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Objective To assess collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane–like interface tissue around aseptically loosened hip replacement implants. Methods The medical indication for the primary total hip replacement was osteoarthritis in all study patients. Samples from the study patients were compared with control synovial membranes obtained from trauma (hip fracture) patients. Proteoglycans were extracted with 4M guanidinium chloride. Denatured collagen in the remaining matrix was solubilized with α-chymotrypsin. Nonsoluble matrix and supernatant fractions were acid hydrolyzed before measurement of hydroxyproline. The proportion of soluble (in vivo–degraded) collagen of the total sample collagen content was calculated. Proteinases were stained using the avidin–biotin–peroxidase complex method. Results Collagen in the interface membrane from the implants was highly degraded (mean ± SEM 20 ± 3%) compared with that in the control synovial membranes (12 ± 1%; P = 0.007). In controls, the degree of collagen degradation did not correlate with levels of matrix metalloproteinase 1 (MMP-1), MMP-13, or cathepsin K, although MMP-1 approached statistical significance. In interface membranes, the correlations were r = 0.88 (P = 0.002), r = 0.92 (P = 0.001), and r = 0.98 (P < 0.0001) for MMP-1, MMP-13, and cathepsin K, respectively. Conclusion In normal synovial membrane, collagen matrix remodeling may be mainly an intracellular process. In contrast, pathologic tissue destruction in the interface membrane from prosthetic hip joints is associated with a shift toward MMP-13 and cathepsin K, which become activated and overcome their endogenous inhibitors (tissue inhibitors of metalloproteinases and cystatin C). The highly significant correlation between collagen degradation and cathepsin K indicates an extracellular role of this acidic endoproteinase, consistent with previous observations concerning the acidity of the interface membrane.

Objective. Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting the elderly. Its etiology is largely unknown, but age is the most prominent risk factor. The current study was designed to test whether accumulation of advanced glycation end products (AGEs), which are known to adversely affect cartilage turnover and mechanical properties, provides a molecular mechanism by which aging contributes to the development of OA. Methods. The hypothesis that elevated AGE levels predispose to the development of OA was tested in the canine anterior cruciate ligament transection (ACLT) model of experimental OA. Cartilage AGE levels were enhanced in young dogs by intraarticular injections of ribose. This mimics the accumulation of AGEs without the interference of other age-related changes. The severity of OA was then assessed 7 weeks after ACLT surgery in dogs with normal versus enhanced AGE levels. Results. Intraarticular injections of ribose enhanced cartilage AGE levels ∼5-fold, which is similar to the normal increase that is observed in old dogs. ACLT surgery resulted in more-pronounced OA in dogs with enhanced AGE levels. This was observed as increased collagen damage and enhanced release of proteoglycans. The attempt to repair the matrix damage was impaired; proteoglycan synthesis and retention were decreased at enhanced AGE levels. Mankin grading of histology sections also revealed more-severe OA in animals with enhanced AGE levels. Conclusion. These findings demonstrate increased severity of OA at higher cartilage AGE levels and provide the first in vivo experimental evidence for a molecular mechanism by which aging may predispose to the development of OA.

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.

Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers. A metabolic fingerprint consists of a typical combination of metabolites in a biological fluid and is identified by a combination of (1)H NMR spectroscopy and multivariate data analysis (MVDA). The current feasibility study was aimed at identifying a metabolic fingerprint for OA and applying this in a nutritional intervention study. Urine samples were collected from osteoarthritic male Hartley guinea pigs (n = 44) at 10 and 12 mo of age, treated from 4 mo onward with variable vitamin C doses (2.5-3, 30 and 150 mg/d) and from healthy male Strain 13 guinea pigs (n = 8) at 12 mo of age, treated with 30 mg vitamin C/d. NMR measurements were performed on all urine samples. Subsequently, MVDA was carried out on the data obtained using NMR. An NMR fingerprint was identified that reflected the osteoarthritic changes in guinea pigs. The metabolites that comprised the fingerprint indicate that energy and purine metabolism are of major importance in OA. Metabolic fingerprinting also allowed detection of differences in OA-specific metabolites induced by different dietary vitamin C intakes. This study demonstrates the feasibility of metabolic fingerprinting to identify disease-specific profiles of urinary metabolites. NMR fingerprinting is a promising means of identifying new disease markers and of gaining fresh insights into the pathophysiology of disease.

