Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes

Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-alpha (100 ng mL(-1)), IL-1beta (100 ng mL(-1)), IGF-I (20 ng mL(-1)), TGF-beta (20 ng mL(-1)) and insulin (12.5 microg mL(-1)) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-beta, IL-1beta and TNF-alpha resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors.     

Effects of insulin-like growth factor-II on the mitogenic and metabolic activities of equine articular cartilage with and without interleukin 1-beta

OBJECTIVE: To investigate the effects of insulin-like growth factor-II (IGF-II) on DNA and glycosaminoglycan (GAG) synthesis and the expression of matrix-related genes in equine articular cartilage explants and chondrocytes, respectively, with and without interleukin 1-beta (IL1-beta). SAMPLE POPULATION: Articular cartilage from 12 adult horses. PROCEDURE: Articular cartilage was incubated in standard media with and without equine IL1-beta (10 ng/mL) containing various concentrations of IGF-II for 72 hours. Synthesis of DNA and GAG was determined by incorporation of thymidine labeled with radioactive hydrogen (3H) and sulfate labeled with radioactive sulfur (35S), respectively. Total GAG content of the explants and spent media was determined by use of the 1,9-dimethylmethylene blue assay. Northern blots of RNA from cultured equine articular cartilage chondrocytes were hybridized with cDNA of major matrix molecules. RESULTS: Insulin-like growth factor-II stimulated DNA and GAG synthesis at concentrations of 25 and 50 ng/mL, respectively. In cartilage explants conditioned with IL1-beta, IGF-II stimulated DNA and GAG synthesis at concentrations of 500 and 50 ng/mL, respectively. Insulin-like growth factor-II had no effect on total GAG content as determined by the 1,9-dimethylmethylene blue assay. No specific effects on steady-state levels of messenger RNAs were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Insulin-like growth factor-II stimulated DNA and GAG synthesis in equine adult cartilage and may have potential application in vivo.     

Homeostasis of intracellular Ca2+ in equine chondrocytes: response to hypotonic shock

REASONS FOR PERFORMING STUDY: Ca2+ homeostasis in articular chondrocytes affects synthesis and degradation of the cartilage matrix, as well as other cellular functions, thereby contributing to joint integrity. Although it will be affected by mechanical loading, the sensitivity of intracellular Ca2+ concentration ([Ca2+]i) in equine articular chondrocytes to many stimuli remains unknown. HYPOTHESIS: An improved understanding of Ca2+ homeostasis in equine articular chondrocytes, and how it is altered during joint loading and pathology, will be important in understanding how joints respond to mechanical loads. METHODS: [Ca2+]i was determined using the fluorophore fura-2. We examined the effects of hypotonic shock, a perturbation experienced in vivo during mechanical loading cycles. We used inhibitors of Ca2+ transporters to ascertain the important factors in Ca2+ homeostasis. RESULTS: Under isotonic conditions, [Ca2+]i was 148 +/- 23 nmol/l, increasing by 216 +/- 66 nmol/l in response to reduction in extracellular osmolality of 50%. Resting [Ca2+]i, and the increase following hypotonic shock, were decreased by Ca2+ removal; they were both elevated when extracellular [Ca2+] ([Ca2+]o) was raised or following Na+ removal. The hypotonicity-induced rise in [Ca2+]i was inhibited by exposure of cells to gadolinium (Gd3+; 10 micromol/l), an inhibitor of mechanosensitive channels. [Ca2+]i was also elevated following treatment of cells with thapsigargin (10 micromol/l), an inhibitor of the Ca2+ pump of intracellular stores. CONCLUSIONS: A model is presented which interprets these findings in relation to Ca2+ homeostasis in equine articular chondrocytes, including the presence of mechanosensitive channels allowing Ca2+ entry, a Na+/Ca2+ exchanger for removal of intracellular Ca2+ and intracellular stores sensitive to thapsigargin. POTENTIAL RELEVANCE: A more complete understanding of Ca2+ homeostasis in equine chondrocytes may allow development of future therapeutic regimes to ameliorate joint disease.     

Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.     

Immunolocalization of cathepsin B in equinedyschondroplastic articular cartilage

A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses.In normal articular cartilage (n=6 animals), significant amounts of enzyme were detected only in hypertrophicchondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n=5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n=6), but increased progressively during serial subculture, reaching a maximum at passage 5–6. In contrast, primary cultures of dyschondroplastic chondrocytes (n=3) expressed abundant cathepsin B.     

