J. DeGroot

Collagenase-1 (MMP-1) activity in equine synovial fluid: influence of age, joint pathology, exercise and repeated arthrocentesis

REASONS FOR PERFORMING STUDY Matrix metalloproteinases (MMPs) are considered candidate biomarkers for both physiological and pathological tissue remodelling because of their key role in articular cartilage homeostasis. As disruption of the collagenous architecture is thought to be pivotal in chronic degenerative diseases such as osteoarthritis (OA), the collagenases form an interesting subset of the MMPs. The significance of any biomarker in synovial fluid (SF) can be assessed properly only when fluctuations in patterns induced by physiological processes such as development and growth, and by external influences and interventions such as exercise and repeated arthrocentesis, are known and taken into account. OBJECTIVES To investigate the activity of MMP-1 in equine SF at different stages of development and in joints affected by OA, and the influence of exercise and repeated arthrocentesis thereon. METHODS MMP-1 activity was determined in SF of normal joints of fetal, juvenile and mature horses, and in SF of horses suffering from OA, using an internally quenched fluorogenic peptide substrate. MMP-1 activity was also measured in SF from horses subjected to an exercise regimen and those subjected to repeated arthrocentesis. RESULTS An age-related decline in the SF levels of active MMP-1 was observed. MMP-1 activity was 15-fold higher in fetal than in juvenile animals, which showed significantly higher MMP-1 activity levels than mature horses. In SF of OA joints, MMP-1 activity was increased. Exercise did not affect MMP-1 activity in SF, but repeated arthrocentesis (within 60 h) increased MMP-1 activity significantly. CONCLUSIONS The high MMP-1 activity in SF of young individuals parallels the high metabolic activity occurring during rapid growth and differentiation at early age. The elevated MMP-1 activity in SF of OA joints probably reflects pathological matrix degradation, confirming the potential of MMP-1 to serve as a biochemical marker for early joint disease. Moderate exercise is not likely to influence the outcome of MMP-1 activity measurements in equine SF, but arthrocentesis should be taken into account as a possible confounding factor. POTENTIAL RELEVANCE Given the crucial role of the collagen matrix for tissue integrity, MMP-1 activity may be a useful tool in diagnostic, therapeutic or prognostic studies in horses suspected of OA. However, care should be taken to exclude fluctuations in MMP-1 activity induced by physiological processes such as development and growth, and by interventions such as repeated arthrocentesis.

Corticosteroids reduce the tensile strength of isolated collagen fascicles.

Background Overuse tendon injuries are frequent. Corticosteroid injections are commonly used as treatment, although their direct effects on the material properties of the tendon are poorly understood. Purpose To examine the influence of corticosteroids on the tensile strength of isolated collagen fascicles. Study Design Controlled laboratory study. Methods Single strands (300-500 [.mu]m) of rat-tail collagen fascicles were incubated in either high (1 mL of 40 mgmL-1 mixed with 0.5 mL saline 9%) or low (1 mL of 40 mgmL-1 mixed with 2 mL saline 9%) concentration of methylprednisolone acetate (Depomedrol) for 3 or 7 days, while the control segment from the same fascicle was kept in saline (N = 64). After the incubation period, the fascicles underwent displacement to failure in a mechanical test rig at 0.13 mm/s, and thereafter hydroxylysyl pyridinoline and lysyl pyridinoline cross-link content was evaluated in a high-performance liquid chromatography system. Data for each group were analyzed with a 2-way analysis of variance (time × incubation) for ultimate stress (mean ± standard deviation). Results In the high-concentration groups, strength was reduced after 3 (16.6 ± 4.6 MPa) and 7 (8.6 ± 1.7 MPa) days compared to the controls (30.2 ± 5.0 MPa and 25.6 ± 4.6 MPa, respectively; P <. 05). In the low-concentration groups, strength was reduced after 3 (12.0 ± 3.1 MPa) and 7 days (10.9 ± 2.5 MPa) compared to the controls (31.5 ± 5.0 MPa and 32.4 ± 5.6 MPa, respectively; P <. 05). The amount of cross-linking was unaffected by the intervention. Conclusion Data show that the tensile strength of isolated fascicles is markedly reduced after 3-and 7-day incubation in both high and low concentration of corticosteroids, although the observed effect on whole tendon remains unknown. Clinical Relevance Corticosteroids may weaken specific regions of the injected tendon and leave it more prone to rupture. This weakening effect is manifested in the individual collagen fascicles that constitute the tendon.