Objective. To determine whether increasing advanced glycation end products (AGEs) in bovine articular cartilage to levels present in aged human cartilage modulates the tensile biomechanical properties of the tissue. Methods. Adult bovine articular cartilage samples were incubated in a buffer solution with ribose to induce the formation of AGEs or in a control solution. Portions of cartilage samples were assayed for biochemical indices of AGEs and tested to assess their tensile biomechanical properties, including stiffness, strength, and elongation at failure. Results. Ribose treatment of cartilage induced increases in tissue fluorescence, absorbance, and pentosidine content (P < 0.001 for each comparison) by amounts similar to those that occur during aging in humans. Ribose treatment of cartilage also induced an increase in dynamic modulus (60% increase) and strength (35% increase), and a decrease (25% decrease) in strain (P < 0.001 for each comparison). Conclusion. The concomitant increase in AGEs and alteration of tensile properties of cartilage after ribose treatment suggest that aging- associated changes in AGEs have functional consequences for this tissue. The AGE-associated increases in strength and stiffness of cartilage may be beneficial by counteracting the decreases in these properties that are associated with degeneration. Conversely, the AGE-associated decrease in failure length, or increase in brittleness, together with increased stiffness may predispose cartilage to increased stress concentration, fracture, and aging-associated biomechanical dysfunction.

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal ("control") supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by alpha-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of D-aspartic acid (% D-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P

Objective. Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular cartilage. Methods. To increase AGE levels, human adult articular cartilage was incubated with threose. The stiffness of the collagen network was measured as the instantaneous deformation (ID) of the cartilage and as the change in tensile stress in the collagen network as a function of hydration (osmotic stress technique). AGE levels in the collagen network were determined as: Ne-(carboxy[m]ethyl)lysine, pentosidine, amino acid modification (loss of arginine and [hydroxy-]lysine), AGE fluorescence (360/460 nm), and digestibility by bacterial collagenase. Results. Incubation of cartilage with threose resulted in a dose-dependent increase in AGEs and a concomitant decrease in ID (r = -0.81, P < 0.001; up to a 40% decrease at 200 mM threose), i.e., increased stiffness, which was confirmed by results from the osmotic stress technique. The decreased ID strongly correlated with AGE levels (e.g., AGE fluorescence r = -0.81, P < 0.0001). Coincubation with arginine or lysine (glycation inhibitors) attenuated the threose-induced decrease in ID (P < 0.05). Conclusion. Increasing cartilage AGE crosslinking by in vitro incubation with threose resulted in increased stiffness of the collagen network. Increased stiffness by AGE crosslinking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage. Thus, the age-related accumulation of AGE crosslinks presents a putative molecular mechanism whereby age is a predisposing factor for the development of OA.

Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated with a stiffer collagen network in cartilage. However, even in cartilage, pentosidine levels themselves represent < 1 cross-link per 20 collagen molecules, and as such cannot be expected to contribute substantially to the increase in collagen network stiffness. In the present study, we investigated a broad range of Maillard reaction products in cartilage collagen in order to determine whether pentosidine serves as an adequate marker for AGE levels. Not only did the well-characterized AGEs pentosidine, Nε-(carboxymethyl)lysine, and Nε-(carboxyethyl)lysine increase with age in cartilage collagen (all P < 0.0001), but also general measures of AGE cross-linking, such as browning and fluorescence (both P < 0.0001), increased. The levels of these AGEs are all higher in cartilage collagen than in skin collagen. As a functional measure of glycation the digestibility of articular collagen by bacterial collagenase was investigated; digestibility decreased linearly with age, proportional to the extent of glycation. Furthermore, the arginine content and the sum of the hydroxylysine and lysine content of cartilage collagen decrease significantly with age (P < 0.0001 and P < 0.01 respectively), possibly due to modification by the Maillard reaction. The observed relationship between glycation and amino acid modification has not been reported previously in vivo. Our present results indicate that extensive accumulation of a variety of Maillard reaction products occurs in cartilage collagen with age. Altogether our results support the hypothesis that glycation contributes to stiffer and more brittle cartilage with advancing age.

