Evaluation of cartilage damage by measuring collagen degradation products in joint extracts in a traumatic model of osteoarthritis

The objective of this work was to investigate whether collagen degradation products in protein extract from joints could provide quantitative information on cartilage damage. Osteoarthritis (OA) was surgically induced in rat knee joints. Joints were isolated 7, 14 and 28 days after surgery for protein extraction and histology. C-terminal telopeptide of type II collagen (CTX-II), CTX-I and hydroxyproline were measured in protein extracts. Matrix metalloproteinase (MMP)-2 and -9 activity was evaluated by gelatinase zymography and joint pathology was visualized by histology and immunohistochemistry. The results showed that levels of CTX-II were significantly increased in anterior cruciate ligament transection (ACLT)-operated compared with sham-operated knee joints on days 7 and 28, whereas the levels of hydroxyproline and CTX-I epitopes showed no difference. MMP activity was slightly increased in ACLT-operated joints. The CTX-II epitope was highly expressed and co-localized to damaged articular cartilage in ACLT-operated joints. We have therefore demonstrated an increased type II collagen degradation in knees after surgical induction of OA, and propose assessment of collagen degradation epitopes as a quantitative measure of cartilage damage.     

Evaluation of the effect of glucosamine on an experimental rat osteoarthritis model

AimsTo investigate the in vivo effect of glucosamine on articular cartilage in osteoarthritis (OA), we evaluated serum biomarkers such as CTX-II (type II collagen degradation) and CPII (type II collagen synthesis) as well as histopathological changes (Mankin score, toluidine blue staining of proteoglycans in an experimental OA model using rats.Main methodsOA was surgically induced in the knee joint by anterior cruciate ligament transection (ACLT) in rats. Animals were divided into three groups: sham-operated group (Sham), ACLT group without GlcN administration (? GlcN) and ACLT group with oral administration of glucosamine hydrochloride (+ GlcN; 1000 mg/kg/day for 56 days).Key findingsACLT induced macroscopic erosive changes on the surfaces of articular cartilage and histological damages such as increase of Mankin score. Of note, glucosamine administration substantially suppressed the macroscopic changes, although the effect on Mankin score was not significant. In addition, serum CTX-II levels were elevated in ?GlcN group compared to that in Sham group after the operation. Of importance, the increase of CTX-II was significantly suppressed by GlcN administration. Moreover, serum CP-II levels were substantially increased in + GlcN group compared to those in Sham and ? GlcN groups after the operation.SignificanceGlcN has a potential to exert a chondroprotective action on OA by inhibiting type II collagen degradation and enhancing type II collagen synthesis in the articular cartilage.     

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague–Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.     

Hormone replacement therapy,calcium and vitamin D<Subscript>3</Subscript> versus calcium and vitamin D<Subscript>3</Subscript>alone decreases markers of cartilage and bone metabolism in rheumatoid arthritis: a randomized controlled trial [ISRCTN46523456]

This study aimed to evaluate the effects of hormone replacement therapy (HRT), known to prevent osteoporosis and fractures, on markers of bone and cartilage metabolism. Furthermore, we assessed whether changes in these markers corresponded to alterations in bone mineral density and radiographic joint destructions in postmenopausal women with rheumatoid arthritis. Eighty-eight women were randomized to receive HRT, calcium, and vitamin D3, or calcium and vitamin D3 alone, for 2 years. Bone turnover was studied by analyzing serum levels of C-terminal telopeptide fragments of type I collagen (CTX-I), C-terminal telopeptide of type I collagen (ICTP), bone sialoprotein, and C-terminal propeptide of type I procollagen (PICP) and cartilage turnover by urinary levels of collagen type II C-telopeptide degradation fragments (CTX-II) and cartilage oligomeric matrix protein (COMP) in serum. Treatment with HRT resulted in decrease in CTX-I (P < 0.001), ICTP (P < 0.001), PICP (P < 0.05), COMP (P < 0.01), and CTX-II (P < 0.05) at 2 years. Reductions in CTX-I, ICTP, and PICP were associated with improved bone mineral density. Of the markers tested, CTX-I reflected bone turnover most sensitively; it was reduced by 53 +/- 6% in the patients receiving HRT. Baseline ICTP (P < 0.001), CTX-II (P < 0.01), and COMP (P < 0.05) correlated with the Larsen score. We suggest that biochemical markers of bone and cartilage turnover may provide a useful tool for assessing novel treatment modalities in arthritis, concerning both joint protection and prevention of osteoporosis.     