The influence of repeated arthrocentesis and exercise on matrix metalloproteinase and tumour necrosis factor a activities in normal equine joints

REASONS FOR PERFORMING STUDY Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) may be useful as biomarkers of joint disease or inflammation. However, activity of both MMPs and TNF-alpha in synovial fluid (SF) may be influenced by nonpathological factors such as arthrocentesis or exercise. OBJECTIVE To investigate the influence of repeated arthrocentesis and exercise on MMP and TNF-alpha activities in SF from normal equine joints. METHODS SF was collected from the left metacarpophalangeal, radiocarpal and tarsocrural joints of 16 horses. Eight of these horses were subsequently subjected to an exercise programme on a treadmill and 8 were box-rested as controls. Arthrocentesis was repeated 14, 145, 17 and 24 days after the start of the exercise programme. General MMP and TNF-alpha activities were determined in SF. RESULTS Repeated arthrocentesis caused a gradual increase but the exercise regimen no significant increase in MMP activity. There was a significant increase in TNF-alpha activity in SF collected from horses 2 h after cessation of the exercise programme. CONCLUSIONS AND POTENTIAL RELEVANCE When using MMPs as biomarkers for joint disease, at least 14 days should elapse after previous arthrocentesis before subsequent SF collection. Moderate exercise does not increase MMP activity in SF from normal joints and it may be possible to ignore this as a source of error in evaluating MMP activity in diseased joints.

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.

Leflunomide and methotrexate reduce levels of activated matrix metalloproteinases in complexes with α2 macroglobulin in serum of rheumatoid arthritis patients

Objective: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in α2 macroglobulin/MMP (α2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. Methods: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in α2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). Results: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p<0.001). In leflunomide-treated patients a significant reduction of MMP activity levels was observed as early as at the 4 months timepoint persisting thereafter, whereas in methotrexate-treated patients the reduction was seen at 1 year. Conclusion: The results show that systemic levels of activated MMPs are reduced in RA patients upon exposure to leflunomide or methotrexate.

Study of cartilage and bone layers of the bearing surface of the equine metacarpophalangeal joint relative to different timescales of maturation

REASONS FOR PERFORMING STUDY A detailed and comprehensive insight into the normal maturation process of the different tissues that make up functional units of the locomotor system such as joints is necessary to understand the influence of early training on musculoskeletal tissues. OBJECTIVES To study simultaneously the maturation process in the entire composite structure that makes up the bearing surface of a joint (cartilage, subchondral and trabecular bone) in terms of biochemical changes in the tissues of juvenile horses at 2 differently loaded sites of the metacarpophalangeal joint, compared to a group of mature horses. HYPOTHESIS In all the structures described above developmental changes may follow a different timescale. METHODS Age-related changes in biochemical characteristics of the collagen part of the extracellular matrix (hydroxylysine, hydroxyproline, hydroxypyridinum crosslinks) of articular cartilage and of the underlying subchondral and trabecular bone were determined in a group of juvenile horses (n = 13) (Group 1, age 6 months-4 years) and compared to a group of mature horses (n = 30) (Group 2, >4 years). In both bony layers, bone mineral density, ash content and levels of individual minerals were determined. RESULTS In cartilage, subchondral bone and trabecular bone, virtually all collagen parameters in juvenile horses were already at a similar (stable) level as in mature horses. In both bony layers, bone mineral density, ash- and calcium content were also stable in the mature horses, but continued to increase in the juvenile group. For magnesium there was a decrease in the juvenile animals, followed by a steady state in the mature horses. CONCLUSIONS In horses age 6 months-4 years, the collagen network of all 3 layers within the joint has already attained a mature biochemical composition, but the mineral composition of both subchondral and trabecular bone continues to develop until approximately age 4 years. POTENTIAL RELEVANCE The disparity in maturation of the various extracellular matrix components of a joint can be assumed to have consequences for the capacity to sustain load and should hence be taken into account when training or racing young animals.