Objective. To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. Methods. The proteoglycan-synthesis rate (35SO42- incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography measurement of pentosidine) were evaluated in human articular cartilage from 129 donors, varying in age from 25 to 88 years, and in cartilage with enhanced levels of advanced glycation end-products (AGEs) resulting from incubation with ribose. Results. Cartilage showed a strong age-related increase in pentosidine levels (r = 0.97, P < 0.0005) and, concomitantly, a decrease in proteoglycan synthesis (r = -0.98, P < 0.0002). This decrease in proteoglycan synthesis correlated with the increase in pentosidine (r = -0.95, P < 0.02). Moreover, the elevation of pentosidine levels in the in vitro-ribosylated cartilage was proportional with the decrease in proteoglycan synthesis (r = -0.95, P < 0.005). Conclusion. In both aged and in vitro AGE-enriched cartilage, the rate of proteoglycan synthesis was negatively correlated with the degree of glycation. This suggests that the age-related increase in cartilage AGE levels may be responsible, at least in part, for the age-related decline in the synthetic capacity of cartilage.

The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically. Predominantly type H collagen was present in the alginate bead, as reflected by the unique extent of lysyl hydroxylation, glycosylation, and pyridinoline crosslink formation measured. Collagen crosslinks, predominantly hydroxylysylpyridinoline (>93%), were observed after 7 to 11 days of culture and their formation was effectively blocked by β-aminopropionitrile (BAPN). Unexpectedly, BAPN treatment resulted in a 100% increase of collagen levels, without influencing cell proliferation and proteoglycan levels. In control cultures 90% of the synthesized collagen was retained in the cell-associated matrix, while in BAPN-treated cultures half of the collagen was found in the interterritorial matrix compartment further removed from the cells. This suggests that impaired crosslinking of collagen interferes with pericellular collagen deposition, causing upregulation of collagen synthesis by impaired cell-matrix interactions. Integrins are likely to be involved in this feedback inhibition by extracellular collagen since the cyclic RGD-containing peptide CGRGDSPC downregulated collagen synthesis by 37%. Chemicals/CAS: Alginates; alginic acid, 9005-32-7; Amino Acids; Aminopropionitrile, 151-18-8; arginyl-glycyl-aspartic acid, 99896-85-2; Collagen, 9007-34-5; DNA, 9007-49-2; Glucuronic Acid, 576-37-4; glycyl-arginyl-glycyl-aspartyl-seryl-proline, 91037-75-1; Hexuronic Acids; Oligopeptides; Proteoglycans; pyridinoline, 63800-01-1

Objective: To analyse matrix metalloproteinases (MMPs) and tissue inhibitor-1 of MMPs (TIMP-1) levels in the systemic circulation and synovial fluid (SF) of patients with RA and to compare these levels with inflammatory and collagen degradation markers. Methods: ProMMP-1, -2, -3, -8, -9, TIMP-1, levels of MMP/ α2-macroglobulin complexes, and collagen degradation products were measured by sandwich ELISA, activity assays, and HPLC in paired SF and serum samples from 15 patients with RA and 13 with OA. Results: MMPs were higher in SF of patients with RA than in OA or controls. MMP levels in SF of patients with OA were higher than in controls. In serum, levels of proMMP-3, -8 and -9 were higher in patients with RA than in OA or controls, whereas only proMMP-8 and -9 were higher in serum of patients with OA than in controls. A strong correlation was seen between serum and SF levels of MMP-8 and -9 in RA. Increased levels of MMP/α2-macroglobulin complexes indicated an MMP/TIMP imbalance in serum and SF in RA. SF hydroxyproline correlated significantly with SF levels of proMMP-9 in RA. Conclusions: Systemic MMP-8 and -9 levels represent the situation in the inflamed joint; MMP-9 is likely to be involved in degradation of joint collagen. The hypothesis of MMP/ TIMP imbalance in RA is strengthened. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; hydroxyproline, 51-35-4, 6912-67-0; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 1, 140208-24-8; alpha-Macroglobulins; Collagenases, EC 3.4.24.-; Enzyme Precursors; Hydroxyproline, 51-35-4; Matrix Metalloproteinase 8, EC 3.4.24.34; Matrix Metalloproteinase 9, EC 3.4.24.35; Matrix Metalloproteinases, EC 3.4.24.-; Metalloendopeptidases, EC 3.4.24.-; MMP8 protein, human, EC 3.4.24.-; pro-matrix metalloproteinase 9, EC 3.4.24.-; prostromelysin, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