Matrix Metalloproteases and Tissue Inhibitors of Metalloproteinases in Medial Plica and Pannus-like Tissue Contribute to Knee Osteoarthritis Progression

Osteoarthritis (OA) is characterized by degradation of the cartilage matrix, leading to pathologic changes in the joints. However, the pathogenic effects of synovial tissue inflammation on OA knees are not clear. To investigate whether the inflammation caused by the medial plica is involved in the pathogenesis of osteoarthritis, we examined the expression of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the medial plica and pannus-like tissue in the knees of patients with medial compartment OA who underwent either arthroscopic medial release (stage II; 15 knee joints from 15 patients) or total knee replacement (stage IV; 18 knee joints from 18 patients). MMP-2, MMP-3, MMP-9, IL-1β, and TNF-α mRNA and protein levels measured, respectively, by quantitative real-time PCR and Quantibody human MMP arrays, were highly expressed in extracts of medial plica and pannus-like tissue from stage IV knee joints. Immunohistochemical staining also demonstrated high expression of MMP-2, MMP-3, and MMP-9 in plica and pannus-like tissue of stage IV OA knees and not in normal cartilage. Some TIMP/MMP ratios decreased significantly in both medial plica and pannus-like tissue as disease progressed from stage II to stage IV. Furthermore, the migration of cells from the pannus-like tissue was enhanced by IL-1β, while plica cell migration was enhanced by TNF-α. The results suggest that medial plica and pannus-like tissue may be involved in the process of cartilage degradation in medial compartment OA of the knee.     

Early and stable upregulation of collagen type II, collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond-Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.     

Early and stable upregulation of collagen type II,collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond–Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.     

Cartilage oligomeric matrix protein and hyaluronic acid are sensitive serum biomarkers for early cartilage lesions in the knee joint

The purpose of this study was to evaluate the relationship between five previously established serum osteoarthritis biomarkers and the severity of cartilage lesions in the knee. Cartilage damage (classified according to the Outerbridge scoring system) and serum concentrations of cartilage oligomeric matrix protein (COMP), collagen type II C-telopeptide (CTX-II), matrix metalloproteinase-3 (MMP-3), collagen type III N-propeptide, (PIIINP), and hyaluronic acid (HA) were determined in 79 patients who underwent knee arthroscopy or total knee replacement. HA and COMP concentrations were significantly higher in the Outerbridge score 1 and 2 groups, respectively. These results suggest that serum COMP and HA concentrations can be used to predict early cartilage lesions in the knee.     

Alleviation of osteoarthritis by Trichostatin A, a histone deacetylase inhibitor, in experimental osteoarthritis

This study investigated the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on cartilage degradation in an experimental model of osteoarthritis (OA). Thirty-two male New Zealand rabbits underwent unilateral anterior cruciate ligament transection (ACLT) on left knee joints to induce OA and were randomly divided into two groups (n = 16), the TSA group was injected intra-articularly with 0.3 ml TSA [250 ng/ml in the dimethylsulphoxide (DMSO)], the OA group received DSMO since 4 weeks after operation once a week for 5 weeks. Rabbits were killed seven days after the last injection. Left knee cartilage was harvested for morphological, histological and genetic analysis. Another ten rabbits were used for normal control and received no injection. The TSA group showed less cartilage degradation as compared to the OA group assessed by morphological and histological evaluation. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, and interleukin-1 (IL-1) was increased significantly in the OA group compared to the normal group. The elevated expression was reduced by TSA. Our results suggest that TSA could be considered as a potential agent for treatment for OA.     

Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs

Objective

To investigate the pathogenesis of late phase osteoarthritic (OA) synovial fluid (SF) on normal articular cartilage in vivo and provide an understanding of degenerative cartilage extending in OA joint.

