Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid

In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented.Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative–dissociative cesium chloride density gradient centrifugation (A1D1–D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions.The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800–1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample.This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.     

Osteoblast-like cells and fluid flow: cytoskeleton-dependent shear sensitivity

The cytoskeleton is thought to play a central role in cellular mechanotransduction. However, the specific mechanisms operative in bone cells have not yet been clearly elucidated. Isolating the roles of the specific cytoskeletal elements could ultimately aid in development of treatments for conditions related to the mechanoresponsiveness of bone (e.g. osteoporosis, space flight). Using an osteoblast-like cell line, the minimum doses of nocodazole (microtubules) and cytochalasin D (actin filaments) that would partially disrupt the cytoskeleton while leaving some elements intact were determined. Cultures were exposed to fluid flow shear, and loaded in the presence or absence of inhibitory drugs at the previously established doses. In untreated cultures, shear stress was associated with significant increases in mRNA levels for collagen I and matrix metalloproteinases 1 and 3. These increases were maintained in cytochalasin D-treated cultures, but were almost completely abrogated by nocodazole treatment. These results suggest that some mechanotransduction pathways related to bone matrix metabolism are primarily dependent on the microtubule network.     

Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid

IgG Fc- cytotoxic cells found in the synovial fluid of patients with rheumatoid arthritis have natural killer (NK)-like characteristics but can kill NK-resistant cell lines as well. The phenotype of these cells was defined by complement-mediated lysis with monoclonal antibodies. The synovial fluid killer cell activity was significantly reduced by treatment with complement and OKT11 and 4F2, but the cytotoxic T cells did not express the NK-related antigens OKM1 and Leu-7, nor the cytotoxic T lymphocyte-specific antigen, OKT8. These results demonstrate that the synovial fluid killer cells resemble the activated T cells generated in an autologous mixed leukocyte reaction or in the treatment of peripheral blood mononuclear cells with interleukin 2, and they are distinct from the conventional NK cells found in blood.     

Characterization of f-Met-Leu-Phe-stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry

N-formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.     

Generalized dispersion in a synovial fluid of human joints

An unsteady convective diffusion in a synovial fluid of human joints modeled as a power-law fluid is studied using the generalized dispersion model of Gill and Sankara-subramanian [12]. The contributions of convection and diffusion, and pure convection on the dispersion of nutrient are investigated in detail. It is shown that the effect of decrease in non-Newtonian parameter is to decrease the dispersion coefficient. The mean concentration distribution appears to increase as the non-Newtonian parameter decreases upto a certain value of the axial distance. Beyond this point, however, the reverse pattern is observed.     

Liquid chromatographic determination of diclofenac in human synovial fluid

A simple, rapid and sensitive HPLC method for the determination of diclofenac in synovial fluid is described. Special attention was paid to the procedure of sample preparation since gel formation may sometimes occur in synovial samples. With a one-step extraction procedure good and reproducible recovery of diclofenac was obtained. A subsequent HPLC assay was adjusted so as to achieve adequate sensitivity and precision needed for analysis of true samples. The results obtained by the described procedure proved the method to be suitable for monitoring concentrations of diclofenac in synovial fluid.     

Some properties of latent collagenase from human synovial fluid

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.     

Rheology of hyaluronan solutions under extensional flow

The extensional viscosity and the steady shear viscosity of sodium type hyaluronan (NaHA) in water with sodium chloride and/or sucrose and in DMSO solvent were measured. The extensional viscosities for HA in aqueous solution (0.05, 0.1, 0.3 w/v%) were constant at lower extensional rates, and then became strain thinning above a critical extensional rate. However, on adding sodium chloride, the extensional viscosity decreased and became strain thickening at higher extensional rates. Sodium ions shield the electrostatic repulsion between carboxyl residues of HA molecules and constrict the coil dimensions. The strain thickening of HA solution in the presence of sodium chloride at higher extension rates is due to the coil stretching. The addition of sucrose increased the extensional viscosity and shifted the critical extensional rate to lower strain rates. With increasing strain (shear) rates, extensional (shear) viscosities for HA aqueous solutions remained constant up to a critical extension (shear) rate; but they showed no plateau and decreased linearly in DMSO. It is clear that molecular interaction of HA in DMSO is stronger than that in aqueous solution. This should be attributed to the different conformations of HA in DMSO and in aqueous solutions.     

Depolymerization of sodium hyaluronate during freeze drying

Freeze drying of sodium hyaluronate causes free radical induced depolymerization of the polysaccharide which may be inhibited by such free radical scanvengers as Cl^?, I^?, alcohols and sugars. The free radical process is probably induced by mechanical stress factors. Such effects are important in freeze drying of all biopolymer solutions.     

Growth factors with heparin binding affinity in human synovial fluid

Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of 3H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.     

Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid.

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.     

Macroscopic assessment of cartilage shear: Effects of counter-surface roughness,synovial fluid lubricant,and compression offset

During joint articulation, cartilage is subjected to compression, shear, and sliding, mechanical factors that regulate and affect cartilage metabolism. The objective of this study was to use an in vitro material-on-cartilage shear test to elucidate the effects of counter-surface roughness (Polished, Mildly rough, and Rough), lubricants (phosphate buffered saline (PBS) and bovine synovial fluid (bSF)), and compression offset on the shearing and sliding of normal human talar cartilage under dynamic lateral displacement. Peak shear stress (σ_xz,m) and strain (E_xz,m) increased with increasing platen roughness and compression offset, and were 30% higher with PBS than with bSF. Compared to PBS, bSF was more effective as a lubricant for P than for M and R platens as indicated by the higher reduction in kinetic friction coefficient (?60% vs. ?20% and ?19%, respectively), σ_xz,m (?50% vs. ?14% and ?17%) and E_xz,m (?54% vs. ?19% and ?17%). Cartilage shear and sliding were evident for all counter-surfaces either at low compression offset (10%) or with high lateral displacement (70%), regardless of lubricant. An increase in tissue shear occurred with either increased compression offset or increased surface roughness. This material and biomechanical test system allow control of cartilage σ_xz,m and E_xz,m, and hence, sliding magnitude, for an imposed lateral displacement. It therefore can facilitate study of cartilage mechanobiological responses to distinct regimes of cartilage loading and articulation, such as shear with variable amounts of sliding.     

Tube flow of human blood at near zero shear

Pressure-velocity relations were obtained in vertical and horizontal glass tubes (I.D. 26 to 83 micron) perfused with normal human blood at feed hematocrits between 0.25 and 0.65. Perfusion pressures used corresponded to wall shear stresses up to 0.27 dyn cm-2. Red cell velocity measurements were made both immediately following implementation of perfusion pressure (with red cells still disaggregated) and in a steady state situation (with red cells aggregated). Analysis of the slopes of the linear relations between perfusion pressure and velocity showed apparent viscosity to decrease with the manifestation of red cell aggregation. In horizontal tubes, sedimentation and aggregation occurred simultaneously, and apparent viscosity increased due to axial asymmetry of cell concentration. Evidence for a yield shear stress (flow stagnation at positive driving pressure) was not observed.