Objective The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) could be one of these mechanisms. We undertook this study to investigate the role of the receptor for AGEs (RAGE) in mediating the cellular effects of AGEs on chondrocytes and fibroblast-like synoviocytes (FLS). Methods AGE levels in human cartilage were determined by fluorescence, browning, and pentosidine levels. Chondrocyte activation by AGEs was assessed as the release of proteoglycans and the synthesis of matrix metalloproteinase 1 (MMP-1) and type II collagen messenger RNA (mRNA). The activation of FLS by AGEs was measured by MMP-1 production and invasion through matrix proteins. Results Patients with focal degeneration of cartilage showed increased AGE levels in their healthy cartilage compared with the levels in healthy cartilage from donors without cartilage degeneration (P < 0.01 for both fluorescence and browning; P not significant for pentosidine content). Stimulation of bovine chondrocytes with glycated albumin increased the release of proteoglycans by 110% (P < 0.001) and the production of MMP-1 mRNA by 200% (P = 0.028). In addition, OA FLS produced 240% more MMP-1 when stimulated with glycated albumin (P < 0.001). Glycated matrix or albumin increased the catabolic activity of OA FLS, which was assessed as invasive behavior, by 150% and 140% (P = 0.001 and P = 0.010), respectively. Effects of stimulation with AGEs were blocked by a neutralizing antibody against RAGE, but not by an isotype control. Conclusion This study shows that AGEs trigger RAGE on chondrocytes and FLS, leading to increased catabolic activity and therefore to cartilage degradation. AGEs, via RAGE, could therefore contribute to the development and/or progression of OA.

Objective: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). Methods: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n=283), osteoarthritis (n=105), and pseudogout (n=65), and in healthy controls (n=35). Activity of MMP-1 and MMP-3 in α2 macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. Results: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. Conclusions: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; alpha-Macroglobulins; Matrix Metalloproteinases, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

Nonenzymatic glycosylation of low-density lipoprotein (LDL) has been suggested to be involved in the development of atherosclerosis in patients with diabetes. Since α-dicarbonyl compounds were identified as intermediates in nonenzymatic glycosylation, we investigated the effect of the physiological α-dicarbonyl compound methylglyoxal (MG) on the physico-chemical and biological properties of LDL. MG modifies LDL in a timeand concentration-dependent manner as indicated by the production of fluorescent products and the increase in net-negative charge. Despite the production of superoxide anion radicals, LDL modification by MG is not accompanied by peroxidation of the polyunsaturated fatty acids. MG-LDL showed impaired recognition by the LDL receptor, but increased binding to scavenger receptors. However, MG-LDL did not enhance cholesteryl ester synthesis in murine macrophages.