Methods

A random knee, each of 8 beagle dogs, received anterior cruciate ligament transection (ACLT) and was confirmed to have late phase OA degenerative changes at 24 weeks after operation. Thereafter, one random elbow of each canine was injected with autologous late phase OA knee SF. The contralateral elbow was injected with normal saline (NS) of the same volume as SF aspirated from ACLT knee. These two groups of elbows were labeled “SF” and “NS”. 8 other beagle dogs were left intact and placed in Group Control. After aseptic arthrocentesis was performed weekly on both elbows for 24 weeks, morphological changes were observed in the cartilage of the elbows, and expressions of 7 biological etiological factors of chondrocytes of the elbows were determined in Group SF, Group NS and Group Control, respectively.

Results

Morphological changes were observed in articular cartilage of the elbows in Group SF. Levels of unit area of collagen type I in the noncalcified, calcified and full zones of articular cartilage of the elbows in Group SF increased significantly. Level of unit area of collagen type III in the calcified zone of articular cartilage of the elbows in Group SF remained unchanged. Meanwhile, expressions of MMP-1 and MMP-3 of chondrocytes of the elbows in Group SF increased significantly. There was almost no difference between articular cartilage in Group NS and Group Control.

Conclusion

Based on these results, we conclude that OA degeneration of normal articular cartilage can be independently induced by late phase OA SF. Endogenous OA biological etiological factor may be one of the reasons causing degenerative cartilage extending in OA joint.     

Cdkn2a示踪小鼠:研究创伤性骨关节炎软骨退变细胞衰老的新模型

骨关节炎(osteoarthritis,OA)是临床上最为常见的老年性运动系统疾病之一。研究表明,衰老是OA发生发展的重要影响因素之一,但其具体作用及机制尚未完全清楚。本研究通过CRISPR/Cas9技术,建立了Cdkn2a-e(Luc-2A-tdTomato-2A-CreERT2-WPRE-pA)1定点敲入的杂合子小鼠模型,可在小鼠活体内追踪衰老经典标记物Cdkn2a(p16, p16INK4a)的表达情况,结合前交叉韧带横断术(ACLT)诱导OA小鼠模型,将OA病理进程中的衰老变化在体外直观呈现,明确衰老与OA之间的关系。本研究选取10 ～ 12周龄Cdkn2a小鼠,随机分为非手术对照组、假手术组和ACLT组,通过ACLT手术在小鼠中构建OA的模型,术后4周收取动物进行活体荧光成像检测显示,ACLT组术后4周的小鼠膝关节局部Cdkn2a荧光表达升高(P＜0.05),小鼠膝关节组织切片的番红O固绿染色显示,4周时ACLT组膝关节软骨出现退变(P＜0.05),对小鼠膝关节组织进行Cdkn2a免疫组织化学染色,相较其他2个组,ACLT组的小鼠膝关节组织软骨表面Cdkn2a染色更深。研究结果显示,通过手术诱导的OA模型在局部具有衰老的表现,这进一步验证了衰老和OA的关系。同时,该Cdkn2a示踪小鼠模型能够在活体小鼠内体现衰老的进展。结合影像学检查,可以实时观察衰老和OA发生、进展的关系,为衰老与OA疾病机制的研究提供了良好的模型,也为今后进行靶向衰老进行OA的治疗提供了很好的研究工具。     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling

Bone morphogenetic protein-2 (BMP-2) has been proposed as a tool for cartilage repair and as a stimulant of chondrogenesis. In healthy cartilage, BMP-2 is hardly present, whereas it is highly expressed during osteoarthritis. To assess its function in cartilage, BMP-2 was overexpressed in healthy murine knee joints and the effects on proteoglycan (PG) synthesis and degradation were evaluated. Moreover, the contribution of BMP in repairing damage induced by interleukin-1 (IL-1) was investigated. Ad-BMP-2 was injected intra-articularly into murine knee joints, which were isolated 3, 7, and 21 days after injection for histology, immunohistochemistry, and autoradiography. In addition, patellar and tibial cartilage was isolated for RNA isolation or measurement of PG synthesis by means of ³⁵SO₄ ^2- incorporation. To investigate the role for BMP-2 in cartilage repair, cartilage damage was induced by intra-articular injection of IL-1. After 2 days, Ad-BMP-2, Ad-BMP-2 + Ad-gremlin, Ad-gremlin, or a control virus was injected. Whole knee joints were isolated for histology at day 4 or patellae were isolated to measure ³⁵SO₄ ^2- incorporation. BMP-2 stimulated PG synthesis in patellar cartilage on all days and in tibial cartilage on day 21. Aggrecan mRNA expression had increased on all days in patellar cartilage, with the highest increase on day 7. Collagen type II expression showed a similar expression pattern. In tibial cartilage, collagen type II and aggrecan mRNA expression had increased on days 7 and 21. BMP-2 overexpression also induced increased aggrecan degradation in cartilage. VDIPEN staining (indicating matrix metalloproteinase activity) was elevated on day 3 in tibial cartilage and on days 3 and 7 in patellar cartilage, but no longer was by day 21. Increased NITEGE staining (indicating aggrecanase activity) was found on days 7 and 21. In IL-1-damaged patellar cartilage, BMP-2 boosted PG synthesis. Blocking of BMP activity resulted in a decreased PG synthesis compared with IL-1 alone. This decreased PG synthesis was associated with PG depletion in the cartilage. These data show that BMP-2 boosts matrix turnover in intact and IL-damaged cartilage. Moreover, BMP contributes to the intrinsic repair capacity of damaged cartilage. Increased matrix turnover might be functional in replacing matrix molecules in the repair of a damaged cartilage matrix.     

