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Abstract
To investigate the secretion profiles of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in synovial fluid-derived fibroblasts and to compare them with those of tissue-derived fibroblasts.
Fibroblast cultures established from synovial tissues (TSC) and fluids (FSC) of the same OA patients were stimulated with tumor necrosis factor(TNF)-alpha, interleukin(IL)-1alpha, IL-1beta, IL-6 and a combination of TNFalpha and IL-1beta. Cocultures of fibroblasts and cartilage were stimulated either with the cytokine combination or with osteoarthritic synovial fluid. Secretion of MMP-1, MMP-3, MMP-8, MMP-13, TIMP-1, and TIMP-2 was measured by enzyme-linked immunosorbent assay. Gelatin zymography and immunoblotting were performed to demonstrate enzyme activity.
TNFalpha, IL-1alpha, and IL-1beta led to marked increases in MMP-1 and MMP-3 release (up to 4.2-fold and 547-fold, respectively) by synovial fibroblasts, whereas secretion of MMP-13 was induced by concomitant administration of TNFalpha and IL-1beta. Expression of intracellular MMP-8 was stimulated by cytokines, but adhesion of synovial fibroblasts to cartilage was required for the release. Throughout the study, significantly higher levels of secreted MMPs were observed in stimulated FSC compared to TSC cultures. Furthermore, increases in MMP secretion were not accompanied by increases in secreted TIMP-1 and TIMP-2, resulting in marked imbalances between enzyme and inhibitor levels.
The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.
... Also, our results show that IL-6, MMP-1 and MMP-3 are produced constitutively by FD-FLS and TD-FLS and their synthesis is up-regulated by IL-1β and TNF-α. IL-1β and TNF-α are considered important players in OA because they stimulate the synthesis and secretion of many proteases and MMPs (7). IL-1β is related to the destruction of cartilage, and TNF-α is associated to induction of downstream inflammatory cascade. ...
... MMP-1 and MMP-3 are expressed by TD-FLS and production of these enzymes increases markedly when exposed to these pro-inflammatory cytokines (9). Previous studies demonstrated that both tissue and fluid synovial fibroblasts secrete metalloproteinases, which it is most likely to be important in the process of cartilage degradation (7,10). They found consistent secretion of MMP-1 and MMP-3 in all synovial fibroblast cultures with greater increased levels after cytokine stimulation (8). ...
... They found consistent secretion of MMP-1 and MMP-3 in all synovial fibroblast cultures with greater increased levels after cytokine stimulation (8). MMP-3 levels are found increased in OA synovial fluids (7) and the significant concentrations of MMP-3 released at baseline and after stimulation in our study and theirs indicate a synovia inflammation as a source of this protease (7). In contrast to our study, some differences were found in production of MMP-1 and -3 after stimulation with IL1-β and TNF-α, but with no difference at MMP-3 baseline levels (7). ...
Objectives:
The objective of the study was to compare the production of metalloproteinases (MMP)-1, -3 and interleukin (IL)-6 by fibroblast-like synoviocytes (FLS) derived from synovial fluid (FD-FLS), and FLS derived from synovial tissue (TD-FLS) of patients with primary osteoarthritis (OA). The more accessible FD-FLS could facilitate the study of the role of these cells in OA pathophysiology.
Methods:
MMP-1, MMP-3, and IL-6 levels were measured in the supernatant culture at baseline and 22 hours after stimulation with TNF-α and IL-1 β.
Results:
There was no difference at baseline between MMP-1, MMP-3 and IL-6 production by FD-FLS and TDFLS. Analogous to baseline, stimulation of FD-FLS and TD-FLS with IL-1β and TNF-α did not result in difference on MMP-3 and IL-6 production. However, TD-FLS produced more MMP-1 than FD-FLS after stimulation with IL-1β (p=0.01). Additionally, there was a positive correlation for production of MMP-1, MMP-3 and IL-6 between FD-FLS and TD-FLS (r=0.40 and p<0.0008; r=0.66 and p<0.0001; r=0.76 and p<0.0001, respectively). Supporting this statistical significant positive correlation, the Bland-Altman plotting, showed a homogeneous distribution of the values and low mean disagreement rates between all results of FD-FLS and TD-FLS (23.1%, 56.8% and 48.1%, respectively).
Conclusions:
Our data demonstrated functional similarity between FD-FLS and TD-FLS and support the use of a more accessible source of FLS for the study of the pathogenesis of joint destruction and therapeutic targets in primary OA.