Objective: To analyse the relation between systemic levels of pro-MMP-3, -8, and -9 matrix metalloproteinase (MMP) activity in α2 macroglobulin (α2M)/MMP complexes and the progression of joint destruction in patients with recent onset rheumatoid arthritis (RA). Methods: 109 patients with RA of recent onset were entered into this longitudinal study. Patients were followed up for two years; clinical data, blood samples, and radiographs were obtained at baseline and at 1 and 2 years. Serum levels of MMPs were measured by sandwich ELISA and MMP activity assays. Results: During the two years joint damage progressed from 0 to 10 (median Sharp score, p

Purpose of review: Across the world, osteoarthritis is the most commonly occurring musculoskeletal disease of the elderly, affecting more than 25% of the population older than 60 years of age. By far the single greatest risk factor for the development of osteoarthritis is age, but a mechanism to explain this relation has not yet been identified. If such a mechanism is identified, this potentially also provides a novel target for osteoarthritis therapy. The identification of new therapeutic targets is of utmost importance, because a disease-modifying treatment for osteoarthritis is not available and, because of the graying of the population, the number of patients with osteoarthritis will continue to increase, which will pose an enormous social and economic burden on society. Recent findings: Advanced glycation end products accumulate in human articular cartilage with increasing age, and affect biomechanical, biochemical, and cellular characteristics of the tissue. As an illustration, accumulation of advanced glycation end products increase cartilage stiffness and brittleness while decreasing the synthesis and degradation of cartilage matrix constituents. Articular cartilage becomes more prone to damage, and thus osteoarthritis, at elevated concentrations of advanced glycation end products. Summary: The reviewed literature demonstrates that the age-related accumulation of advanced glycation end products in articular cartilage may provide a molecular mechanism capable of (at least in part) explaining the age-related increase in the incidence of osteoarthritis. This conclusion paves the way for new strategies to prevent or treat osteoarthritis via inhibition and/or reversal of this process. © 2003 Lippincott Williams & Wilkins. Chemicals/CAS: Glycosylation End Products, Advanced

Objective The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects of accumulation of AGEs on the turnover of the extracellular matrix of human articular cartilage. Design Chondrocyte mediated cartilage degradation (GAG release, colorimetric) was measured in human articular cartilage of donors aged 19–82 years ( N =30, 4-day culture). In addition, to mimic the age-related increase in AGE levels in vitro , cartilage was cultured in the absence or presence of glucose, ribose or threose. Cartilage degradation and proteoglycan synthesis ( 35 SO2−4 incorporation) were measured and related to the degree of cartilage AGE levels (fluorescence at 360/460nm). Results Chondrocyte-mediated degradation of articular cartilage (i.e. GAG release) decreased with increasing age of the cartilage donor ( r =−0.43, P 0.02). In vitro incubation of cartilage with glucose, ribose or threose resulted in a range of AGE levels that was highly correlated to the chondrocyte-mediated cartilage degradation ( r =−0.77, P 0.001, N =26). In addition, in these in vitro glycated cartilage samples, a decrease in proteoglycan synthesis was observed at increasing AGE levels ( r =−0.54, P 0.005, N =25). Conclusions This study shows that an increase in AGE levels negatively affects the proteoglycan synthesis and degradation of articular cartilage. In combination, these two effects reduce the turnover of the cartilage and thereby the maintenance and repair capacity of the tissue. By this mechanism, the age-related increase in cartilage AGE levels may contribute to the development of OA.

Osteoarthritis is a disabling joint disease that is characterized by the progressive destruction of articular cartilage. Diagnosis is based on clinical symptoms 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. Such molecular markers are essential for diagnosis, prognosis, and monitoring of disease progression and efficacy of therapy that is targeted at joint destruction. In addition, these markers are important for the development of new disease-modifying therapies. This concise review discusses the developments over the past 2 years in the field of molecular markers for osteoarthritis. © 2002 Lippincott Williams & Wilkins, Inc. Chemicals/CAS: cartilage oligomeric matrix protein, 161028-53-1; chondroitin sulfate, 9007-28-7, 9082-07-9; collagen, 9007-34-5; hyaluronic acid, 31799-91-4, 9004-61-9, 9067-32-7; keratan sulfate, 69992-87-6, 9056-36-4; osteocalcin, 136461-80-8; tissue inhibitor of metalloproteinase, 97837-28-0; Matrix Metalloproteinases, EC 3.4.24.-; Collagen Type II; Cross-Linking Reagents; Genetic Markers; Proteoglycans