Therapeutic effects of bone marrow mesenchymal stem cells-derived exosomes on osteoarthritis

Mesenchymal stem cells (MSCs) have shown chondroprotective effects in clinical models of osteoarthritis (OA). However, effects of MSC-derived exosomes on OA remain unclear. The study aimed to investigate the therapeutic potential of exosomes from human bone marrow MSCs (BM-MSCs) in alleviating OA. The anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery were performed on the knee joints of a rat OA model, followed by intra-articular injection of BM-MSCs or their exosomes. In addition, BM-MSC-derived exosomes were administrated to primary human chondrocytes to observe the functional and molecular alterations. Both of BM-MSCs and BM-MSC-derived exosomes alleviated cartilage destruction and subchondral bone remodelling in OA rat model. Administration of BM-MSCs and exosomes could reduce joint damage and restore the trabecular bone volume fraction, trabecular number and connectivity density of OA rats. In addition, in vitro assays showed that BM-MSCs-exosomes could maintain the chondrocyte phenotype by increasing collagen type II synthesis and inhibiting IL-1β–induced senescence and apoptosis. Furthermore, exosomal lncRNA MEG-3 also reduced the senescence and apoptosis of chondrocytes induced by IL-1β, indicating that lncRNA MEG-3 might partially account the anti-OA effects of BM-MSC exosomes. The exosomes from BM-MSCs exerted beneficial therapeutic effects on OA by reducing the senescence and apoptosis of chondrocytes, suggesting that MSC-derived exosomes might provide a candidate therapy for OA treatment.     

1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)₂D₃ in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)₂D₃ on chondrocytes in OA remains obscure. Effect of 1,25-(OH)₂D₃ on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)₂D₃ activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)₂D₃-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)₂D₃ on MMP13 expression, transfection assays were used to show that 1,25-(OH)₂D₃ activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)₂D₃ in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)₂D₃ activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)₂D₃ to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.     

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G₁₇₇/V₁₇₈) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.     

Leptin plays a catabolic role on articular cartilage

Leptin has been shown to play a crucial role in the regulation of body weight. There is also evidence that this adipokine plays a key role in the process of osteoarthritis. However, the precise role of leptin on articular cartilage metabolism is not clear. We investigate the role of leptin on articular cartilage in vivo in this study. Recombinant rat leptin (100 μg) was injected into the knee joints of rats, 48 h later, messenger RNA (mRNA) expression and protein levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), cathepsin D, and collagen II from articular cartilage were analyzed by real-time quantitative polymerase chain reaction (PCR) and western blot. Two important aggrecanases ADAMTS-4 and -5 (a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5) were also analyzed by real-time quantitative PCR. Besides, articular cartilage was also assessed for proteoglycan/GAG content by Safranin O staining. Leptin significantly increased both gene and protein levels of MMP-2, MMP-9, cathepsin D, and collagen II, while decreased bFGF markedly in cartilage. Moreover, the gene expression of ADAMTS-4 and -5 were markedly increased, and histologically assessed depletion of proteoglycan in articular cartilage was observed after treatment with leptin. These results strongly suggest that leptin plays a catabolic role on cartilage metabolism and may be a disadvantage factor involve in the pathological process of OA.     

The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

Introduction  

The urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue.