J. Catterall

Changes in serum and synovial fluid biomarkers after acute injury (NCT00332254)

IntroductionAcute trauma involving the anterior cruciate ligament is believed to be a major risk factor for the development of post-traumatic osteoarthritis 10 to 20 years post-injury. In this study, to better understand the early biological changes which occur after acute injury, we investigated synovial fluid and serum biomarkers.MethodsWe collected serum from 11 patients without pre-existing osteoarthritis from a pilot intervention trial (5 placebo and 6 drug treated) using an intra-articular interleukin-1 receptor antagonist (IL-1Ra) therapy, 9 of which also supplied matched synovial fluid samples at presentation to the clinic after acute knee injury (mean 15.2 ± 7.2 days) and at the follow-up visit for reconstructive surgery (mean 47.6 ± 12.4 days). To exclude patients with pre-existing osteoarthritis (OA), the study was limited to individuals younger than 40 years of age (mean 23 ± 3.5) with no prior history of joint symptoms or trauma. We profiled a total of 21 biomarkers; 20 biomarkers in synovial fluid and 13 in serum with 12 biomarkers measured in both fluids. Biomarkers analyzed in this study were found to be independent of treatment (P > 0.05) as measured by Mann-Whitney and two-way ANOVA.ResultsWe observed significant decreases in synovial fluid (sf) biomarker concentrations from baseline to follow-up for sfC-Reactive protein (CRP) (P = 0.039), sflubricin (P = 0.008) and the proteoglycan biomarkers: sfGlycosaminoglycan (GAG) (P = 0.019), and sfAlanine-Arginine-Glycine-Serine (ARGS) aggrecan (P = 0.004). In contrast, we observed significant increases in the collagen biomarkers: sfC-terminal crosslinked telopeptide type II collagen (CTxII) (P = 0.012), sfC1,2C (P = 0.039), sfC-terminal crosslinked telopeptide type I collagen (CTxI) (P = 0.004), and sfN-terminal telopeptides of type I collagen (NTx) (P = 0.008). The concentrations of seven biomarkers were significantly higher in synovial fluid than serum suggesting release from the signal knee: IL-1β (P < 0.0001), fetal aggrecan FA846 (P = 0.0001), CTxI (P = 0.0002), NTx (P = 0.012), osteocalcin (P = 0.012), Cartilage oligomeric matrix protein (COMP) (P = 0.0001) and matrix metalloproteinase (MMP)-3 (P = 0.0001). For these seven biomarkers we found significant correlations between the serum and synovial fluid concentrations for only CTxI (P = 0.0002), NTx (P < 0.0001), osteocalcin (P = 0.0002) and MMP-3 (P = 0.038).ConclusionsThese data strongly suggest that the biology after acute injury reflects that seen in cartilage explant models stimulated with pro-inflammatory cytokines, which are characterized by an initial wave of proteoglycan loss followed by subsequent collagen loss. As the rise of collagen biomarkers in synovial fluid occurs within the first month after injury, and as collagen loss is thought to be irreversible, very early treatment with agents to either reduce inflammation and/or reduce collagen loss may have the potential to reduce the onset of future post-traumatic osteoarthritis.Trial registrationThe samples used in this study were derived from a clinical trial NCT00332254 registered with ClinicalTrial.gov.

Protein Modification by Deamidation Indicates Variations in Joint Extracellular Matrix Turnover

As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage.

Post-translational aging of proteins in osteoarthritic cartilage and synovial fluid as measured by isomerized aspartate.

IntroductionAging proteins undergo non-enzymatic post-translational modification, including isomerization and racemization. We hypothesized that cartilage with many long-lived components could accumulate non-enzymatically modified amino acids in the form of isomerized aspartate and that its liberation due to osteoarthritis (OA)-related cartilage degradation could reflect OA severity.MethodsArticular cartilage and synovial fluid were obtained from 14 randomly selected total knee arthroplasty cases (56 to 79 years old) and non-arthritis cartilage from 8 trauma cases (51 to 83 years old). Paired lesional cartilage and non-lesioned OA cartilage were graded histologically using a modified Mankin system. Paired cartilage and synovial fluids were assayed for isomerized aspartate, phosphate-buffered saline/EDTA (ethylenediaminetetraacetic acid) extractable glycosaminoglycans, and total protein. Macroscopically normal non-lesioned OA cartilage was separated into superficial and deep regions when cartilage thickness was at least 3 mm (n = 6).ResultsNormalized to cartilage wet weight, normal cartilage and deep non-lesioned OA cartilage contained significantly (P < 0.05) more isomerized aspartate than superficial non-lesioned OA cartilage and lesioned cartilage. Synovial fluid isomerized aspartate correlated positively (R2 = 0.53, P = 0.02) and glycosaminoglycans correlated negatively (R2 = 0.42, P = 0.04) with histological OA lesion severity. Neither synovial fluid isomerized aspartate nor glycosaminoglycans nor total protein correlated with histological scores of non-lesioned areas.ConclusionsWe show for the first time that human cartilage and synovial fluid contain measurable quantities of an isomerized amino acid and that synovial fluid concentrations of isomerized aspartate reflected severity of histological OA. Further assessment is warranted to identify the cartilage proteins containing this modification and to assess the functional consequences and biomarker applications of this analyte in OA.

142 A UNIQUE DEAMIDATED CARTILAGE OLIGOMERIC MATRIX PROTEIN (COMP) BIOMARKER PREFERENTIALLY IDENTIFIES HIP OSTEOARTHRITIS

Results: Cartilage pentosidine (a marker of AGE) levels were ~5 fold enhanced (p =0.001 vs. PBS injected joints). The macroscopic and microscopic damage was similar for the AGEd and PBS injected joints (macroscopic score of 1 of a max score of 4 and histological OARSI score of 3–5 of a max score of 36, respectively). For the PG synthesis (5.09±0.29 vs 4.64±0.32) % total and newly formed PG (23.29±1.88 vs 23.66±2.02 and 43.02±2.23 vs 43.75±2.4), as well as the PG content (31.20±1.17 vs 30.83±0.98) no statistically difference could be found between both groups. This was not expected and therefore no control (untouched) and OA (experimentally induced according to the classic Groove model) joint were included in the study. Therefore, the biochemical data were compared to historical controls of the most recent study using the Groove model. Comparing these results, it demonstrates that the AGEd joints are in general in between the PBS injected joints and the experimental early OA joints (for example PG content 31.20±1.17 vs 30.83±0.98 vs 30.44±1.52). The PBS injected joints are in between the normal healthy joints and the AGEd joints. Assuming a stepwise increase in severity of damage there was a clear correlation between severity of damage and the different conditions from healthy control joints, via PBS injected joints with minimally surgically damage, AGEd joints with minimal surgical damage, to experimentally induced OA joints according to the Groove model (PG synthesis r = −0.873, % total and newly formed PG release r = 0.971, p = 0.029 and r = 0.954, p = 0.046, and PG content r = 0.836). Conclusions: Despite the fact that enhanced cross-linking of macromolecules by the AGEs restrains loss of proteoglycans, corroborating the diminished turnover of old cartilage, and a tendency toward enhanced cartilage damage in the artificially aged joints, the present data do not clearly support a role for enhanced cartilage AGE levels in development of joint degeneration.