Objective: The collagen network in human articular cartilage experiences a large number of stress cycles during life as it shows hardly any turnover after adolescence. We hypothesized that, to withstand fatigue failure, the physical condition of the collagen network laid down at adolescence is of crucial importance for the age of onset of osteoarthritis (OA). Methods: We have compared the lysyl hydroxylation level and pyridinoline cross-link level of the collagen network of degenerated (DG) cartilage of the femoral knee condyle (representing a preclinical early stage of OA) with that of normal cartilage from the contralateral knee. The biological age of the collagen network was determined by means of pentosidine levels. For each donor, collagen modifications of normal cartilage were compared with DG cartilage that showed no significant remodeling of the collagen network (as evidenced by identical pentosidine levels). Results: DG cartilage contained significantly more hydroxylysine residues per collagen molecule in comparison with healthy cartilage from the same donor, both in the upper and lower half (the region near the articular surface and adjacent to bone, respectively). In addition, a significantly higher level of pyridinoline cross-linking was observed in the upper half of DG cartilage. Considering the biological age of the collagen network, the changes observed in DG cartilage must have been present several decades before cartilage became degenerated. Conclusions: The data suggest that high levels of lysyl hydroxylation and pyridinoline cross-linking result in a collagen network that fails mechanically in long term loading. Areas containing collagen with low hydroxylysine and pyridinoline levels are less prone to degeneration. As such, this study indicates that post-translational modifications of collagen molecules synthesized during adolescence are causally involved in the pathogenesis of OA. © 2002 OsteoArthritis Research Society International. Chemicals/CAS: Amino Acids; Arginine, 74-79-3; Collagen, 9007-34-5; Cross-Linking Reagents; Hydroxylysine, 28902-93-4; Lysine, 56-87-1; pentosidine, 124505-87-9; pyridinoline, 63800-01-1

Animal models for osteoarthritis (OA) face the difficulty to display the key pathological events of a naturally slowly progressing disease in a protracted time frame. To serve as pharmacological models, in addition, these changes need to be meaningful and synchronous enough to allow predictions regarding the potential effects of new therapies. Therefore, high expectations have been placed in models where the OA-like pathology develops spontaneously, without any defined stimulus potentially limiting the true disease relevance of the tissue reactions thus induced.

During aging, non-enzymatic glycation results in the formation and accumulation of the advanced glycation endproduct pentosidine in long-lived proteins, such as articular cartilage collagen. In the present study, we investigated whether pentosidine accumulation also occurs in cartilage aggrecan. Furthermore, pentosidine levels in aggrecan subfractions of different residence time were used to explore pentosidine levels as a quantitative measure of aggrecan turnover. In order to compare protein turnover rates, protein residence time was measured as racemization of aspartic acid. As has previously been shown for collagen, pentosidine levels increase with age in cartilage aggrecan. Consistent with the faster turnover of aggrecan compared to collagen, the rate of pentosidine accumulation was three fold lower in aggrecan than in collagen. In the subfractions of aggrecan, pentosidine levels increased with protein residence time. These pentosidine levels were used to estimate the half-life of the globular hyaluronan-binding domain of aggrecan to be 19.5 years. This value is in good agreement with the half-life of 23.5 years that was estimated based on aspartic acid racemization. In aggrecan from osteoarthritic (OA) cartilage, decreased pentosidine levels were found compared with normal cartilage, which reflects increased aggrecan turnover during the OA disease process. In conclusion, we showed that pentosidine accumulates with age in aggrecan and that pentosidine levels can be used as a measure of turnover of long-lived proteins, both during normal aging and during disease. © 2001 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved. Chemicals/CAS: Aggrecans; Arginine, 74-79-3; Extracellular Matrix Proteins; Glycosylation End Products, Advanced; Lectins, C-Type; Lysine, 56-87-1; pentosidine, 124505-87-9; Proteochondroitin Sulfates; Proteoglycans

