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Abstract

Objective

To determine the influence of activated equine neutrophils on sulfated glycosaminoglycan metabolism of equine articular cartilage in vitro.

Animals

Articular cartilage explants harvested from the metacarpophalangeal joints of 7 horses.

Procedure

Proteoglycan degradation and synthesis were measured bv release of glycosaminoglycan from the explants, and incorporation of [35Slsulfate into newly synthesized glycosaminoglycan.

Results

Activated equine neutrophils significantly increased the release of glycosaminoglycan from explant matrix and the magnitude of that response was influenced by duration of exposure. This response varied significantly between horses, but was detected as early as 3 hours after co-cultures were initiated. In addition to enhancing degradation, incubation of explants with activated neutrophils for 3 days caused significant inhibition of glycosaminoglycan synthesis during a subsequent 3-hour pulse-labeling period. This response varied significantly between individual animals, but age was not a predictive factor.

Conclusion

Neutrophils may have a critical role in the process of cartilage degradation during equine inflammatory joint disease. (Am J Vet Res 1996;57:1738–1747)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes.

Sample Population—Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease.

Procedure—Chondrocytes were isolated, plated at a high density (105 cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1α (rhIL-1α). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as µmol of nitrite in the supernatant/µg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline.

Results—Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1α-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187. Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity.

Conclusions and Clinical Relevance—Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease. (Am J Vet Res 2002;63:204–210)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine the response of equine articular cartilage cells to heat and calcium stresses.

Design

Analysis of newly synthesized, [35S]methionine-labeled proteins after treatment of isolated primary equine chondrocytes.

Procedure

Primary cultures of equine articular chondrocytes were incubated at temperatures ranging from 37 to 42 C for heat stress experiments or incubated in the presence or absence of the intracellular calcium pump inhibitor, thapsigargin, for calcium stress experiments. Patterns of new protein synthesis were determined by incubating with [35S]methionine followed by separation of proteins by use of one- or two-dimensional gel electrophoresis and visualization of labeled proteins by use of fluorography.

Results

Equine chondrocytes cultured at temperature of 42 C had increased synthesis of specific proteins, compared with the profile of protein synthesis in control chondrocytes cultured at 37 C. These changes were characteristic of the heat shock stress response described in a number of other mammalian cell-types. Equine chondrocytes cultured in the presence of thapsigargin also had increased synthesis of specific proteins. Two-dimensional gel electrophoresis of these newly synthesized proteins revealed the changes to be consistent with the induction of the glucose-regulated protein family of stress proteins.

Conclusions

Changes in the pattern of new protein synthesis can be induced in differentiated equine articular chondrocytes by heat shock or calcium stress. These responses are characteristic of a widely described mammalian stress response that has been postulated to be involved in cellular protective mechanisms. The ability of equine chondrocytes to mount a robust stress response may be important in the processes of tissue damage and recovery in articular joints of horses. (Am J Vet Res 1996;57:860–865)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate whether recombinant human bone morphogenetic protein-2 (rhBMP-2) regulates glycosaminoglycan (GAG) synthesis and release from equine articular cartilage explant cultures.

Design

Equine articular cartilage explants were maintained in vitro for 7 days in the presence of 0 (control), 1, 10, or 100 ng of rhBMP-2/ml. Synthesis and release of GAG were assessed as measures of production and degradation of the extracellular matrix, respectively.

Animals

6 horses (age range, 2 to 25 years old) without clinically detectable musculoskeletal abnormalities.

Procedure

Rate of synthesis of GAG was assessed by incorporation of [35S]sulfate during the final 24 hours of the 7-day incubation period. Release of GAG was assessed on days 3, 6, and 7, using 1,9-dimethylmethylene blue

Results

Explants from all 6 horses had a significant (P = 0.05) increase in release of GAG in response to incubation with 100 ng of rhBMP-2/ml. There was a significant (P = 0.05) decrease in GAG synthesis in explants from only 2 of the 6 horses at the same concentration of rhBMP-2. There was no significant age correlation between responsive and nonresponsive horses.

Conclusions

A concentration of 100 ng of rhBMP-2/ml stimulates GAG release from explant cultures of equine articular cartilage. The data suggest that bone morphogenetic proteins may be potential regulators of equine cartilage degradation and repair.

Clinical Relevance

Surgical procedures that damage subchondral bone may stimulate generation of improved cartilage-like tissue. It is, therefore, crucial to understand how bone-derived factors may influence cartilage metabolism in horses. (Am J Vet Res 1996;57:554–559)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes.

Sample Population—Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses.

Procedure—Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis.

Results—Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7. Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes.

Conclusions and Clinical Relevance—Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints. ( Am J Vet Res 2005;66:984–990)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To investigate accumulation of extracellular adenosine (ADO) by equine articular chondrocytes and to compare effects of adenosine kinase inhibition and adenosine deaminase inhibition on the amount of nitric oxide (NO) produced by lipopolysaccharide (LPS)-stimulated chondrocytes.

