Cytokine and catabolic enzyme expression in synovium, synovial fluid and articular cartilage of naturally osteoarthritic equine carpi

Reasons for performing study: Understanding the expression of catabolic and anabolic genes during osteoarthritis progression should help to identify the major mediators of the disease. Objective: To compare the cytokine and anabolic marker concentrations in synovium, synovial fluid and cartilage between normal and osteoarthritic joints. Methods: Carpi from horses age 2–11 years were used. Tissues were harvested at the time of surgery or euthanasia, and RNA was isolated for RT‐PCR analysis. Tumour necrosis factor alpha (TNFα), interleukin‐1beta (IL‐1β), aggrecanase 1 (ADAMTS‐4), aggrecanase 2 (ADAMTS‐5), matrix metalloproteinase‐13 (MMP‐13), interleukin 17 (IL‐17) and collagen type I alpha 1(Col‐1) expression were determined in synovium. TNFα, IL‐1β, ADAMTS‐4, ADAMTS‐5, MMP‐13, IL‐17, collagen type IIB (Col‐2B), and aggrecan expression were determined in cartilage. TNFα concentration in the synovial fluid was determined by enzyme‐linked immunosorbent assay (ELISA). Results: Expression of TNFα, ADAMTS‐5 and MMP‐13 was significantly increased in synovial tissue from OA joints. Synovial membrane IL‐1β abundance showed only moderate elevations in OA, without reaching significant levels. Cytokine expression was increased significantly in OA cartilage samples, particularly TNFα, IL‐1β, ADAMTS‐4 and MMP‐13; and collagen type I expression was significantly increased in synovial tissues from OA groups. Collagen type II message was diminished in mild and moderate stages of OA, but rebounded to significant elevations in severely degenerate joints. Conversely, aggrecan levels significantly declined in cartilage from all OA groups. Synovial fluid TNFα peptide concentration was significantly increased in severe OA cases. Conclusion: TNFα was increased in all degrees of equine OA, and was abundantly expressed in synovial membrane and cartilage. IL‐1β was overexpressed in OA cartilage, but not to a significant extent in synovium. Potential relevance: Control of TNFα should be considered further as a target in the treatment of OA. ADAMTS‐4 may be the primary aggrecanase causing cartilage breakdown in OA.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.     

Expression of fibrosis-related genes in canine chronic hepatitis

Molecular regulation of fibrosis in chronic canine hepatitis is poorly understood. The authors employed quantitative polymerase chain reaction (PCR) to determine the expression levels of genes reported to be related to fibrosis in other species (human, mouse, and rat) and to elucidate the relationship of these genes with the degree of fibrosis and the presence or absence of ascites and/or jaundice in dogs with hepatitis. Nine fibrosis-related genes were assayed: PDGFB, PDGFD, MMP2, TIMP1, THBS1, COL1A1, COL3A1, TGFB1, and TGFB2. Liver samples of 15 dogs with chronic hepatitis and 4 healthy control dogs were obtained via laparoscopic biopsy and subjected to histologic and quantitative PCR analyses. The expression of all 9 genes showed significant positive correlation (P<.01, r>.70) with the degree of fibrosis. Furthermore, the expression levels of all genes except TGFB1 were significantly higher (P<.05) in dogs with hepatic failure-related symptoms (ascites/jaundice). Results suggest that these 9 genes are integral to the development of fibrosis in canine chronic hepatitis.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

An immunohistochemical study of the role of matrix metalloproteinases and their tissue inhibitors in chronic mitral valvular disease (valvular endocardiosis) in dogs

While the pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, alterations in the activity of specific metalloproteinase enzymes and their inhibitors within the valve stroma are suspected of having a role. This study describes the immunohistochemical distribution pattern of matrix metalloproteinase (MMP) types 2, 9 and 14 and their tissue inhibitors, termed tissue inhibitors of metalloproteinase (TIMP), types 2 and 3, in normal canine mitral valves (MVs) (n=10) and in dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal MVs, MMP-2 and -14, and TIMP-2 were expressed in isolated stromal cells. Tissue inhibitor of metalloproteinase-3 exhibited moderate intracellular and mild extracellular expression. With increasing severity of CVD, the expression of MMP-2 decreased. The number of stromal cells expressing MMP-14 increased, predominantly in the margins of the nodular lesions. Tissue inhibitor of metalloproteinase-2 and -3 expression increased both intra- and extracellularly. Matrix metalloproteinase-9 was not detected in normal or diseased valves. In conclusion, CVD was characterised by alterations in the distribution and intensity of valvular MMP and TIMP expression, suggesting that depressed catabolism and the accumulation of extracellular matrix components within affected valves contributes to their structural alteration and consequent loss of function.     