Characterisation of reconstituted equine cartilage formed in vitro

Lesions in cartilage of equine weightbearing joints commonly result in lameness. Cell-based resurfacing techniques are currently being developed for human and veterinary applications. Biopsies of stifle joint cartilage (1 g) were harvested aseptically and chondrocytes were isolated by sequential enzyme digestion. The cells were grown in vitro on filter inserts. Analysis of cultures 8 weeks later showed that the cells had accumulated extracellular matrix and formed a continuous layer of cartilagenous tissue as determined histologically. The cells maintained their phenotype as they synthesised type II collagen and proteoglycans similar in size to those synthesised by chondrocytes in native cartilage, but this reconstituted tissue had more sulphated glycosaminoglycan and lower collagen content than native cartilage. This experiment tests the feasibility of growing equine cartilagenous tissue in vitro. This tissue may be useful in the management of chondral injuries in the horse in a scenario where the patient donates cells, the cells are propagated under laboratory conditions and the resulting tissue becomes the therapeutic agent.     

Effects of enrofloxacin and magnesium deficiency on matrix metabolism in equine articular cartilage

OBJECTIVE: To investigate the effects of enrofloxacin and magnesium deficiency on explants of equine articular cartilage. SAMPLE POPULATION: Articular cartilage explants and cultured chondrocytes obtained from adult and neonatal horses. PROCEDURE: Full-thickness explants and cultured chondrocytes were incubated in complete or magnesium-deficient media containing enrofloxacin at concentrations of 0, 1, 5, 25, 100, and 500 microg/ml. Incorporation and release of sulfate 35S over 24 hours were used to assess glycosaminoglycan (GAG) synthesis and degradation. An assay that measured binding of dimethylmethylene blue dye was used to compare total GAG content between groups. Northern blots of RNA from cultured chondrocytes were probed with equine cDNA of aggrecan, type-II collagen, biglycan, decorin, link protein, matrix metalloproteinases 1, 3, and 13, and tissue inhibitor of metalloproteinase 1. RESULTS: A dose-dependent suppression of 35S incorporation was observed. In cartilage of neonates, 35S incorporation was substantially decreased at enrofloxacin concentrations of 25 mg/ml. In cartilage of adult horses, 35S incorporation was decreased only at enrofloxacin concentrations of > or =100 microg/ml. Magnesium deficiency caused suppression of 35S incorporation. Enrofloxacin or magnesium deficiency did not affect GAG degradation or endogenous GAG content. Specific effects of enrofloxacin on steady-state mRNA for the various genes were not observed. CONCLUSION AND CLINICAL RELEVANCE: Enrofloxacin may have a detrimental effect on cartilage metabolism in horses, especially in neonates.     

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.     

Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes

OBJECTIVE: To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. STUDY DESIGN: In vitro experiment using equine chondrocyte cultures. SAMPLE POPULATION: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). METHODS: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and ubiquitin (house keeping gene) mRNA expression were investigated by real-time RT-PCR. RESULTS: IL-1beta, IL-6, and IL-8 mRNA were expressed in unstimulated chondrocytes from macroscopically normal joints and were significantly up-regulated after stimulation (5/5 horses). IL-4 mRNA was not detected in any samples (0/5 horses). TNF-alpha mRNA, by comparison, was expressed in 2/5 unstimulated samples and in all stimulated samples but a considerable sample variation in response to IL-1beta stimulation was observed. CONCLUSIONS: Equine chondrocytes express mRNA for several proinflammatory cytokines and chemokines and IL-1beta modulates their expression. CLINICAL RELEVANCE: Chondrocytes express proinflammatory cytokines and chemokines capable of modulating a local inflammatory cascade in articular cartilage, which could potentially lead to focal degradation and osteoarthritis.     

Proteoglycan metabolism of equine articular cartilage and its modulation by insulin-like growth factors

The effect of human recombinant insulin-like growth factor 1 (rhIGF-1) on proteoglycan (PG) metabolism of full thickness equine articular cartilage explants was investigated. PG synthesis was stimulated at all ages, but higher concentrations of rhIGF-1 were required for maximal stimulation of adult cartilage. There were no changes in the hydrodynamic size, electrophoretic heterogeneity or composition of proteoglycans isolated from rhIGF-1-stimulated cartilage. rhIGF-1 reduced the rate of turnover of both newly synthesized and endogenous proteoglycans in all ages of cartilage investigated. The structure of proteoglycan fragments retained within the matrix and those released into the culture medium was unaffected by IGF-1 stimulation, suggesting that this peptide is a key regulator of the proteoglycan composition of equine articular cartilage extracellular matrix.     