Leflunomide and methotrexate reduce levels of activated mmps in complexes with α2macroglobulin in serum of rheumatoid arthritis patients

Objective: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in α2 macroglobulin/MMP (α2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. Methods: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in α2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). Results: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p<0.001). In leflunomide-treated patients a significant reduction of MMP activity levels was observed as early as at the 4 months timepoint persisting thereafter, whereas in methotrexate-treated patients the reduction was seen at 1 year. Conclusion: The results show that systemic levels of activated MMPs are reduced in RA patients upon exposure to leflunomide or methotrexate.

Skin and urine pentosidine weakly correlate with joint damage in a cohort of patients with early signs of osteoarthritis (CHECK).

OBJECTIVES Age-related changes in articular cartilage are likely to play a role in the aetiology of osteoarthritis (OA). One of the major age-related changes in cartilage is the accumulation of advanced-glycation-endproducts (AGEs). Since, cartilage tissue is not readily available from patients for studying AGE levels, alternative approaches such as analyzing skin and urine are needed to study the role of cartilage AGE levels in OA. METHODS Paired human skin and cartilage samples were obtained post mortem. Paired skin and urine samples were obtained from the CHECK cohort (early OA patients). Pentosidine levels were measured by high-performance liquid chromatography (HPLC). As marker of cumulative cartilage damage X-rays of both knees and hips were scored. Urinary CTXII (uCTXII) levels were measured, to assess current cartilage breakdown. RESULTS Cartilage and skin pentosidine correlate well (R=0.473, P=0.05). Skin pentosidine was higher in mild (summed (Kellgren & Lawrence K&L) over four large joints ≥4) compared to no (summed K&L≤3) radiographic OA (P=0.007). Urinary pentosidine was not different between these two groups. Skin pentosidine levels were not related to cartilage breakdown (highest vs lowest tertile of uCTXII). Urinary pentosidine, however, was higher in the highest compared to the lowest uCTXII tertile (P=0.009). Multiple regression analysis showed age to be the only predictor of the summed K&L score and age, creatinine clearance and urinary pentosidine as predictors of uCTXII. CONCLUSION The higher skin and urinary pentosidine levels in those with mild compared to no radiographic joint damage and low vs high cartilage breakdown respectively suggest that AGEs may contribute to disease susceptibility and/or progression. However, relations are weak and cannot be used as surrogate markers of severity of OA.

Skin pentosidine in very early hip/knee osteoarthritis (CHECK) is not a strong independent predictor of radiographic progression over 5 years follow-up

OBJECTIVES Age-related changes in articular cartilage are likely to play a role in the etiology of osteoarthritis (OA). One of the major age-related changes in cartilage is the accumulation of advanced glycation end products (AGEs). The present study evaluates whether pentosidine can predict radiographic progression and/or burden over 5 years follow-up in a cohort of early knee and/or hip OA. DESIGN The 5 years follow-up data of 300 patients from cohort hip & cohort knee (CHECK) were used. Radiographic progression and burden were assessed by X-rays of both knees and hips (Kellgren and Lawrence (K&L) and Altman scores). Baseline pentosidine levels (and urinary CTXII as a comparator) were measured by high-performance-liquid-chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA). Univariable and multivariable associations including baseline radiographic damage, age, gender, body mass index (BMI) and kidney function were performed. RESULTS Both pentosidine and urinary C-terminal telopeptide of type II collagen (uCTXII) correlated with radiographic progression and burden. In general pentosidine did not have an added predictive value to uCTXII for progression nor burden of the disease. The best prediction was obtained for burden of radiographic damage (R(2) = 0.60-0.88), bus this was predominantly determined by baseline radiographic damage (without this parameter R(2) = 0.07-0.17). Interestingly, pentosidine significantly added to prediction of osteophyte formation, whereas uCTXII significantly added to prediction of JSN in multivariable analysis. CONCLUSION Pentosidine adds to prediction of radiographic progression and burden of osteophyte formation and uCTXII to radiographic progression and burden of JSN, but overall skin pentosidine did not perform better that uCTXII in predicting radiographic progression or burden. Burden of damage over 5 years is mainly determined by radiographic joint damage at baseline.

Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin‐like growth factor 1 (IGF‐1; increases proteoglycan and collagen synthesis), β‐aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para‐nitrophenyl‐β‐D‐xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF‐1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used. Copyright