Hypoxia induces FGF2 production by vascular endothelial cells and alters MMP9 and TIMP1 expression in extravillous trophoblasts and their invasiveness in a cocultured model

The role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during trophoblast invasion was assessed. The human extravillous trophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were cocultured under normal and hypoxic conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells were analyzed using quantitative RT-PCR and Western blot analyses. TEV-1 cell invasion was also examined. FGF2 expression in the HUVE-12 cells cocultured with TEV-1 cells was significantly increased under hypoxic conditions. In the TEV-1 cells cocultured with HUVE-12, hypoxia reduced MMP9 expression and increased TIMP1 expression; it also reduced cell invasion by 43%. However, the expression of MMP9 and TIMP1 and ratio of MMP9/TIMP1 were increased when the TEV-1 cells were cultured alone under hypoxic conditions. These findings suggest that FGF2 release by stressed endothelial cells of uterine spiral arteries play roles in decreasing MMP9 and increasing TIMP1 production in extravillous trophoblasts (EVT) in response to stress, resulting in reduced EVT invasion and possibly shallow implantation of the placenta.     

Differences in metabolic parameters and gene expression related to osteochondrosis/osteoarthrosis in pigs fed 25-hydroxyvitamin D3

Osteochondrosis/osteoarthrosis (OC/OA) are common terms for various joint pathologies that occur in pigs. Pathologies that may contribute to these disorders have been described, but the primary cause(s) remain unknown. We hypothesised that as OC has some similarities to dyschondroplasia, which involves a failure of growth plate chondrocytes to fully differentiate and hypertrophy, treatment with 25-hydroxyvitamin D3 (25-D) might reduce the incidence and/or severity of lesions in pigs, as it does in chickens with dyschondroplasia. Control pigs were fed a commercial diet ad libitum. In the treated group this diet was supplemented with 25-D at 0.1 mg/kg. Ten pigs from each of the control and treated groups were sampled at 7, 12, 16 and 21 weeks. Treatment with 25-D had no effect on the incidence or severity of OC/OA lesions. Cartilage dry weight, total collagen content and proteoglycan content, and plasma levels oftotal calcium, inorganic phosphorous, vitamin C, insuline-like growth factor-I, parathyroid hormone and tumour necrosis factor alpha were unaffected by treatment. In addition, none of these parameters were correlated with the incidence or severity of OC/OA lesions. The mRNA expression levels of 21 out of 23 genes assayed by RT-PCR were unaltered in articular cartilage from OA lesion samples as compared to normal articular cartilage. However, collagen type II was reduced and collagen type X increased in OA lesion and near lesion samples. These results suggest that OA in pigs may share some features of osteoarthritis in other mammalian species.     

Relationship between synovial fluid levels of glycosaminoglycans, hydroxyproline and general MMP activity and the presence and severity of articular cartilage change on the proximal articular surface of P1

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting horses and leads to degeneration of articular cartilage. Diagnosis is based on clinical signs in combination with radiography, which is relatively insensitive and provides only an indication of accumulated damage. Alternative methods, such as molecular markers, are therefore needed that can quantitatively, reliably and sensitively detect osteoarthritic changes in the joints at an early stage of the disease. If such markers are to be used reliably, it is important to know the relationship between marker concentration and cartilage composition. OBJECTIVES: To study the relationship between cartilage composition, synovial fluid levels of glycosaminoglycans (GAGs), hydroxyproline (Hyp) and general matrix metalloproteinase (MMP) activity, and the presence and severity of articular cartilage damage on the articular surface of P1. METHODS: Synovial fluid (SF) was collected from the metacarpophalangeal joints of 60 mature horses, and levels of GAGs, Hyp and general MMP activity were determined. Further, GAG and denatured collagen content of the articular cartilage were determined at the dorsal articular margin of P1 (site 1) and central cavity (site 2). The presence and severity of cartilage change was quantified using the cartilage degeneration index (CDI), measured at the same 2 sites. Correlations between SF parameters, cartilage composition and degree of cartilage degeneration were sought using correlation analysis. RESULTS: There was no correlation between GAG or Hyp content of SF and the amount of GAGs or denatured collagen, respectively, in cartilage. In joints with moderate to severe cartilage damage, the GAG content of site 1 was significantly lower than in joints with no to minimal cartilage change (P = 0.005) and there was a negative correlation between the amount of denatured collagen and GAG content at site 1 in all joints (r = -039, P = 0.002). Further, in joints with moderate to severe cartilage damage, there was a significant positive correlation between MMP activity in SF and Hyp levels in SF (r = 0.72, P < 0.001) and CDI at sites 1 (r = 0.46, P = 0.03) and 2 (r = 0.43, P = 0.04). CONCLUSIONS: General MMP activity in joints with moderate to severe cartilage damage is related to the severity of those cartilage changes and to Hyp levels in SF. Glycosaminoglycan levels in SF are not directly related to MMP activity, GAG content of articular cartilage or severity of cartilage change. POTENTIAL RELEVANCE: Glycosaminoglycan levels in SF are not helpful for the early detection of cartilage lesions. In damaged joints, Hyp levels may give an indication of the severity of cartilage change as they are strongly related to MMP activity, but do not qualify as markers for the presence or absence of cartilage lesions.     

