkers and demographic variables were measured at baseline. Statistical association analysis was conducted between the potential predictor factors measured at baseline and documentation of RKOA at 10-year follow-up. RESULTS: Age and body mass index (BMI), were significant predictors of incidence of RKOA as assessed by K/L and OSP. Considering biomarkers, independent significant association was found between COMP circulating levels and K/L scores (Odd Ratio (OR) = 2.87, 95% Confidence Interval (CI) = 1.19-6.89, P = 0.018). Significant negative association was detected between aggrecan plasma concentrations and JSN, with OR = 0.37 (95% CI 0.15-0.89), P = 0.026. CONCLUSIONS: Aggrecan and COMP circulating levels contribute to identification of phenotype-specific RKOA incidence. These data suggest potentially protective role of aggrecan in cartilage loss, as measured by JSN. High COMP levels are risk factors for development of RKOA, as assessed by K/L scores.

Association of serum levels of aggrecan ARGS, NITEGE fragments and radiologic knee osteoarthritis in Tunisian patients.

OBJECTIVES: Proteolytic degradation of aggrecan is a hallmark of the pathology of osteoarthritis. The aim of this study was to develop enzyme-linked immunosorbent assay (ELISA) to quantify the serum levels of specific aggrecan fragments generated by aggrecanases-mediated cleavage. We investigated the relationships between these two aggrecan degradations fragments and urinary CTX-II levels. METHODS: The competitive ELISAs employ a polyclonal antibody raised against the aggrecan fragments containing two neoepitopes NITEGE(373)and (374)ARGSVI. We measured serum levels of ARGSV and NITEGE in 125 women with knee osteoarthritis (mean±SD age of 53.6±7.6 years, mean±SD disease duration of 3.6±3.8 years), and 57 women age-matched controls. RESULTS: Aggrecan neoepitopes assays showed an intra- and inter-assay imprecision (CV) lower than 20% for both tests and good linearity. Median serum ARGSVI (by 18%; P=0.002), and NITEGE (36.4%; P<0.001) levels were significantly decreased in patients with knee osteoarthritis compared with controls. Minimal joint space width was negatively correlated with ARGSVI (r=-0.368, P=0.04) and NITEGE (r=-0.274, P=0.038) in knee osteoarthritis patients. Median urinary CTX-II levels were significantly increased by 39.5% (P=0.001) in knee OA patients compared with controls. CONCLUSION: Markers of degradation aggrecan were analyzed for the first time in an African osteoarthritis population. These markers can be used to monitor aggrecanase activity in human joint disease. Their combination with CTX-II can improve clinical investigation of patients with osteoarthritis patients.

[Effect of cartilage protective agents on histopathological, histochemical features of articular cartilage and serum level of aggrecan in Hartley guinea pigs].

OBJECTIVE: To observe the effect of glucosamine (GS) and chondroitin sulfate (CS) on histopathological, histochemical features of articular cartilage and on the aggrecan serum level in Hartley guinea pigs: a kind of primary OA animal model. METHODS: 120 female Hartley guinea pigs aged 2 months were randomly divided into 3 test groups and a control group, 30 animals for each group. The three test groups refer to GS group, CS group and combined group. GS group has been administrated with 1 g/kg bw GS, CS group administrated with 0.5 g/kg bw CS, combined group with 1 g/kg bw GS + 0.5 g/kg bw CS and a control group administrated with distilled water. The above four substances were treated via ad limbitum for a period of 5 months. Before dosing and after each monthly treatment during the five months, knee joints Cartilage specimens from 5 guinea pigs each group were examined through histopathological method (H. E stain) and histopathological method (Alcain Blue, PAS and Mallory stain), and the serum levels of aggrecan were detected synchronously. RESULTS: During the entire 5 months period, distinct pathological lesions appeared just after the first month in control group. And the distinct pathological lesions didn't appear until the third month ended in GS group. In CS group, moderate pathological lesions were observed in the fourth month, while there were almost no obviously pathological changes in combined group during the whole test period. The serum levels of aggrecan in all three test groups were all decreased slower after 4 months treatment than those in the control group, with a significant difference (P < 0.05). CONCLUSION: GS and CS can postpone and inhibit the pathological changes of articular cartilage, as well as serum aggrecan levels decrease in Hartley guinea pigs. The effect of GS and CS used in combination was stronger than individual GS and CS, showing a repair property.

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic auto-immune disease with extensive articular cartilage destruction. Aggrecan depletion, mediated by aggrecanases is one of the first signs of early cartilage erosion. We investigated, whether measurement of aggrecan and fragments thereof in serum, could be used as biomarkers for joint-disease in RA patients and furthermore characterized the fragments found in the circulation. METHODS: The study consisted of 38 patients, 12 males (62.2 +/- 16.0 years) and 26 females (59.8 +/- 20.7 years) diagnosed with RA: 41.5 +/- 27.5 mm/h erythrocyte sedimentation rate (ESR), 38.4 +/- 34.7 mg/ml C-reactive protein (CRP) and 4.8 +/- 1.7 disease activity score (DAS) and 108 healthy age-matched controls. Aggrecan levels were measured using two immunoassays, i.e. the (374)ARGSVI-G2 sandwich ELISA measuring aggrecanase-mediated aggrecan degradation and the G1/G2 sandwich assay, detecting aggrecan molecules containing G1 and/or G2 (total aggrecan) We further characterized serum samples by western blots, by using monoclonal antibodies F-78, binding to G1 and G2, or by BC-3, detecting the aggrecanase-generated N-terminal 374ARGSVI neo-epitope. RESULTS: Total aggrecan levels in RA patients were significantly decreased from 824.8 +/- 31 ng/ml in healthy controls to 570.5 +/- 30 ng/ml (31% decrease, P < 0.0001), as measured by the G1/G2 ELISA. Western blot analysis with F-78 showed one strong band at 10 kDa, and weaker bands at 25 and 45 kDa in both healthy controls and RA patients. In contrast, staining for aggrecanase-activity revealed only one strong band in RA patients of 45 kDa. CONCLUSION: This is the first study, which characterizes different aggrecan fragments in human serum. The data strongly suggests that total aggrecan levels, i.e. aggrecan molecules containing G1 and/or G2 are lower in RA patients, and that RA patients have at least one specific subpopulation of aggrecan fragments, namely aggrecanse generated 374ARGSVI fragments. Further clinical studies are needed to investigate the potential of G1/G2 as a structure-related biochemical marker in destructive joint-diseases.