M.-F. Hsueh

Biomarkers and proteomic analysis of osteoarthritis.

Our friend and colleague, Dr. Dick Heinegård, contributed greatly to the understanding of joint tissue biochemistry, the discovery and validation of arthritis-related biomarkers and the establishment of methodology for proteomic studies in osteoarthritis (OA). To date, discovery of OA-related biomarkers has focused on cartilage, synovial fluid and serum. Methods, such as affinity depletion and hyaluronidase treatment have facilitated proteomics discovery research from these sources. Osteoarthritis usually involves multiple joints; this characteristic makes it easier to detect OA with a systemic biomarker but makes it hard to delineate abnormalities of individual affected joints. Although the abundance of cartilage proteins in urine may generally be lower than other tissue/sample sources, the protein composition of urine is much less complex and its collection is non-invasive thereby facilitating the development of patient friendly biomarkers. To date however, relatively few proteomics studies have been conducted in OA urine. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA. Further targeted approaches to validate the role of these proteins in OA are needed. Herein we summarize recent proteomic studies related to joint tissues and the cohorts used; a clear understanding of the cohorts is important for this work as we expect that the decisive discoveries of OA-related biomarkers rely on comprehensive phenotyping of healthy non-OA and OA subjects. Besides the common phenotyping criteria that include, gender, age, and body mass index (BMI), it is essential to collect data on symptoms and signs of OA outside the index joints and to bolster this with objective imaging data whenever possible to gain the most precise appreciation of the total burden of disease. Proteomic studies on systemic biospecimens, such as serum and urine, rely on comprehensive phenotyping data to unravel the true meaning of the proteomic results.

Xanthine oxidase injurious response in acute joint injury

BACKGROUND While acute trauma is a major cause of osteoarthritis, its etiology is poorly understood. We sought to determine whether xanthine oxidase (XO), a major producer of reactive oxygen species, plays a role in the early events of acute joint injury. METHODS We analyzed synovial fluid from 23 subjects with recent severe acute knee injury. As a control we evaluated SF from 23 individuals with no or minimal knee osteoarthritis. We measured XO activity, reactive oxygen+reactive nitrogen species (ROS+RNS), protein oxidative damage (carbonyl), the type II collagen synthesis marker procollagen II c-propeptide (CPII) and the type II collagen degradation marker collagen type II telopeptide (CTx-II). We also measured the proinflammatory cytokine IL-6. RESULTS XO and ROS+RNS were higher (p=0.02 and p=0.001 respectively) in acute injury than control and were strongly positively associated (r=0.62, p=0.004). Carbonyl was higher in acute injury than control (p=0.0002) and was positively correlated with XO (r=0.68, p=0.0007) as well as with ROS+RNS (r=0.71, p=0.004). CPII was higher in acute injury than control (p<0.0001) and was negatively correlated with XO (r=-0.49, p=0.017). While CTxII was not significantly higher in acute injury than control, it was positively correlated with CPII (r=0.71, p=0.0002). IL-6 was higher in acute injury than control (p<0.0001). CONCLUSIONS These results are consistent with a potentially injurious effect of XO activity in acute joint injury characterized by excess free radical production and oxidative damage. These effects are associated with an inhibition of type II collagen production that may impede the ability of the injured joint to repair.

Cartilage biomarkers in the osteoarthropathy of alkaptonuria reveal low turnover and accelerated ageing

Objective. Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. Methods. With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann–Whitney U test. Results. Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. Conclusions. These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins.

Cartilage matrix remodelling differs by disease state and joint type

Dramatic alterations in mechanical properties have been documented for osteoarthritic (OA) cartilage. However, the matrix composition underlying these changes has not been mapped and their aetiology is not entirely understood. We hypothesised that an understanding of the cartilage matrix heterogeneity could provide insights into the origin of these OA-related alterations. We generated serial transverse cryo sections for 7 different cartilage conditions: 2 joint sites (knee and hip), 2 disease states (healthy and OA) and 3 tissue depths (superficial, middle and deep). By laser capture microscopy, we acquired ~200 cartilage matrix specimens from territorial (T) and interterritorial (IT) regions for all 7 conditions. A standardised matrix area was collected for each condition for a total of 0.02 ± 0.001 mm3 (corresponding to 20 µg of tissue) from a total of 4800 specimens. Extracted proteins were analysed for abundance by targeted proteomics. For most proteins, a lower IT/T ratio was observed for the OA disease state and knee joint type. A major cause of the altered IT/T ratios was the decreased protein abundance in IT regions. The collagenase-derived type III collagen neo-epitope, indicative of collagen proteolysis, was significantly more abundant in OA cartilage. In addition, it was enriched on average of 1.45-fold in IT relative to T matrix. These results were consistent with an elevated proteolysis in IT regions of OA cartilage, due to degenerative influences originating from synovial tissue and/or produced locally by chondrocytes. In addition, they offered direct evidence for dynamic remodelling of cartilage and provided a cogent biochemical template for understanding the alterations of matrix mechanical properties.

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.