Expression of Matrix Metalloproteinases (MMP‐2, MMP‐9) and their Inhibitors (TIMP‐1, TIMP‐2) in Canine Testis,Epididymis and Semen

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP‐2, MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1) and TIMP‐2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP‐2 and MMP‐2 activity in high than low sperm concentration samples (p < 0.001). ProMMP‐9 and MMP‐9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP‐2 and MMP‐2 were associated with high sperm motility (≥70%, p < 0.001). Sperm‐rich fraction showed significantly (eight‐fold) higher proMMP‐9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP‐2‐specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP‐9, TIMP‐1 and TIMP‐2 were absent in these cells. Matrix metalloproteinase‐9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP‐1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP‐2 localization along acrosomal region of sperm, while MMP‐9, TIMP‐1 and TIMP‐2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP‐2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP‐2 and MMP‐9 may serve as an alternative biomarker in determining semen quality.     

Effects of autologous stem cells on immunohistochemical patterns and gene expression of metalloproteinases and their tissue inhibitors in doxorubicin cardiomyopathy in a rabbit model

This study aims to investigate the expression of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in chronic doxorubicin cardiomyopathy in a rabbit model and to evaluate the effects of bone marrow-derived mesenchymal stem cell (MSC) transplantation in this disease. Thirty-nine 3-month-old New Zealand rabbits were divided into 4 groups: group 1 (n = 9) was the untreated control. Groups 2-4 were treated with 6 weeks of doxorubicin (3 mg/kg). Group 2 (n = 6) received no further treatment. In group 3 (n = 9), animals were treated with culture medium (CM) alone. In group 4 (n = 15), autologous MSCs (1.5-2.0 x 10(6)/ml) were injected in the left ventricular (LV) wall. Hearts were stained with HE and picrosirius red. MMP-1, -2, -3 and -9 and TIMP-2 and -3 were detected immunohistochemically. The mRNA levels were determined by real-time polymerase chain reaction. The results confirmed that doxorubicin treatment resulted in minimal myocardial fibrosis and showed that expression of MMPs increased and TIMP-3 decreased. The injection procedure resulted in increased myocardial fibrosis in groups 3 and 4. After MSC injection, MMP-1, MMP-2, and TIMP-3 expression was higher than that in group 2. CM injection led to more fibrosis, elevated TIMP-3, but diminished MMP-1 and MMP-2 expression compared with MSC injection. The mRNA levels of MMPs and TIMPs were not significantly different among all groups. In conclusion, chronic doxorubicin cardiomyopathy was characterized by increased MMP and decreased TIMP-3 expression. MSCs injection into the LV resulted in marked differences of collagen content and MMP/TIMP expression in the whole heart, although significant numbers of living MSCs were not detected after 4 weeks.     

Evaluation of candidate genes as a cause of chondrodysplasia in Labrador retrievers

Chondrodysplasia (CD) is a disabling, hereditary disease in Labradors with short limbs, warranting genetic screening in families at risk. Segregation analysis of eight litters with 13 affected dogs showed that autosomal recessive inheritance was consistent with the observed incidence of CD in the litters. Possible involvement of eight candidate collagen genes (COL9A1, COL9A2, COL9A3, COMP, MATN3, COL2A1, COL11A1 and COL11A2) and of a sulfate transporter glycoprotein (SLC26A2) gene in eight affected dogs and in 14 related control Labradors was investigated. Assuming recessive inheritance, the candidate genes could not be implicated in CD.     

Assessment of collagen genes involved in fragmented medial coronoid process development in Labrador Retrievers as determined by affected sibling-pair analysis

OBJECTIVE: To evaluate the involvement of various collagen genes in the development of fragmented medial coronoid process (FCP) in Labrador Retrievers. SAMPLE POPULATION: 93 dogs originating from 13 litters were used in the study; FCP was diagnosed in 35 dogs, and each affected dog had at least 1 sibling that was also affected. Twelve dams and sires were included in the analysis. All dogs were purebred Labrador Retrievers except for 2 litters (offspring of a female Golden Retriever-Labrador Retriever mixed-breed dog). PROCEDURES: For each dog, DNA was isolated from blood samples. Polymorphic microsatellite markers adjacent to 14 candidate genes (ie, COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A3, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, and COL24A1) were analyzed by use of PCR assays; genotypes were determined via automated detection of DNA products. The level of allele sharing between pairs of affected siblings was assessed. RESULTS: Among the 93 dogs, allele sharing of the 14 collagen genes was determined as follows: COL1A1, 45%; COL1A2, 47%; COL2A1, 37%; COL3A1, 32%; COL5A1, 43%; COL5A2, 32%; COL6A3, 36%; COL9A1, 45%; COL9A2, 49%; COL9A3, 38%; COL10A1, 46%; COL11A1, 52%; COL11A2, 47%; and COL24A1, 47%. CONCLUSIONS AND CLINICAL RELEVANCE: Because siblings share 50% of their genome at random, the fact that the percentages of allele sharing among the analyzed collagen genes were not significantly > 50% indicates that these genes are not determinant candidates for FCP in Labrador Retrievers. The gene for the vitamin D receptor could also be excluded because of its proximity to COL2A1.