... Also, our results show that IL-6, MMP-1 and MMP-3 are produced constitutively by FD-FLS and TD-FLS and their synthesis is up-regulated by IL-1β and TNF-α. IL-1β and TNF-α are considered important players in OA because they stimulate the synthesis and secretion of many proteases and MMPs (7). IL-1β is related to the destruction of cartilage, and TNF-α is associated to induction of downstream inflammatory cascade. ...
... MMP-1 and MMP-3 are expressed by TD-FLS and production of these enzymes increases markedly when exposed to these pro-inflammatory cytokines (9). Previous studies demonstrated that both tissue and fluid synovial fibroblasts secrete metalloproteinases, which it is most likely to be important in the process of cartilage degradation (7,10). They found consistent secretion of MMP-1 and MMP-3 in all synovial fibroblast cultures with greater increased levels after cytokine stimulation (8). ...
... They found consistent secretion of MMP-1 and MMP-3 in all synovial fibroblast cultures with greater increased levels after cytokine stimulation (8). MMP-3 levels are found increased in OA synovial fluids (7) and the significant concentrations of MMP-3 released at baseline and after stimulation in our study and theirs indicate a synovia inflammation as a source of this protease (7). In contrast to our study, some differences were found in production of MMP-1 and -3 after stimulation with IL1-β and TNF-α, but with no difference at MMP-3 baseline levels (7). ...
... Changes occurring in the joint synovium during OA generally involve an inflammatory process termed synovitis, including a synovial lining hyperplasia (Scanzello 2012) with an increase in the number of activated macrophage-like and fibroblast-like synoviocytes (MLS and FLS, respectively) (Fuchs 2004;Bian 2012). FLS are the most abundant cell type in synovial tissue, playing a key role by producing cytokines that perpetuate inflammation, and proteases that contribute to cartilage destruction (Bartok 2010). ...
... Moreover, we showed that FLS produced MMP-13 constitutively and after IL-1β stimulation. Fuchs et al. had demonstrated MMP-13 production only after IL-1β combined with TNFα in FLS (Fuchs 2004). Discrepancies among these results might be attributed to the IL-1 β exposure time. ...
Plasminogen activators are specific proteolytic enzymes implicated in a variety of basic biological processes. The expression of the urokinase plasminogen activator system components is increased in some human diseases, including osteoarthritis. We sought to study the effect of two components of the inflamed synovial microenvironment on this system, IL-1β and fibronectin fragments, elucidating whether corticotropin-releasing factor (CRF) and vasoactive intestinal peptide (VIP) neuropeptides modulate it, and analyzing the physiological consequences in joint destruction by measuring matrix metalloproteinases-9 and metalloproteinases-13 levels in osteoarthritis fibroblast-like synoviocytes. We showed that IL-1β and fibronectin fragments stimulated urokinase system contributing to the perpetuation of the destructive cascade in joint. VIP modulated, even at constitutive level, this system, also counteracting the effect of both inflammatory stimuli. However, CRF seemed to be ineffective in controlling the production of these proteinases. Moreover, VIP was able to reduce the constitutive expression of matrix metalloproteinase-13 and the levels of both matrix metalloproteinases after stimulation with the pro-inflammatory stimuli. Our results suggest that the presence of early and later inflammatory mediators, such as IL-1β and fibronectin fragments, increases the urokinase system and the matrix metalloproteinases levels. Whereas CRF did not affect this system, VIP counteracts these actions supporting its therapeutic potential for the treatment of osteoarthritis.
... In addition, 3–4% of children with osteosarcoma carry a mutation in p53 (Mcintyre et al. 1994). Several other conditions have been associated with osteosarcoma such as Paget´s disease, chronic osteomyelitis, osteochondroma, enchondroma, fibrous dysplasia (Skubitz & D'Adamo 2007), Li-Fraumeni syndrome, Rothmund-Thomsom syndrome, RAPADILINO syndrome, and Werner syndrome (Fuchs et al. 2004). In addition, tumour site, size, grade, and patient age are demonstrated to be risk factors in osteosarcoma (Davis et al. 1994), as well as, altered levels of alkaline phosphatase (Bramer et al. 2005), telomerase (Ulaner et al. 2003), and P-glycoprotein (Pakos & Ioannidis 2003). ...