Progressive destruction of articular cartilage is a hallmark of osteoarthritis (OA) and rheumatoid arthritis (RA). Age-related changes in cartilage may influence tissue destruction and thus progression of the disease. Therefore, the effect of age-related accumulation of advanced glycation end products (AGEs) on cartilage susceptibility to proteolytic degradation by matrix metalloproteinases (MMPs) present in synovial fluid (SF) of OA and RA patients was studied. Cartilage was incubated with APMA-activated SF obtained from OA or RA patients, and tissue degradation was assessed by colorimetric measurement of glycosaminoglycan (GAG) release. Cartilage degradation was related to the level of AGEs in cartilage from donors of different ages (33-83 years) and in cartilage with in vitro-enhanced AGE levels (by incubation with ribose). MMP activity in SF was measured using a fluorogenic substrate. AGE levels were assessed by high-performance liquid chromatography measurement of the glycation product pentosidine. In cartilage from donors ages 33-83 years, a strong correlation was found between the age-related increase in pentosidine and the decrease in MMP-mediated tissue degradation (r = -0.74, P < 0.0005). Multiple regression analysis showed pentosidine to be the strongest predictor of the decreased GAG release (P < 0.0005); age did not contribute (P > 0.8). In addition, decreased MMP-mediated GAG release was proportional to increased pentosidine levels after in vitro enhancement of glycation (r = -0.27, P < 0.01). This was demonstrated for both OA and RA SF (for control versus glycated, P < 0.002 for all SF samples tested). Increased cartilage AGEs resulted in decreased cartilage degradation by MMPs from SF, indicating that aged cartilage is less sensitive than young cartilage to MMP-mediated cartilage degradation, such as occurs in OA and RA. Therefore, the level of cartilage glycation may influence the progression of these diseases

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase ∼50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis. © 2001 Academic Press.

Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens

To determine the role of interleukin-1 beta (IL-1 beta) in the degradation of proteoglycans and collagen by articular chondrocytes.Chondrocytes were cultured in alginate beads for 2 weeks to produce extracellular matrix, followed by the addition of IL-1 beta for 1 or 2 days. Breakdown of extracellular matrix (with and without activation of pro-matrix metalloproteinases (MMPs) by APMA) was monitored by release of glycosaminoglycans (GAG, proteoglycans) and hydroxyproline (collagen) from the beads into the medium, and by the amount of damaged collagen in the bead. Levels of (pro)MMPs in the beads were assayed by zymography and their activity was quantified fluorometrically.IL-1 beta induced a profound GAG release (approximately 80% after 2 days at 20 ng/ml IL-1 beta) that was both time and IL-1 beta concentration dependent. Under these conditions no increase in collagen release or damaged collagen in the bead was detected. Zymography demonstrated that the synthesis of a variety of proMMPs was induced by IL-1 beta, without a detectable increase of MMP-activity as measured in the activity assay. After activation of the proMMPs by APMA, a time and IL-1 beta concentration-dependent increase in MMP-activity was found, which resulted in almost complete deterioration of collagen already after 18 h of incubation. In the presence of APMA, GAG release from IL-1 beta treated beads was significantly increased from 24 to 31%.Our data suggest that proteoglycan and collagen degradation are regulated through different mechanisms: IL-1 beta induces the synthesis of active enzymes that degrade proteoglycans, such as 'aggrecanase', and inactive proMMPs. Thus, IL-1 beta alone is not sufficient to result in collagen-degrading MMPs. Once activated, MMPs may account for up to a quarter of the aggrecan degradation in this model.

Doxycycline (DOX) profoundly inhibited collagen synthesis by differentiated articular chondrocytes. At 25 μM, the rate of collagen synthesis was suppressed by more than 50% without affecting cell proliferation (DNA levels) and general protein synthesis (35S-Met and 35S-Cys incorporation). Steady-state mRNA levels of type II collagen were also reduced, indicating that DOX may have an effect at the transcriptional level of type II collagen. The IC50 value of DOX to downregulate collagen synthesis (17 μM) is close to DOX levels attained in vivo (< 10 μM), and it is more than ten-fold lower than the IC50 values to inhibit the activity of most matrix metalloproteinases (MMPs). As such, these findings support the hypothesis that the reduced severity of OA observed in the dog anterior cruciate ligament model resulting from prophylactic treatment with DOX may involve mechanisms other than MMP inhibition alone. Our findings suggest that prevention of changes in the chondrocyte phenotype may be involved in the beneficial effect of doxycycline in experimental osteoarthritis. for differentiated chondrocytes in early stages of osteoarthritis exhibit elevated collagen synthesis.

