Early elevation in circulating levels of C-telopeptides of type II collagen predicts structural damage in articular cartilage in the rodent model of collagen-induced arthritis

OBJECTIVE: To investigate changes in the circulating levels of the C-telopeptide of type II collagen (CTX-II) with relation to disease onset and structural damage of cartilage in a rodent model of collagen-induced arthritis (CIA), and to investigate immunolocalization of the CTX-II epitope in the articular cartilage of affected joints. METHODS: Seven-week-old female Lewis rats were immunized with type II collagen and monitored using blood sampling at weekly intervals. At study termination (day 23), the animals were killed, synovial fluid was collected, and the affected joints were scored macroscopically for disease severity and underwent immunohistochemical evaluation. RESULTS: At the time of disease onset (day 15), which was characterized by redness and swelling of the affected joints (mean +/- SD macroscopic severity score 9.1 +/- 1.6), there was a 355% increase in serum CTX-II levels. The early change in serum CTX-II from day 0 to day 15 showed a significant association with the severity of cartilage damage (r = 0.61, P < 0.01). Immunostaining revealed extensive presence of the CTX-II epitope in the damaged, uncalcified cartilage tissue. CONCLUSION: The elevation in serum CTX-II concomitant with the onset of disease and proportional to cartilage damage demonstrates that CTX-II is a sensitive diagnostic tool for monitoring joint disease in the rodent model of CIA. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum CTX-II is the damaged articular cartilage.     

Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation

OBJECTIVE: To investigate how the time of initiation influences the effects of estrogen therapy on type II collagen (CII) turnover and the structural integrity of articular cartilage in ovariectomized rats and to determine whether estrogen exerts direct effects on the catabolic function of chondrocytes ex vivo. METHODS: A total of 46 Sprague-Dawley rats were distributed into 1 of the following treatment groups: 1) ovariectomy, 2) ovariectomy plus early estrogen therapy, 3) ovariectomy plus delayed estrogen therapy, or 4) sham operation. Cartilage turnover was estimated by measuring the serum levels of C-telopeptide of type II collagen (CTX-II). Cartilage lesions at week 9 were quantified using a published scoring technique. The presence of the CTX-II epitope in articular cartilage was assessed by immunohistochemistry. The effects of estrogen (1-100 nM) on chondrocytes were investigated in bovine cartilage explants subjected to catabolic cytokines (tumor necrosis factor alpha [TNFalpha] and oncostatin M [OSM]). RESULTS: In ovariectomized rats, estrogen therapy evoked significant decreases in serum CTX-II independently of the time of initiation; yet, delayed initiation resulted in diminished efficacy in terms of preventing cartilage lesions. CTX-II fragments were present in articular cartilage, colocalizing with early lesions at the cartilage surface. In untreated animals, the early relative increases in serum CTX-II were proportional to the severity of cartilage lesions at week 9 (r = 0.73, P < 0.01). Estrogen significantly and dose-dependently countered CTX-II release from TNFalpha plus OSM-stimulated cartilage explants ex vivo. CONCLUSION: Our results suggest that estrogen counters the acceleration of CII degradation and related structural alterations, and these benefits can be maximized by early initiation after menopause. The protective effect of estrogen seems to involve direct inhibition of the catabolic function of chondrocytes.     

Arthritis instantaneously causes collagen type I and type II degradation in patients with early rheumatoid arthritis: a longitudinal analysis

BACKGROUND: Markers of collagen type I (CTX-1) and type II (CTX-II) degradation, reflecting bone and cartilage breakdown, appear to predict long term radiographic progression in chronic persistent arthritis. OBJECTIVE: To analyse longitudinally whether changes in arthritis severity are linked to immediate changes in the level of CTX-I and CTX-II degradation. METHODS: CTX-I and CTX-II were measured in urine samples from 105 patients with early rheumatoid arthritis who had participated in the COBRA trial at baseline and at 3, 6, 9, and 12 months after the start of treatment. The course of the biomarkers over time was compared with the course of ESR, swollen and tender joint counts, and 28 joint disease activity score (DAS28), measured at the same time points, with adjustment for rheumatoid factor, treatment, and baseline radiographic damage, by generalised estimating equations (GEE) with first order autoregression. RESULTS: GEE showed that CTX-I was longitudinally associated with DAS28, but not with ESR, swollen joint count, or tender joint count. CTX-II, however, was longitudinally associated with ESR, swollen joint count and DAS28, but not with tender joint count. The longitudinal association implies that an increase in the extent of arthritis is immediately followed by an increase in collagen type II degradation, and to a lesser extent collagen type I degradation. CONCLUSIONS: Cartilage degradation as measured by CTX-II and to a lesser extent bone degradation as measured by CTX-I closely follows indices of arthritis. Clinically perceptible arthritis is responsible for immediate damage, which will become visible on plain x rays only much later.     

Association of baseline levels of markers of bone and cartilage degradation with long-term progression of joint damage in patients with early rheumatoid arthritis: the COBRA study

OBJECTIVE: The known risk factors for radiologic progression in rheumatoid arthritis (RA) are not optimally discriminative in patients with early disease who do not have evidence of radiologic damage. We sought to determine whether urinary C-terminal crosslinking telopeptide of type I (CTX-I) and type II (CTX-II) collagen (markers of bone and cartilage destruction, respectively) are associated with long-term radiologic progression in patients with early RA. METHODS: This was a prospective study of 110 patients with early RA who were participating in the COBRA (Combinatietherapie Bij Reumato?de Artritis) clinical trial and followup study, a randomized controlled trial comparing the efficacy of oral pulse prednisolone, methotrexate, plus sulfasalazine with sulfasalazine alone. We investigated the relationship between baseline levels of urinary CTX-I and CTX-II and the mean annual progression of joint destruction over a median of 4 years, as measured by changes in the modified Sharp score (average of 2 independent readers). RESULTS: In multivariate logistic regression analysis, baseline urinary CTX-I and CTX-II levels in the highest tertile were the strongest predictors of radiologic progression (Sharp score increase >2 units/year; odds ratio 7.9 and 11.2, respectively), independently of treatment group, erythrocyte sedimentation rate (ESR), Disease Activity Score in 28 joints, rheumatoid factor (RF), and baseline joint damage (Sharp score). The likelihood ratios for a positive test were 3.8 and 8.0 for CTX-I and CTX-II, respectively, which compared favorably with the likelihood ratios for the ESR (3.0), baseline joint damage (1.6), and RF (1.8). When patients were grouped according to the presence (Sharp score >/=4, n = 49) and absence (Sharp score <4, n = 61) of joint damage at baseline, CTX-I and CTX-II levels were predictive only in those without baseline joint damage (odds ratio 14.9 and 25.7, respectively). CONCLUSION: High baseline levels of urinary CTX-I and CTX-II independently predict an increased risk of radiologic progression over 4 years in patients with early RA, especially those without radiologic joint damage. Urinary CTX-I and CTX-II may be useful for identifying individual RA patients at high risk of progression very early in the disease, before erosions can be detected radiographically. Such patients may be in special need of treatments that inhibit bone and cartilage degradation.     

Effect of bisphosphonates on cartilage turnover assessed with a newly developed assay for collagen type II degradation products

BACKGROUND: Animal studies of arthritis have suggested that bisphosphonates may have chondroprotective abilities. OBJECTIVE: To evaluate the effect of bisphosphonate treatment on cartilage degradation. METHODS: Type II collagen is almost exclusively localised in cartilage, where it is the major structural component of the tissue. Hence fragments derived from this protein should represent a specific index for cartilage degradation. The urinary concentration of collagen type II C-telopeptide degradation products (CTX-II) was measured by a new immunoassay (enzyme linked immunosorbent assay (ELISA)). The serum concentration of collagen type I C-telopeptide degradation products (CTX-I), a marker of bone degradation, was also measured by ELISA. PARTICIPANTS: Two groups were studied. The alendronate group included 63 healthy postmenopausal women aged 45-54 randomly allocated to receive three years' treatment with 1 mg, 5 mg, 10 mg, or 20 mg alendronate daily or placebo. In the third year the women receiving 20 mg were switched to placebo. The ibandronate group included 119 women at least 10 years after the menopause aged <75 randomly allocated to receive 12 months' treatment with 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5 mg ibandronate daily or placebo followed by 12 months without treatment. RESULTS: 20 mg of alendronate and 2.5 and 5 mg of ibandronate treatment produced significant decreases in urinary CTX-II to about 50% of baseline. The level reached after three months of treatment remained practically constant during the following 12-36 treatment months. When treatment was withdrawn CTX-II values returned towards baseline. Serum CTX-I also decreased rapidly within three months, but to a level of about 30% of baseline. CONCLUSIONS: The urinary excretion of CTX-II, a new marker of cartilage degradation, decreases significantly in response to bisphosphonate. This suggests that bisphosphonates may have chondroprotective effects in humans. By measurement of CTX-II it should be possible to monitor the effects of drugs that potentially inhibit cartilage destruction.     

