alpha-chymotrypsin and Osteoarthritis

To determine if type III collagen is concentrated in the chymotrypsin-extractable collagen pool from osteoarthritic articular cartilage to assess its potential as a biomarker of Osteoarthritis (OA) pathogenic mechanisms.. Full thickness articular cartilage from grossly normal surfaces was analyzed from femoral heads, obtained at hip replacement surgery, from OA (n = 10) and fracture (n = 10) patients. Collagen, extracted by α-chymotrypsin, was characterized by SDS-PAGE/Western blot analysis, ELISA and immunohistochemistry using monoclonal antibodies specific to collagens types II and III.. α-Chymotrypsin extracted more collagen from OA than control cartilage. The extractable pool included collagen types II and III from both OA and control hips. Importantly, OA cartilage contained 6-fold more collagen type III than control cartilage, based on ELISA. The estimated total tissue ratio of collagen III/II was in the 1-10% range for individual OA cartilage samples, based on pepsin-solubilized collagen using SDS-PAGE densitometry. Collagen type III N-propeptide trimers were the main molecular fragments seen on Western blot analysis of OA and control extracts. The chymotrypsin-extracted type II collagen gave primarily full-length α1(II) chains and chain fragments of α1(II) on Western blot analysis from both OA and control tissues. Immunohistochemistry showed that type III collagen was more concentrated in the upper half of OA cartilage and in the territorial matrix around individual chondrocytes and chondrocyte clusters.. The findings confirm that collagen type III deposition occurs in adult articular cartilage but significantly more pronounced in osteoarthritic joints, presenting a potential marker of matrix repair or pathobiology.

Topics: Cartilage, Articular; Chondrocytes; Chymotrypsin; Collagen Type II; Collagen Type III; Humans; Osteoarthritis

To evaluate the effects of the proteasome inhibitor MG-132 on the expression of nuclear factor (NF)-κB p65, inhibitor (I)-κB, tumour necrosis factor (TNF)-α, and interleukin (IL)-1β in the cartilage and synovial tissues of rats with osteoarthritis (OA), and to investigate the role that the ubiquitin/proteasome system (UPS) plays in the OA process.. A total of 144 adult male Sprague Dawley rats were randomly assigned to four groups: anterior cruciate ligament transaction (ACLT) + MG-132 (ACLT/M), ACLT + dimethylsulfoxide (ACLT/D), sham surgery (Sham), and naïve + MG-132 (naïve/M). Pathological morphology was undertaken. mRNA expression levels of NF-κB p65, I-κB, TNF-α, and IL-1β were determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes were measured using fluorospectrophotometry.. The Mankin scores at all time points in ACLT/M rats were significantly lower than those in ACLT/D rats (p < 0.05). Despite the NF-κB p65 in the synovial tissue at 2 weeks after surgery and IL-1β in the cartilage tissue at 12 weeks after surgery, mRNA expression levels of NF-κB p65, IL-1β, and TNF-α at other time points in ACLT/M were significantly lower than those in ACLT/D (p < 0.05). mRNA levels of I-κB in the cartilage tissue in ACLT/M were significantly higher than those in ACLT/D at 2 weeks after surgery (p < 0.05). mRNA levels of I-κB in the synovial tissue in ACLT/M were higher than those in ACLT/D at all time points, and the difference was significant at 4 weeks after surgery (p < 0.05). MG-132 decreased the activities of the 20S proteasome chymotrypsin-like and peptidylglutamyl-peptide hydrolase-like enzymes in the cartilage and synovial tissues of rats.. The proteasome inhibitor MG-132 delays the progress of OA by alleviating synovial inflammation and protecting the articular cartilage tissue.

Topics: Animals; Anterior Cruciate Ligament; Cartilage, Articular; Chymotrypsin; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endopeptidases; I-kappa B Proteins; Interleukin-1beta; Leupeptins; Male; Osteoarthritis; Physical Conditioning, Animal; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Signal Transduction; Synovial Membrane; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simplified method for the quantification of the amount of degraded collagen in the collagen network of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by alpha-chymotrypsin, hydrolysis in 6 M HCl of the released fragments as well as the residual tissue, and then measurement of the amount of hydroxyproline in both pools. Since the digestion of degraded collagen by alpha-chymotrypsin and measurement of hydroxyproline is not restricted to a specific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investigations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy cartilage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in osteoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.