A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 μm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra- assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well. Chemicals/CAS: Arginine, 74-79-3; Cross-Linking Reagents; Lysine, 56-87-1; pentosidine, 124505-87-9; Pyridinium Compounds

Doxycycline is known for its ability to inhibit matrix metalloproteinases (MMPs), a family of enzymes that play a role in cartilage breakdown in arthritides. Its prophylactic effect in reducing joint degradation in osteoarthritis is mainly attributed to this property. In this study, we show that doxycycline exhibits a profound inhibition of collagen synthesis by bovine articular chondrocytes cultured in alginate. At 25 μM doxycycline, collagen synthesis was decreased by 50%; no effect on cell proliferation (DNA levels) or general protein synthesis (35S-Met and 35S-Cys incorporation) was observed. Messenger RNA levels of type II collagen were also reduced, indicating an effect of doxycycline at the transcriptional level. The concentration of doxycycline needed to downregulate collagen synthesis was > 10-fold lower than that needed to inhibit most of the MMPs. Inasmuch as differentiated chondrocytes in the early stages of osteoarthritis exhibit increased collagen synthesis, the beneficial effect of doxycycline in vivo may involve prevention of changes in chondrocyte phenotype. Chemicals/CAS: alginic acid, 28961-37-7, 29894-36-8, 9005-32-7, 9005-38-3; cysteine, 4371-52-2, 52-89-1, 52-90-4; doxycycline, 10592-13-9, 17086-28-1, 564-25-0; methionine, 59-51-8, 63-68-3, 7005-18-7; sulfur 35, 15117-53-0; Alginates; alginic acid, 9005-32-7; Collagen, 9007-34-5; Cysteine, 52-90-4; DNA, 9007-49-2; Doxycycline, 564-25-0; Glucuronic Acid, 576-37-4; Hexuronic Acids; Methionine, 63-68-3; Protein Synthesis Inhibitors; RNA, Messenger; Sulfur Radioisotopes

Objective: Recently we described a canine model of osteoarthritis (OA), the groove model with features of OA at 10 weeks after induction, identical to those seen in the canine anterior cruciate ligament transection (ACLT) model. This new model depends on cartilage damage accompanied by transient intensified loading of the affected joint. The present study evaluates this groove model at 20 and 40 weeks after induction, to assess whether the osteoarthritic features progress in time. Methods: Grooves were made in the femoral condyles of one knee without damaging the subchondral bone. After surgery the dogs were forced to load the experimental joint 3 days per week (4 hours/day) for 20 weeks by fixing the contralateral control limb to the trunk. After 20 weeks and 40 weeks (the last 20 weeks normal loading) joints were analysed for biochemical and histological features of OA. Results: All biochemical cartilage parameters were indicative of OA and all these parameters suggested a slow progression of degeneration over time from 20 to 40 weeks after induction, statistically significant for synthesis and content of proteoglycans as well as Mankin grade. Synovial inflammation, which was mild, diminished slightly in time. Conclusion: The degenerative joint damage in the canine groove model is slowly progressive over time in the first year. The cartilage degeneration is induced by a one-time trauma and is not primarily mediated by synovial inflammation, which gives this model unique characteristics compared to presently available models for studying early osteoarthritic features in vivo. In the groove model the effect of treatment of cartilage damage is not counteracted by permanent joint instability or hampered by inflammation. Therefore, the model might be more sensitive to detect effects of therapy, aimed at cartilage protection and repair. © 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.Chemicals/CAS: Proteoglycans