Urinary markers of altered collagen metabolism in fibromyalgia patients

OBJECTIVE: To assess the metabolism of collagen in fibromyalgia (FM) patients, and to compare the occurrence of collagen metabolism markers to the severity of FM symptoms. METHODS: Morning urine was collected from 27 FM women fulfilling the American College of Rheumatology (ACR) criteria for FM, and from seven controls. FM patients completed the Fibromyalgia Impact Questionnaire (FIQ). Bone mineral density (BMD), isokinetic muscle strength in knee and elbow, and hand-grip strength were measured. Urinary concentrations of collagen type I cross-linked C-telopeptide (CTX-I) and collagen type II cross-linked C-telopeptide (CTX-II) were determined by enzyme-linked immunosorbent assay (ELISA). Pyridinoline (Pyd) and deoxypyridinoline (Dpd) were determined by liquid chromatography, and hydroxyproline (Hyp) by spectrophotometry. All concentration data were normalized to creatinine. RESULTS: Mean values in the FM group and the control group, respectively, were: urinary CTX-I 246.8 and 337.5 microg/mmol (p = 0.060); CTX-II 110.4 and 185.1 ng/mmol (p = 0.035); Pyd 56.1 and 52.3 nmol/mmol (NS); Dpd 15.1 and 14.0 nmol/mmol (NS); Pyd : Dpd ratio 4.05 and 3.96 (NS); Hyp 26.1 and 21.1 micromol/mmol (NS). Significant inverse correlations were seen between CTX-I and the intensity of fatigue, and between CTX-II and anxiety. An inverse correlation between CTX-I and muscle strength was apparent, but relied on extreme values from one patient, and no significant correlation was found between CTX-I or CTX-II and tender points or BMD in the FM group. CONCLUSIONS: Low urinary concentrations of CTX-II and CTX-I and normal levels of Pyd and Dpd were found in FM, but their relationship to the intensity of FM symptoms was unclear.     

Urinary collagen type II C-telopeptide fragments are sensitive markers of matrix metalloproteinase-dependent cartilage degradation in rat adjuvant-induced arthritis

OBJECTIVE: To assess the relevance of collagen type II C-telopeptide fragments (CTX-II) as markers of cartilage degradation during adjuvant-induced arthritis in rats. METHODS: Rats were injected with Freund's adjuvant on day 0 and treated orally for 21 days twice a day with vehicle or 10 or 20 mg/kg of a newly designed matrix metalloproteinase inhibitor (MMP-Inh). Urine samples were collected for 24 h between days 19 and 20 and the concentration of the cartilage-derived CTX-II was measured with a 2-site, sandwich-type ELISA. To assess arthritis, inflammatory scores were determined, and changes in paw volumes were measured by plethysmography. RESULTS: On day 21, the inflammation was generalized in rats injected with Freund's adjuvant. The urinary concentration of CTX-II was significantly higher in arthritic rats than in control non-injected rats. Oral treatment of arthritic rats with MMP-Inh dramatically decreased the concentration of CTX-II in urine, with values returning to those of controls. Treatment simultaneously reduced the clinical variables of the disease. CONCLUSION: These results demonstrate that fragments of type II collagen in urine can be used as a measure of cartilage degradation in arthritic rats as well as potent non-invasive markers of the efficacy of chondroprotective treatments.     

Markers for type II collagen breakdown predict the effect of disease-modifying treatment on long-term radiographic progression in patients with rheumatoid arthritis