Sample Population—Articular cartilage from metacarpophalangeal and metatarsophalangeal joints of 14 horses.

Procedure—Chondrocytes were cultured as monolayers, and cells were incubated with LPS, the adenosine kinase inhibitor 5'-iodotubercidin (ITU), or the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3- nonyl)adenine hydrochloride (EHNA). Concentrations of ADO in cell supernatants were measured by use of reverse-phase high-performance liquid chromatography. Effect of inhibition of enzymatic metabolism of ADO on induced NO production was evaluated by exposing cells to a combination of LPS and ITU or LPS and EHNA.

Results—Articular chondrocytes accumulated extracellular ADO when exposed to LPS or ITU. Chondrocytes exposed to ITU accumulated ADO in a time-dependent manner. Unstimulated chondrocytes did not accumulate ADO. Similarly, EHNA alone did not produce detectable ADO concentrations; however, addition of EHNA and ITU resulted in a synergistic effect on accumulation of ADO. Lipopolysaccharideinduced NO production was more effectively suppressed by exposure to ITU than to EHNA

Conclusions and Clinical Relevance—Equine articular chondrocytes release ADO in response to the proinflammatory stimulus of bacterial LPS. Inhibition of the metabolism of ADO increases accumulation of extracellular ADO. Autocrine release of ADO from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions, and pharmacologic modulation of these pathways in joints of arthritic horses could be a potential method of therapy. (Am J Vet Res 2002;63:1512–1519)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether the nonsteroidal anti-inflammatory drug carprofen directly influences canine chondrocyte metabolism.

Animals

Cartilage from the femoral heads of 13 dogs undergoing total hip replacement.

Procedure

Rates of glycosaminoglycan (GAG) synthesis and degradation, protein synthesis, cell viability, and prostaglandin release were determined in canine expiant cartilage or monolayer canine chondrocyte cultures in the presence of 0 to 100 µg of carprofen/ml. Rate of GAG synthesis was assessed as incorporation of [35S]sulfate into cartilage matrix during a 3-hour pulse label. Degradation of cartilage GAG was assessed as rate of release of [35S]sulfate from prelabeled expiant cultures. Rates of total protein synthesis were assessed as incorporation of [35S]methionine into trichloracetic acid precipitable material during a 3-hour pulse label. Radiolabeled chondrocyte proteins were separated by Polyacrylamide gel electrophoresis and visualized by fluorography. Rates of prostaglandin E2 release were assessed by radioimmunoassay.

Results

Carprofen stimulated a significant increase in the rate of GAG synthesis at concentrations of 1 and 10 µg/ml, with no change in total protein synthesis, pattern of new protein synthesis, or cell viability. At concentration ≥ 20 µg/ml, inhibition of GAG synthesis and total protein synthesis was observed. There was no significant change in rate of release of GAG from cartilage explants, but potent inhibition of prostaglandin release was observed.

Conclusions

Carprofen has a direct influence on chondrocyte activity, resulting in changes in rate of production of cartilage matrix.

Clinical Relevance

In determining the optimal therapeutic dose of carprofen for arthritic conditions in dogs, it is important to consider potential influences on cartilage, as well as anti-inflammatory actions. (Am J Vet Res 1997;58:286–292)

Free access
in American Journal of Veterinary Research

Summary

Explant cultures were set up, using articular cartilage obtained from metatarsophalangeal joints of 11 horses. Explants from 2 horses were used to determine culture conditions appropriate for tissue viability. The cartilage explants maintained steady-state metabolism of proteoglycans during a 13-day evaluation period. The metabolic response of equine articular cartilage to incubation with recombinant human interleukin 1 (0.01 to 100 ng/ml) was studied, using cartilage obtained from the remaining 9 horses, age of which ranged from 3 months to 20 years. Interleukin 1 induced a dose-dependent release of glycosaminoglycan from the matrix during a 3-day incubation period. It also caused dose-dependent inhibition of glycosaminoglycan synthesis during a 3-hour pulse-labeling period. Expiants obtained from older horses were significantly (P < 0.05) less responsive to interleukin 1, with respect to synthesis and release of glycosaminoglycan.

Free access
in American Journal of Veterinary Research

Summary

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (lps) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of lps from 0 (control) to 100 μg/ml. Glycosaminoglycan (gag) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-di-methylmethylene blue. Newly synthesized gag content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (pge 2) synthesis was quantified, using a [3H]pge 2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to lps, and explants from 1 horse were used to compare responses to stimulation with lps derived from 2 bacterial sources.

Equine explants cultured with bacterial lps had a dose-dependent decrease in synthesis and increase in release of gag, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of pge 2 released into the culture medium in response to incubation with lps.

Comparison of data for gag synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to lps between the 2 species. Equine explants tended to have a greater suppression of gag synthesis in response to incubation with increasing concentrations of lps than did age-corrected bovine samples. However, similar analysis of data on gag release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of lps derived from different bacterial sources made a significant difference in the response of explants to incubation with lps.

Free access
in American Journal of Veterinary Research