Topics: Adult; Aged; Aged, 80 and over; Cartilage, Articular; Chromatography, High Pressure Liquid; Chymotrypsin; Collagen; Female; Fluorenes; Humans; Middle Aged; o-Phthalaldehyde; Osteoarthritis; Reproducibility of Results

To characterize the effect of added fibronectin (Fn) on human rheumatoid synoviocytes (RAS).. Early passage cultured RAS were studied by fluorescent imaging microscopy with multiple parameter overlaying and confocal laser scanning microscopy.. Added Fn induced a localized decrease in matrix Fn at sites of cell overlap. Similar matrix depletion could be induced by collagen VI, cell binding (120 k) and heparin binding (60 k) fragments of Fn, but not by gelatin binding fragment (45 k).. The decrease in matrix Fn was associated with the induction of a transformation-like phenotype of overlapping cell growth. Both phenomena were inhibited by serine protease inhibitors.

Topics: Arthritis, Rheumatoid; Cell Division; Cells, Cultured; Chymotrypsin; Collagen; Culture Media; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Image Processing, Computer-Assisted; Microscopy, Confocal; Middle Aged; Osteoarthritis; Peptide Fragments; Serine Proteinase Inhibitors; Synovial Membrane; Time Factors; Trypsin Inhibitors

To determine if differential glycosylation of fibronectin (Fn) in inflammatory synovial fluid (SF) included expression of an oncofetal epitope (Onf Fn) heretofore detected only on Fn derived from embryonal or neoplastic tissue.. Fn were purified from plasma, SF and synoviocyte conditioned medium by affinity chromatography and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting utilizing a monoclonal antibody (FDC-6) specifically recognizing the Onf Fn.. The Onf Fn was not expressed on Fn isolated from normal or RA plasma but was strongly expressed on Fn from rheumatoid arthritis (RA) SF and to a lesser extent osteoarthritis SF. Onf Fn was also detected on Fn secreted by cultured RA synoviocytes.. Fn present in the SF but not plasma of patients with rheumatic disease contains an epitope previously thought to be restricted to Fn produced by embryonal or malignant tissue.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Autoradiography; Blotting, Western; Cells, Cultured; Chromatography, Affinity; Chymotrypsin; Culture Media, Conditioned; Densitometry; Electrophoresis, Polyacrylamide Gel; Epitopes; Fibronectins; Glycosylation; Humans; Osteoarthritis; Rheumatic Diseases; Synovial Fluid; Thermolysin

The link protein components of proteoglycan aggregates in adult human articular cartilage show heterogeneity due to proteolysis. Cleavages near the N-terminus of the intact link proteins, before residues 17, 19 and 24, generate three proteins of slightly diminished size (LP3). Cleavages within the N-terminal disulphide-bonded loop, before residues 66 and 73 of the intact link proteins, generate proteins that yield smaller degradation products upon reduction (LP fragments). In vitro, modified link protein components of a similar size to LP3 can be generated by a variety of proteinases, but of the physiologically relevant enzymes only stromelysin, cathepsin B and cathepsin G have the ability to yield modified link proteins with N-termini identical with those observed in situ. None of the proteolytic agents tested was able to produce LP fragments with N-termini identical with those observed in situ, and the majority of proteinases were not able to cleave within the disulphide-bonded loops. Cathepsin L and hydroxyl radicals can cleave within the N-terminal disulphide-bonded loop, and have the potential of initially opening the loop to allow further proteolytic processing by other agents to generate the native cleavage sites.

Topics: Adult; Amino Acid Sequence; Arthritis, Rheumatoid; Cartilage, Articular; Cathepsin B; Cathepsin G; Cathepsin L; Cathepsins; Chymotrypsin; Cysteine Endopeptidases; Endopeptidases; Extracellular Matrix Proteins; Humans; Infant, Newborn; Matrix Metalloproteinase 3; Metalloendopeptidases; Middle Aged; Molecular Sequence Data; Osteoarthritis; Peptide Fragments; Proteins; Proteoglycans; Serine Endopeptidases

The concentrations of five normally occurring protease inhibitors in serum and synovial fluid were compared in patients with rheumatoid arthritis, osteoarthrosis, and normal controls. The patients with rheumatoid arthritis showed a significant rise in alpha1-antitrypsin, alpha1-antichymotrypsin, and inter-alpha-trypsin inhibitor (in decreasing order) in serum as well as in synovial fluid. In synovial fluid the inhibitors were present in their native form and bound to hyaluronate. A large molecular protein with immunological specificity of alpha1-antitrypsin, presumably a complex of alpha1-antitrypsin and a protease, could be shown in synovial fluid of all patients with classical and probable rheumatoid arthritis and not in that of the other subjects studied.23Author

Topics: alpha 1-Antitrypsin; Arthritis, Rheumatoid; Chromatography, Gel; Chymotrypsin; Female; Humans; Hyaluronoglucosaminidase; Immunoelectrophoresis; Male; Molecular Weight; Osteoarthritis; Protease Inhibitors; Synovial Fluid; Trypsin Inhibitors

Topics: Animals; Cartilage, Articular; Chymotrypsin; Humans; Microscopy, Electron; Osteoarthritis; Rabbits