OBJECTIVE: To investigate in a randomized clinical trial setting with an aggressive combination-therapy arm and a mild-monotherapy arm, whether therapy-induced changes in urinary C-terminal crosslinking telopeptide of type I collagen (CTX-I) and type II collagen (CTX-II) predict 5-year radiographic progression in patients with rheumatoid arthritis (RA). METHODS: Patients had participated in the COBRA (Combinatietherapie Bij Reumato?de Artritis) trial comparing aggressive step-down combination therapy (the COBRA regimen, including temporary high-dose prednisolone, temporary low-dose methotrexate, and sulfasalazine [SSZ]) and mild monotherapy (SSZ). Urinary CTX-I and CTX-II levels were measured at baseline and 3, 6, 9, and 12 months after initiation of treatment. Radiographs were scored according to the modified Sharp/van der Heijde method (mean of 2 independent readers who were aware of the sequence). Individual long-term radiographic progression was estimated, using baseline radiographs and all radiographs obtained during the followup period, by simple linear regression analysis (curve fitting). RESULTS: Both COBRA therapy and SSZ monotherapy produced a significant decrease in urinary CTX-I and CTX-II levels at 3 months, and this decrease was amplified at 6 months. COBRA therapy suppressed CTX-II (change from baseline levels -36% and -43% at 3 and 6 months, respectively), but not CTX-I, significantly better than did SSZ (-17% and -21% at 3 and 6 months, respectively) at 3 and 6 months. The magnitude of the decrease in urinary CTX-II levels at 3 months significantly predicted long-term (5-year) radiographic progression (beta = 0.48 [95% confidence interval (95% CI) 0.13, 0.83]). This effect was independent of the change in disease activity and inflammation indices at 3 months. Patients whose CTX-II levels were normalized (<150 ng/mmoles of urinary creatinine) at 3 months had a significantly higher chance of radiographic stability (no progression over 5 years) than did patients whose CTX-II levels were increased both at baseline and at 3 months (odds ratio 4.5 [95% CI 1.5, 13]). CONCLUSION: The individual CTX-II response measured after 3 months of therapy in patients with active RA who had increased CTX-II levels at baseline independently predicts long-term radiographic progression. Urinary CTX-II levels may be used as early markers of treatment efficacy in patients with RA.     

Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis: an ultrastructural demonstration of an increased superficial chondroprotective layer

OBJECTIVE: To determine the ultastructural changes of Taxol (paclitaxel) involution of articular cartilage destruction in collagen induced arthritis (CIA) and to compare with articular cartilage from normal rats. METHODS: Forty-five Louvain rats were randomized to one of 3 protocols for structural analysis: (1) control group, (2) CIA group, and (3) Taxol treated CIA group. The latter group received 10 mg/kg body weight of Taxol at Days 10, 12, and 14 and 7.5 mg/kg body weight of Taxol on Days 16, 18, and 20 postimmunization with collagen type II. Eight days later, each group was examined by light microscopy and scanning and transmission electron microscopy. RESULTS: In Taxol treated rats, the morphology of the articular cartilage reverted to that observed in naive rats except for a striking increase in the thickness of the superficial amorphous layer covering the articular surface. CONCLUSION: The involution of CIA by Taxol suggests that this agent may be useful in the clinical treatment of RA.     

Appearance of calpain correlates with arthritis and cartilage destruction in collagen induced arthritic knee joints of mice.

OBJECTIVES--To determine the relevance of calpain in murine collagen induced arthritis (CIA) and to correlate the presence of m-calpain with the appearance of arthritis and cartilage destruction. METHODS--The immunohistochemical appearance and localisation of m-calpain at different stages of arthritis were analysed and compared with the histological changes occurring during type II CIA. The arthritic knee joint lavage was also examined for m-calpain by immunoelectrophoretic blotting. RESULTS--Immunohistochemical staining demonstrated a clear positive correlation between the appearance of m-calpain and both a histological grade of arthritis and an acute phase of cartilage destruction. Further development of the disease showed continual presence of m-calpain but with reduced intensity. Intra-articular inflammatory cells (mainly polymorphonuclear leucocytes, synovial lining cells, and sublining fibroblasts) were found to be the most positively stained, but extracellular localisation of m-calpain on the surface of cartilage and synovium, and in the articular cartilage matrix and chondrocyte lacunae, was also observed. In the knee joint lavage obtained at the most intensive stage of acute arthritis, m-calpain was detectable by immunoelectrophoretic blotting. CONCLUSIONS--The findings suggest that m-calpain may act at an early phase of CIA as a matrix proteinase and take part in the destruction of articular cartilage or activate other destructive enzymes.     