The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses

OBJECTIVE: To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). STUDY DESIGN: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effect of corticosteroid treatment on proteoglycan metabolism was studied in normal explants. ANIMALS: Two young cross-breed horses and 3 young Labrador retrievers. METHODS: Equine articular cartilage explants were incubated in medium containing methylprednisolone sodium succinate (MPS) at 0, .001, .01, .1, 1, and 10 mg/mL (final concentration) for 1 day and then in fresh medium without MPS. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate at 1, 3, 7, 10, and 13 days after initial treatment with MPS. Proteoglycan release was measured from separate explants prelabeled with sodium [35S]sulfate and treated similarly. Equine articular cartilage explants were treated with equine MCM simultaneously with, and 24 hours before MPS, at 0, 0.01, 0.1, 1, or 5 mg/mL for 72 hours. Proteoglycan synthesis and degradation in these explants was compared. Proteoglycan synthesis and degradation were measured similarly in canine articular cartilage explants treated simultaneously with canine MCM and MPS at 0, 0.001, 0.01, 0.1, 1 and 10 mg/mL for 72 hours. Equine articular cartilage explants treated with 0, 0.01, 0.1, 1, and 5 mg/mL of MPS for 72 hours were evaluated histologically. RESULTS: Proteoglycan synthesis in normal equine articular cartilage was severely depressed by 10 mg/mL MPS for 24 hours, and proteoglycan synthesis failed to recover after 13 days of culture in medium without MPS. Cartilage treated with 5 mg/mL MPS had pyknotic chondrocyte nuclei and empty lacunae. Concentrations of 1 and 0.1 mg/mL MPS depressed proteoglycan synthesis in normal equine cartilage explants. For these 2 concentrations, proteoglycan synthesis recovered 2 days after MPS removal and increased significantly (P < .05) 7 days after treatment with MPS compared with controls without MPS. Concentrations of 0.001 and 0.01 mg/mL MPS did not significantly affect proteoglycan synthesis in normal equine cartilage explants. Cumulative proteoglycan loss over 13 days in culture from normal equine explants treated for 24 hours with different concentrations of MPS was not significantly different between treatment groups at any time point. MCM significantly depressed proteoglycan synthesis in both canine and equine articular cartilage explants and significantly increased proteoglycan release. These effects were prevented in the canine explants by simultaneous treatment with MPS at 1 and 0.1 mg/mL, and proteoglycan release induced by MCM in equine articular cartilage was inhibited by 1 mg/mL MPS. CONCLUSIONS: Concentrations of 1.0 and 0.1 mg/mL MPS alleviated articular cartilage degradation in MCM-treated articular cartilage in vitro. These concentrations of MPS in contact with normal cartilage explants for 24 hours are unlikely to be detrimental in the long term to proteoglycan synthesis. The response of articular cartilage to MPS was affected by treatment with MCM so that results of experiments with normal articular cartilage explants may not reflect results obtained with abnormal cartilage. CLINICAL RELEVANCE: It may be possible to find an intraarticular concentration of corticosteroid that protects articular cartilage against cytokine-induced matrix degradation yet not have prolonged or permanent detrimental effects on chondrocyte matrix synthesis.     

Distribution of beta-endorphin and substance P in the shoulder joint of the dog before and after a low impact exercise programme

Beta-endorphin and substance P were immunolocalized in the articular cartilage, synovial membrane and fibrous joint capsule of dogs. Twelve adult greyhounds were randomly assigned to one of three groups: control, limited exercise, or regimented exercise. On day 0, biopsies of articular cartilage and joint capsule were obtained from the left shoulder joints of dogs receiving limited and regimented exercise. On day 72, biopsies of joint capsule from right and left shoulders and articular cartilage from the right shoulder joint were analysed for the presence of glycosaminoglycans (GAG) and for immunolocalization of substance P and beta-endorphin. Regimented exercise increased the presence of GAGs and immunolocalization of substance P and beta-endorphin in articular cartilage and synovial membrane compared to day 0 biopsies and untreated controls. Localization of beta-endorphin became prominent in and around the chondrocytes. Substance P was increased in chondrocytes and extracellular matrix. Concomitant changes in localization of beta-endorphin and substance P may have a role in the modulation of the microphysiological environment, metabolism, or function of joint tissues in response to low-impact exercise.     

Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage

OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage. SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers. PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment. RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.     

Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days. CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.     

Influence of serotype, cell type, tissue composition, and time after inoculation on gene expression in recombinant adeno-associated viral vector-transduced equine joint tissues

Objective-To evaluate transduction efficiency of gene therapy for treatment of osteoarthritis in horses. Sample-Cartilage and synovial tissues were aseptically collected from the stifle joints of 3 Thoroughbreds; horses were 3, 7, and 12 years old and free from sepsis and long-term drug treatment and were euthanized for reasons unrelated to joint disease. Procedures-Gene transfer experiments were performed with 8 recombinant adeno-associated viral vector (rAAV) serotypes in monolayer-cultured equine chondrocytes, synovial cells, and mesenchymal stromal cells and in cartilage and synovial tissues. Results-Serotypes rAAV2/5 and rAAV2/2 yielded the highest transduction efficiency in cultured cells 6 days after transduction. Synovial cells and mesenchymal stromal cells were more readily transduced than were chondrocytes. Serotype rAAV2/6.2 yielded the highest rate of gene expression in both cartilage and synovial tissues at 6 days after inoculation. However, at 30 and 60 days after inoculation, gene expression of serotypes rAAV2/2 and rAAV2/5 surpassed that of rAAV2/6.2 and all other serotypes. Conclusions and Clinical Relevance-Maximally expressing serotypes changed between 6 and 30 days in tissues; however, the most efficient serotypes for transduction of joint cells over time were also the most efficient serotypes for transduction of joint tissues. In addition, the low transduction efficiency of articular cartilage tissue was paralleled by a low transduction efficiency of isolated chondrocytes. This suggested that the typically low transduction efficiency of articular cartilage may be attributable in part to the low transduction efficiency of the chondrocytes and not solely a result of the dense cartilage matrix.     

Immunohistochemical study of matrix metalloproteinase‐3 (MMP‐3) at the articular cartilage in osteochondrotic pigs

In order to understand the mechanism of osteochondrosis in the pig, articular cartilage was taken from the distal femoral condyles of Duroc pigs exhibiting leg weakness and then examined immunohistochemically for the localization of matrix metalloproteinases‐3 (MMP‐3), one of the enzymes involved in the resolution of cartilage matrix. The articular cartilage had the typical characteristics of osteochondrotic lesions, such as abnormal calcification, clefts of cartilage, disappearance of proteoglycan, and necrotic chondrocytes. The immunoreaction of MMP‐3 was observed in chondrocytes at the boundary between normal cartilage and proteoglycan‐deficient area. Moreover, chondrocytes expressing MMP‐3 showed normal morphology, but the surrounding cartilage matrix did not stain with toluidine blue, which indicated a lack of glycosaminoglycans. These results suggest that MMP‐3 is highly involved in the appearance and expansion of osteochondrotic lesions.     

Patterns of aquaporin expression in the canine eye

Aquaporins (AQPs) function as water channels in many types of cells involved in fluid transport. More than 10 isoforms have been identified, and these are differentially expressed in many types of mammalian cells in the body. Six AQPs (AQP0, AQP1, AQP3, AQP4, AQP5, and AQP9) have been identified in the eyes of humans and/or rodents. The unique permeability characteristics and distribution of AQPs indicate their diverse roles in the regulation of water homeostasis in the eye. The aim of this study was to investigate the localisation of AQPs in normal canine eyes, with AQP0 protein expressed in the crystalline lens and retina. Although AQP1 mRNA was detected in various areas of the canine eye, its protein expression was limited to the cornea, iris and ciliary body. AQP4 was identified in the iris, retina and optic nerve. AQP3 and AQP5 were found in the cornea and conjunctiva, and their expression was particularly high in the limbus. AQP3 and AQP5 were present in the nictitating membrane indicating that they play a role in water transport within the membrane. The observations suggested that several subtypes of the AQP family are involved in the regulation of water homeostasis in the canine eye.     

Effects of an adenosine kinase inhibitor and an adenosine deaminase inhibitor on accumulation of extracellular adenosine by equine articular chondrocytes

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. Lipopolysaccharide-induced 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.     

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes

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 (10(5) 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-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg 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-1alpha-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.