Ronald L. Goldberg

An improved method for determining proteoglycans synthesized by chondrocytes in culture.

An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes.

Enzyme-linked immunosorbent assay for hyaluronate using cartilage proteoglycan and an antibody to keratan sulfate.

An enzyme-linked immunosorbent assay has been developed to measure hyaluronate concentrations in biological samples. The assay is based on the aggregation of hyaluronate with cartilage proteoglycan monomer, followed by binding of a monoclonal antibody to the keratan sulfate on the proteoglycan. The sensitivity of the assay is 10 ng hyaluronate/ml of either serum or conditioned cell culture medium. The coefficient of variability was between 10 and 20%. Hyaluronate added to samples was recovered quantitatively and digestion of cell culture medium with protease did not affect the concentration of hyaluronate. Hyaluronate polysaccharides as small as a decasaccharide can be measured. This sensitive and convenient assay can be used for measuring large numbers of biological samples from a variety of animal and tissue sources.

Inhibition of interleukin‐1α‐induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: Potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Oral administration of a matrix metalloproteinase inhibitor, CGS 27023A, protects the cartilage proteoglycan matrix in a partial meniscectomy model of osteoarthritis in rabbits.

Matrix metalloproteinases (MMP) are elevated in human osteoarthritic cartilage [1] and in cartilage from rabbits with experimental osteoarthritis (OA) following partial meniscectomy [2]. CGS 27023A is a M M P inhibitor that inhibits stromelysin, collagenase and gelatinase. CGS 27023A is an orally active inhibitor of stromelysin. CGS 27023A at 75 gmoles/kg p.o. inhibits release of proteoglycan into synovial fluid following intra-articular injection of stromelysin into rabbit knees. The purpose of these experiments was to determine whether CGS 27023A would inhibit cartilage proteoglycan loss in a partial meniscectomy model of osteoarthritis in rabbits [3]. Doxycycline was used as a reference drug [4].

Development of a High-Throughput Screening Assay for Inhibitors of Aggrecan Cleavage Using Luminescent Oxygen Channeling (AlphaScreen™)

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen™. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report. (Journal of Biomolecular Screening 2003:136-148)

Effect of cytokines and anti-arthritic drugs on glycosaminoglycan synthesis by bovine articular chondrocytes

The effect of cytokines (interleukin-1α, interleukin-1β, and tumor necrosis factor α) and several antiarthritic drugs on glycosaminoglycan synthesis and secretion into medium by bovine articular chondrocytes was examined. Sulfated glycosaminoglycans (S-GAG) were measured by a modified 1,9-dimethylmethylene blue (DMB) dye binding assay. Hyaluronate (HA) was measured by an inhibition ELISA based on specific binding to a proteoglycan. All three cytokines caused a dose-dependent decrease in S-GAG production and a dose-dependent increase in HA production. Non-steroidal antiinflammatory drugs (NSAIDs, indomethacin, naproxen, and piroxicam, 1μM) could not reverse the effect of IL-1 α on inhibiting S-GAG and stimulating HA synthesis. The anti-inflammatory steroid (dexamethasone, 1μM) depressed HA synthesis by 50–70% in the absence or presence of IL-1α. Dexamethasone depressed S-GAG synthesis by 20–30% in the absence or presence of IL-1α. Therefore, none of the tested anti-rheumatic drugs reversed the cytokine mediated changes in glycosaminoglycan synthesis by bovine chondrocytes.

Effect of gold sodium thiomalate on proliferation of human rheumatoid synovial cells and on collagen synthesis in tissue culture

Abstract Synovial tissue obtained from patients with rheumatoid arthritis who were undergoing reconstructive joint surgery was used to obtain explant cultures of synovial cells. The experiments described were performed on growing monolayer cultures during the second to fifth passages. Synovial cells were exposed to gold sodium thiomalate (GST) in concentrations equivalent to levels attained in synovial tissues during chrysotherapy (3–50 μg/ml). After 5 days of exposure of cells to 10 or 50 μg/ml of GST, [ 3 H]thymidine incorporation into DNA was inhibited 94 or 99 per cent, respectively. After 10 days of exposure to 10 μg/ml of GST, cell number was decreased 50 per cent, although no change in cell number was found after a 5-day exposure to 100 μg/ml of GST. The total collagen content of the media was decreased in flasks of cells exposed to 50 μg/ml of GST for 15 days, which reflects the decrease in cell numbers observed at this concentration. However, after 15 days of exposure of synovial cells to 12 μg/ml of GST, incorporation of [ 14 C]proline into total collagen per cell increased 4-fold. This increase in [ 14 C]proline incorporation occurred predominantly in type I collagen. In these experiments, the percentage of type III collagen containing [ 14 C]proline and the amount found in media were suppressed 50 per cent by a fifteen day exposure to 3 μg/ml of GST. The ability of GST to increase the relative commitment of these cultures to make type I collagen is dose dependent in the range from 3 to 12 μg/ml. These data indicate that changes in cell proliferation and in the nature (genetic composition) of the extracellular matrix produced are direct effects of GST on the synovial cell in tissue culture and may represent one important mechanism of action of chrysotherapy in the treatment of patients with rheumatoid arthritis.

Quantitation of collagen in polyacrylamide gels by fluorescent scanning of MDPF1-labeled proteins: An improvement over densitometric scanning of gels stained by coomassie blue

Abstract Purified α chains of collagen were made fluorescent by coupling with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) and then electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. The migration of MDPF-labeled collagen was similar to unlabeled collagen α chains except that MDPF-α1(I) migrated closer to MDPF-α2. The area under the peaks recorded from fluorometric scanning of MDPF-labeled α1(I), α2, and α1(III) was linear from 10−5 to 10−8 g. The standard curves for the three α chains were similar. The results from nonreplicate determinations had ±6% SE. This method is an improvement over staining with Coomassie blue. It has a greater sensitivity, peak area is independent of migration distance, has a wider range of linearity, and permits observation of bands during electrophoresis with quantitation immediately after electrophoresis.

Chondrocyte Culture and Assay

Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage. Cellular, biochemical and pharmacological studies of arthritis and other cartilage disorders have increasingly focused on chondrocyte function. Three methods are presented in this unit for culturing chondrocytes, and two assays are described that characterize proteoglycan synthesis, a key measure of chondrocyte function.Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage.

Comparison of magnetic resonance imaging (MRI) and histopathology in rabbit models of osteoarthritis and immune arthritis

Osteoarthritis was surgically induced in mature male Dutch Belted rabbits by sectioning the fibular collateral and sesamoid ligaments and removal of the anterior horn of the lateral meniscus. The site of surgical intervention was detectable by MRI. Histopathologic analysis revealed severe focal cartilage lesions on opposing surfaces of the tibia and femur. Histology of cartilage adjacent to the osteoarthritic lesions appeared normal. In another animal model, arthritis was induced by immunization against ovalbumin followed by intra-articular injection of ovalbumin. MRI of immune arthritic rabbit knees showed accumulation of synovial fluid and cartilage degradation. Histopathology was characterized by vascular necrosis of the synovium and depletion of cartilage proteoglycan. MRI can be used to non-invasively follow the therapeutic effects of drug treatment on synovial inflammation and cartilage degradation in rabbit knees.