keratan-sulfate and Knee-Injuries

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

Topics: ADAM Proteins; ADAMTS4 Protein; Adolescent; Adult; Aged; Aged, 80 and over; Aggrecans; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Keratan Sulfate; Knee Injuries; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Middle Aged; Osteoarthritis; Peptide Fragments; Procollagen N-Endopeptidase; Protein Interaction Domains and Motifs; Proteolysis; Recombinant Proteins; Substrate Specificity; Synovial Fluid; Young Adult

To find serum markers that may serve as indices for an early diagnosis of degeneration or damage of the articular cartilage.. Twenty-four healthy volunteers, 19 individuals with knee trauma (KT) and 31 with knee osteoarthritis (OA) were evaluated. KT patients were divided into a group (n = 5) with an injury <2 months old (recent KT) and a group (n = 14) with that >2 months old (old KT). Articular cartilage damage was assessed using either arthroscopy or direct observation. Serum concentrations of hyaluronic acid (HA), cartilage proteoglycan aggrecan turnover epitope (CS846) and cartilage oligomeric protein (COMP) were measured using enzyme-linked immunosorbent assay kits and those of keratan sulfate (KS) and chondroitin-6-sulfate (C6S) using high-performance liquid chromatography.. Serum KS in the recent KT group (2095 +/- 594 ng/ml) was significantly higher than that in the old KT group (1373 +/- 418 ng/ml; P = 0.021), and serum COMP in the recent KT group (1572 +/- 182 ng/ml) showed a tendency that was higher than that in the old KT group (1350 +/- 250 ng/ml; P = 0.079). Serum KS in OA patients with Kellgren and Lawrence (KL) grades 0 and I (1456 +/- 334 ng/ml) showed a tendency that was higher than that in OA patients with KL grades II, III and IV (1248 +/- 220 ng/ml; P = 0.084).. The serum concentration of KS correlated with the damage of the articular cartilage and it was significantly increased even at an early stage after the injury.

Topics: Adult; Aged; Arthroscopy; Biomarkers; Cartilage Diseases; Cartilage Oligomeric Matrix Protein; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix Proteins; Female; Glycoproteins; Humans; Keratan Sulfate; Knee Injuries; Male; Matrilin Proteins; Middle Aged; Osteoarthritis, Knee; Radiography

We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls.. CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate.. The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS (P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower (P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher (P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS.. The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.

Topics: Arthritis, Rheumatoid; Cartilage, Articular; Collagen Type II; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Keratan Sulfate; Knee Injuries; Knee Joint; Osteoarthritis, Knee; Pancreatic Elastase; Proteoglycans; Synovial Fluid; Synovitis

Results from several recent studies suggest that the levels of antigenic keratan sulfate (agKS) and hyaluronan (HA) in serum provide useful information about changes taking place in injured or diseased synovial joints. To improve our understanding of the significance of such changes, we investigated the points of entry of these molecules into the blood circulation and their subsequent clearance after experimentally induced injury to rabbit knee joint.. Chymopapain was injected into knee joints of 8 young adult rabbits to induce aggrecan degradation in articular cartilage within the injected joint. Levels of agKS and HA in serum from various blood vessels were measured before and 5 h after the injury. The statistical significance of injury related changes and differences among the different vessels were evaluated.. After the injury, the level of agKS rose most significantly in the popliteal vein draining the injected knee joint and dropped rapidly by the time the blood reached the femoral vein. The level of agKS was similar, although lower, in other blood vessels but, in each case, it was significantly higher than before the injection. The level of HA showed a different pattern of changes after injection. While highest in the popliteal vein draining the injected knee, HA was markedly elevated in the cranial vena cava, close to the entry of lymph into the circulation, and was 50% lower in the hepatic than in the portal vein.. (1) Measurement of agKS and HA in a blood vessel draining or close to an injured/diseased knee joint may provide more specific information about degradative changes taking place in that joint than measurement of levels of these markers in other blood vessels; (2) some HA molecules but no measurable amounts of agKS enter the blood circulation via the lymphatic system: and (3) HA but not agKS is very rapidly cleared from the blood by the liver.

Topics: Animals; Chymopapain; Hyaluronic Acid; Keratan Sulfate; Knee Injuries; Male; Rabbits; Synovial Fluid

To measure the levels of epitope on the chondroitin sulfate (CS) and keratan sulfate (KS) chains of proteoglycan fragments in synovial fluids from injured and contralateral uninjured knees of patients with traumatic cruciate ligament and/or meniscus damage.. Enzyme-linked immunosorbent assays were used to determine the levels of monoclonal antibody epitopes 3-B-3 and 7-D-4 (CS), and 5-D-4 (KS), in paired joint fluids from the injured and uninjured knees of trauma patients.. Levels of the CS epitopes were increased in the trauma joint fluids from most patients, with higher levels of 3-B-3 epitope in 12 of the 16 patients, but the difference did not achieve significance; however, 7-D-4 levels were higher in 15 patients, and the difference was highly significant (P = 0.0005). In contrast, the KS epitope detected by 5-D-4 was decreased in 13 of 15 patients, and the difference was significant (P = 0.0074).. The increased level of 7-D-4 epitope on proteoglycans in joint fluid from injured knees may reflect the response of the articular cartilage to acute trauma resulting in altered expression of specific CS epitopes on cartilage proteoglycans. The fall in KS epitope levels may reflect the synthesis of proteoglycans that have lower KS content.

Topics: Antibodies, Monoclonal; Cartilage, Articular; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycosaminoglycans; Humans; Keratan Sulfate; Knee Injuries; Knee Joint; Synovial Fluid