Therapeutic effect of anti-Fas antibody on a collagen induced arthritis model

OBJECTIVE: To investigate the therapeutic effect of anti-Fas monoclonal antibody (Mab, RK-8) in collagen induced arthritis (CIA). METHODS: CD1F1 mice were immunized with bovine type II collagen to induce CIA and were treated with RK-8 intravenously. The effect of RK-8 was monitored by visual scoring. ELISA to detect serum anti-type II collagen antibody was performed on Day 47 and 70. Histopathological analysis was performed on Days 31 and 72. Digital micrography was performed on Day 72. RESULTS: RK-8 treatment almost completely prevented CIA. This suppressive effect continued after RK-8 was discontinued. RK-8 significantly suppressed the serum anti-type II collagen antibody level on Day 47. Histological analysis revealed that RK-8 significantly reduced joint histopathology, as determined by the infiltration of inflammatory cells and cartilage damage, consistent with digital micrography. CONCLUSION: Administration of anti-Fas Mab may be a useful therapeutic strategy for rheumatoid arthritis if used early in the disease.     

Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture.

Explants of articular cartilage from young pigs were maintained in organ culture for 10--16 days, and degradation of matrix was induced by retinol or complement-sufficient antiserum. The percentage breakdown of proteoglycan and collagen (as hydroxyproline release) was measured. The response of the cartilage depended on whether or not the explants were cut so as to include some of the invading marrow ('invasion zone'). In media containing retinol, cartilage lost up to three-quarters of its proteoglycan whether the invasion zone was present or not, but very little of its collagen unless this region was included. In the presence of complement-sufficient anti-serum, however, cartilage without the invasion zone was virtually unaffected, but both proteoglycan and hydroxyproline were released when invasion zone was included; here proteoglycan release began almost immediately, but there was a time-lag of 6--8 days before a substantial amount of hydroxyproline appeared in the medium. Histological examination of sample explants from the experiments supported the biochemical findings. The possible significance of the results in relation to rheumatoid arthritis is discussed.     

CNI-1493, an inhibitor of proinflammatory cytokines, retards cartilage destruction in rats with collagen induced arthritis

OBJECTIVE: To investigate if administration of CNI-1493, an inhibitor of the synthesis of proinflammatory cytokines and NO, protects against development of joint destruction in collagen induced arthritis (CIA) in rats. METHODS: In a placebo controlled experiment, CNI-1493 was given once daily intraperitoneally after onset of clinical arthritis in DA rats. Disease progression was studied by clinical scoring of arthritis, serial measurement of serum levels of COMP, and histological examination of joints. RESULTS: Clinical signs of arthritis were significantly reduced in the CNI-1493 treated group of rats in comparison with the placebo treated group. Histological examinations of paws demonstrated a significant reduction of cartilage destruction in the CNI-1493 treated group, but marked destruction of cartilage in the placebo group. Serum levels of COMP increased in the placebo group, whereas in the CNI-1493 treated group levels were low and decreased significantly during the observation time. CONCLUSIONS: Treatment with CNI-1493 provides efficient protection against synovial inflammation and cartilage destruction when used therapeutically in CIA. The protective effect against cartilage destruction can be monitored by measuring serum COMP. These observations make CNI-1493 an attractive candidate for therapeutic studies in human arthritis, and COMP an attractive serum marker for monitoring joint protective effects.     

Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage

OBJECTIVE: To analyse the relations between the urinary levels of type II collagen C-telopeptide (CTX-II) and glucosyl-galactosyl pyridinoline (Glc-Gal-PYD)-two newly developed biochemical markers of type II collagen and synovial tissue destruction respectively-disease activity and the severity of joint destruction in patients with knee osteoarthritis (OA). The clinical performance of these two new markers was compared with that of a panel of other established biochemical markers of connective tissue metabolism. METHODS: The following biochemical markers were measured in a group of 67 patients with knee OA (mean age 64 years, median disease duration eight years ) and in 67 healthy controls: for bone, serum osteocalcin, serum and urinary C-telopeptide of type I collagen (CTX-I); for cartilage, urinary CTX-II, serum cartilage oligomeric matrix protein (COMP), and serum human cartilage glycoprotein 39 (YKL-40); for synovium, urinary Glc-Gal-PYD, serum type III collagen N-propeptide (PIIINP), serum hyaluronic acid (HA); and for inflammation, serum C reactive protein. Biochemical markers were correlated with pain and physical function (WOMAC index) and with quantitative radiographic evaluation of the joint space using the posteroanterior view of the knees flexed at 30 degrees. RESULTS: All bone turnover markers were decreased in patients with knee OA compared with controls (-36%, -38%, and -52%, p<0.0001 for serum osteocalcin, serum CTX-I and urinary CTX-I, respectively). Serum COMP (+16%, p=0.0004), urinary CTX-II (+25%, p=0.0009), urinary Glc-Gal-PYD (+18%, p=0.028), serum PIIINP (+33%, p<0.0001), and serum HA (+ 233%, p<0.0001) were increased. By univariate analyses, increased urinary Glc-Gal-PYD (r=0.41, p=0.002) and decreased serum osteocalcin (r=-0.30, p=0.025) were associated with a higher total WOMAC index. Increased urinary CTX-II (r=-0.40, p=0.0002) and Glc-Gal-PYD (r=-0.30, p=0.0046) and serum PIIINP (r=-0.29, p=0.0034) were the only markers which correlated with joint surface area. By multivariate analyses, urinary Glc-Gal-PYD and CTX-II were the most important predictors of the WOMAC index and joint damage, respectively. CONCLUSION: Knee OA appears to be characterised by a systemic decrease of bone turnover and increased cartilage and synovial tissue turnover. CTX-II, Glc-Gal-PYD, and PIIINP may be useful markers of disease severity in patients with knee OA.     