... The high molecular form of MMP-8 (>100kDa) is most likely a complex with its endogenous inhibitors such as α 2 -macroglobulin, TIMPs or a result of dimerisation (Ingman et al. 1996, Chen et al. 1998) and the low molecular form of MMP-8 (<30kDa) is probably a degradation fragment (Apajalahti et al. 2003). Latent MMP-8 can be activated in response to reactive oxygen species (Saari et al. 1990), human trypsinogen-2 (Moilanen et al. 2003), MT1-MMP (Holopainen et al. 2003), MMP-3 (Knauper et al. 1993), several cytokines (Fuchs et al. 2004), and bacterial proteases (Sorsa et al. 1992). Non-PMN leukocyte-type MMP-8 has been detected from rheumatoid 37 synovial fibroblasts and endothelial cells (Hanemaaijer et al. 1997), gingival sulcular epithelial cells (Tervahartiala et al. 2000), oral carcinoma cells (Moilanen et al. 2002), plasma cells (Wahlgren et al. 2001), odontoblasts (Palosaari et al. 2000), melanoma cells (Giambernardi et al. 1998), leukemia cells (Kim et al. 2001), chondrocytes in rheumatoid arthritic and osteoarthritic lesions (Chubinskaya et al. 1999), migratory and proliferating keratinocytes, fibroblasts and inflammatory cells of healing wounds (Pirilä et al. 2001), human atheroma cells (Herman et al. 2001), breast cancer cells (Agarwal et al. 2003), and bronchial epithelial cells (Prikk et al. 2001). ...
... Similarly, elevated levels of MMP1 are often observed in OA, and are recognized to contribute to cartilage degradation. MMP-1 is produced by various cells, including synovial fibroblasts and articular chondrocytes [53], and its activation is considered a key event in the pathological process leading to cartilage breakdown [54]. Altogether, our findings show how each patient-specific model displays a different behavior in terms of cytokine modulation and degradative enzyme production, depending on the patient-specific combination of articular cells and synovial fluid, since the same MSCs were injected in all patients. ...
Osteoarthritis (OA) is a highly disabling pathology, characterized by synovial inflammation and cartilage degeneration. Orthobiologics have shown promising results in OA treatment thanks to their ability to influence articular cells and modulate the inflammatory OA environment. Considering their complex mechanism of action, the development of reliable and relevant joint models appears as crucial to select the best orthobiologics for each patient.
The aim of this study was to establish a microfluidic OA model to test therapies in a personalized human setting. The joint-on-a-chip model included cartilage and synovial compartments, containing hydrogel-embedded chondrocytes and synovial fibroblasts, separated by a channel for synovial fluid. For the cartilage compartment, a Hyaluronic Acid-based matrix was selected to preserve chondrocyte phenotype. Adding OA synovial fluid induced the production of inflammatory cytokines and degradative enzymes, generating an OA microenvironment. Personalized models were generated using patient-matched cells and synovial fluid to test the efficacy of mesenchymal stem cells on OA signatures. The patient-specific models allowed monitoring changes induced by cell injection, highlighting different individual responses to the treatment. Altogether, these results support the use of this joint-on-a-chip model as a prognostic tool to screen the patient-specific efficacy of orthobiologics.
... For instance, Type B broblast-like synovial cells (FLS) are not only the main cell components of synovium but also are crucial in cartilage homeostasis. In the in ammatory state, the FLS of OA patients can secrete a variety of proteolytic enzymes (Fuchs S et al., 2004) that cause cartilage damage and increase the level of nerve growth factor (NGF) (Takano S et al.,2016), thereby promoting the production of various in ammatory mediators that exacerbate the patient's pain. Furthermore, in ltrating immune cells in synovitis (Rosshirt N et al.,2019) will in uence OA progress. ...
Osteoarthritis (OA) is a common degenerative joint disease characterized by articular cartilage destruction and synovial damage. The immune system has an impact on the development of OA. The infiltration of immune cells will cause the autoimmune reaction of osteoarthritis and disrupt the immune equilibrium in bone tissue. Recent studies have demonstrated that copper is crucial in regulating the immune system. Copper can also mediate cell death through a new cell death program by targeting the tricarboxylic acid cycle (TCA) circulating protein. However, no research has been conducted on the copper death gene regulation of OA immune response.The gene expression profiles—GSE55235, GSE82107, and GSE206848—were obtained from the GEO database. A total of 24 healthy and 27 OA joint synovial samples were screened for differentially expressed genes (DEGs) in combination with copper death-related genes and immune-related genes to conduct subsequent analyses.We have selected seven genes—NAMPT, EGFR, ADM, APOD, CD28, CXCL12, and MMP9—as potentially essential marker genes to regulate copper death. These marker genes are enriched in autophagy, lysosome, apoptosis, immune response, polysaccharide synthesis and metabolism, and signaling pathways of related diseases. The proportion of NKT cells in OA samples was significantly higher than that in the control group, whereas neutrophil expression was significantly lower than that in the healthy group. Finally, RT-PCR confirmed that the expressions of NAMPT and EGFR in the disease group were lower than those in the control group.We developed and validated the immune copper death DEGs model, which can accurately diagnose and characterize the biological changes of OA. Seven marker genes may also be potential targets of immunomodulatory therapy, including NAMPT and EGFR, which play an essential role in bone and joint, thereby providing prediction and theoretical support for the regulatory mechanism of copper death in osteoarthritis.