The destruction evaluation in different foot joints: new ideas in collagen-induced arthritis rat model

Collagen-induced arthritis (CIA) has been widely used as the animal model of rheumatoid arthritis since 1977, while till now, no paper has depicted the destruction characteristics in different foot joints. In this study, we observed the differences among the foot joint destruction process of CIA to elucidate further the pathological process of this model. CIA was induced in male Wistar rat immunized with bovine type II collagen and Freund’s incomplete adjuvant. Radiological studies were performed 1, 2, 4, 6, and 8 months after the second immunization to follow the development of disease. At last, all the animals were killed and histological research was performed. In the histological observation, three main types of joint destructions such as subchondral side erosion, external joint erosion and the cartilaginous fusion of articular cartilage were identified. All these destruction forms exist in one joint or several different joints. Furthermore, we found that tartrate-resistant acid phosphatase (TRAP) stain-positive cells participated in the destruction of articular cartilage. These new findings showed that in the disease process of the CIA model, different foot joints show different destruction characteristics and cartilaginous fusion of foot joints is another typical pathological characteristic. Our research was supported by Shanghai Key Laboratory of Orthopaedic Implant (08DZ2230300).     

The effect of oral calcitonin on cartilage turnover and surface erosion in an ovariectomized rat model

OBJECTIVE: To investigate whether oral calcitonin treatment influences the increases in type II collagen (CII) degradation and related surface erosions of articular cartilage in ovariectomized rats. METHODS: Fifty rats were randomly allocated into 1 of the 5 following intervention groups: sham-operated, ovariectomy, ovariectomy plus subcutaneously implanted 17beta-estradiol pellet, ovariectomy plus 2 mg/kg salmon calcitonin plus 50 mg/kg 5CNAC (carrier), or ovariectomy plus 50 mg/kg 5CNAC. Each treatment was administered for 9 weeks after the ovariectomy. Blood samples for biochemical marker analysis were collected from fasting animals at baseline, on day 3, and after 1, 2, 4, 6, and 9 weeks. CII degradation was quantified by specific immunoassay, and the changes in severity scores of articular cartilage erosions were visualized and scored in histologic sections of the knees. RESULTS: Ovariectomy resulted in a marked increase in serum levels of C-telopeptide of type II collagen (CTX-II) (P < 0.001), which could be effectively reversed by 17beta-estradiol supplementation. Oral administration of calcitonin elicited similar decreases in serum levels of CTX-II (P < 0.001). Histologic scoring of cartilage erosion showed significantly less cartilage erosion in calcitonin-treated ovariectomized rats versus control ovariectomized rats that were untreated or treated with 5CNAC alone. (P < 0.01). CONCLUSION: The in vivo effects of calcitonin in rats suggest that calcitonin is able to counteract CII degradation and the accompanying structural disintegration of articular cartilage promoted by estrogen deficiency. Clinical assessment of the chondroprotective potential of calcitonin in postmenopausal women seems warranted.     

Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis

OBJECTIVE: To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium. METHODS: Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry. RESULTS: Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front. CONCLUSION: The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.     

Urinary type II collagen C-telopeptide levels are increased in patients with rapidly destructive hip osteoarthritis

OBJECTIVE: To compare type II collagen degradation using a new urinary specific marker in patients with rapidly destructive and those with slowly progressive hip OA. METHODS: Twelve patients with rapidly destructive and 28 patients with slowly progressive hip OA were included in a prospective, cross sectional case-control study. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTX-II) as a marker of cartilage degradation were measured by an ELISA, and urinary free deoxypyridinoline (free DPD), a marker of bone resorption, was measured by high performance liquid chromatography. One x ray evaluation of the hips and urine samples was made in all patients when the diagnosis of OA was established. RESULTS: Patients with hip OA had higher mean (SD) urinary CTX-II levels than 65 healthy age matched controls (492 (232) v 342 (141), p<0.001), but no significant difference was seen for urinary free DPD (p=0.30). Increased urinary CTX-II, but not urinary free DPD, correlated significantly with decreased minimum joint space width assessed by radiograph of the hip. Mean urinary CTX-II levels were significantly higher in patients with rapidly progressive OA than in the slowly progressive group (612 (218) v 441 (221), p=0.015), whereas no significant difference of urinary free DPD was seen between the two groups (p=0.55). CONCLUSION: Patients with hip OA have increased CTX-II degradation as assessed by a new urinary marker. Increased urinary CTX-II levels are associated with rapidly destructive disease, suggesting that this marker might be useful in identifying patients with hip OA at high risk for rapid progression of joint damage.     

The release of crosslinked peptides from type II collagen into human synovial fluid is increased soon after joint injury and in osteoarthritis

OBJECTIVE: To determine concentrations of crosslinked C-telopeptide fragments of type II collagen (CTX-II) in synovial fluid (SF) from patients with joint injury, osteoarthritis (OA), or other knee arthritides. METHODS: Two study groups were used: a cross-sectional group, which included healthy-knee volunteers (reference group [REF]) and patients with pseudogout (PPA), an anterior cruciate ligament tear with or without a meniscus tear (INJ), or primary knee OA (POA); and a longitudinal group, which included patients with arthroscopic cartilage changes or septic arthritis. CTX-II was quantified by competition enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody that recognized the C-terminus of the peptide EKGPDP as a proteolytic neoepitope. Aggrecan fragments, matrix metalloproteinases 1 and 3, and tissue inhibitor of metalloproteinases 1 were determined by ELISAs. RESULTS: Concentrations of CTX-II in SF were higher in patients with PPA, INJ, and POA than in the REF group (P < 0.001). After joint injury, mean levels of CTX-II in SF were increased above REF levels at all time intervals (P < 0.001), and were highest within hours after trauma. In those in the longitudinal study group with joint cartilage damage, variation coefficients for CTX-II were 81% (between patients) and 64% (within patient), monitored over 1 year. In a patient with septic arthritis, SF CTX-II increased at the onset of symptoms, and peaked 30-fold higher than the baseline. Concentrations of all biomarkers decreased with successful treatment. CONCLUSION: This is the first report to describe the release into SF of soluble molecular fragments specific for the degradation of mature, crosslinked, type II collagen (CII) in human OA and joint injury. The results provide strong evidence that the integrity of the CII network of cartilage is compromised soon after joint injury and in arthritis. This early degradation of CII may represent an important treatment target.     

Angiostatin gene transfer as an effective treatment strategy in murine collagen-induced arthritis

OBJECTIVE: To determine the efficacy of local therapy with human angiostatin gene in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with bovine type II collagen. Before the onset of arthritis, NIH3T3 fibroblasts, transduced with angiostatin-expressing retroviral vectors or control vectors, were transplanted into the knee cavity. The incidence of arthritis in the knee joints was evaluated histologically based on pannus formation and cartilage destruction. Paws were evaluated macroscopically for redness, swelling, and deformities and immunologically for levels of interleukin-1 beta. Angiogenesis in paws and knee joints was studied by immunohistochemistry using anti-CD31 antibody and measurement of von Willebrand factor levels. RESULTS: Pannus formation and cartilage erosion were dramatically reduced in knees transplanted with angiostatin-expressing cells. In addition, the onset of CIA in the ipsilateral paws below the knees injected with the angiostatin gene was significantly prevented. Furthermore, angiostatin gene transfer inhibited arthritis-associated angiogenesis. CONCLUSION: Local production of angiostatin in the knee was able to prevent the onset of CIA not only in the knee injected with genetically engineered cells, but also in the uninjected ipsilateral paw. This suggests that transfer of the angiostatin gene, and potentially also its protein, may provide a new, effective approach to the treatment of rheumatoid arthritis.