... In addition, MMP-3 accelerates cartilage destruction by activating other MMP members via a cascade-amplifying effect. The longer the course of OA, the more MMPs were present in the synovial membrane [31]. ...
Osteoarthritis (OA) is a chronic degenerative disease that primarily includes articular cartilage destruction and inflammatory reactions, and effective treatments for this disease are still lacking. The present study aimed to explore the protective effects of ectoine, a compatible solute found in nature, on chondrocytes in rats and its possible application in OA treatment. In the in vitro studies, the morphology of the chondrocytes after trypsin digestion for 2 min and the viability of the chondrocytes at 50°C were observed after ectoine treatment. The reactive oxygen species (ROS) levels in chondrocytes pretreated with ectoine and post-stimulated with H 2 O 2 were detected using an ROS assay. Chondrocytes were pretreated with ectoine before IL-1β stimulation. RT‒qPCR was used to measure the mRNA levels of cyclooxygenase-2 (COX-2), metallomatrix proteinase-3, -9 (MMP-3, -9), and collagen type II alpha 1 (Col2A1). In addition, immunofluorescence was used to assess the expression of type II collagen. The in vivo effect of ectoine was evaluated in a rat OA model induced by the modified Hulth method. The findings revealed that ectoine significantly increased the trypsin tolerance of chondrocytes, maintained the viability of the chondrocytes at 50°C, and improved their resistance to oxidation. Compared with IL-1β treatment alone, ectoine pretreatment significantly reduced COX-2, MMP-3, and MMP-9 expression and maintained type II collagen synthesis in chondrocytes. In vivo , the cartilage of ectoine-treated rats exhibited less degeneration and lower Osteoarthritis Research Society International (OARSI) scores. The results of this study suggest that ectoine exerts protective effects on chondrocytes and cartilage and can, therefore, be used as a potential therapeutic agent in the treatment of OA.
... Studies have shown that genetic markers associated with apoptosis and senescence in the synovial tissue of OA promote OA progression [13,14]. Scientists have found that OA synoviocytes produce some in ammatory regulators and matrix-degrading enzymes that promote the progression of OA [15,16]. Dysregulation of lncRNAs and mRNA expression in synovial tissues is often associated with the pathogenesis of OA [17]. ...
Background
Osteoarthritis (OA) is a multifactorial disease that places an increasing burden on modern society. Synovial inflammation plays an important role in osteoarthritis. Therefore, new approaches or techniques for the diagnosis of OA are urgently needed. 7-methylguanosine (m7G) is one of the most common forms of base modification in post-transcriptional regulation. Studies have reported that m7G is involved in the development of different diseases. However, the function of m7G RNA methylation regulators in synovial tissue remains unclear, and the expression and predictive value of m7G RNA methylation regulators are rarely reported. So new studies are needed to fill this gap.
Methods
Firstly, 40 m7G RNA methylation-related genes were selected from the four pathways of the Gene Set Enrichment Analysis (GSEA) database and compared with the GSE55457 dataset in the Gene Expression Omnibus (GEO) database to obtain gene expression profiles and clinical information. And 25 related genes were obtained. These genes were analyzed for functional enrichment, protein-protein interactions (PPI), and correlation among regulators. Six differential genes were also selected after the differential analysis of the 25 m7G RNA methylation-related regulators in OA and normal patients. The expression of differential genes in OA patients and the correlation between regulators were analyzed. Finally, the receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to evaluate the diagnostic efficacy of m7G related genes in distinguishing OA patients from healthy population.
Results
We found that these genes are mainly involved in RNA metabolism, RNA cap binding, and the formation of RNA cap-binding complexes, and that the regulation of RNA cap formation has a strong impact on gene regulation. Then we identified 6 differentially expressed genes (DEGs) in 25 genes between OA and normal people, confirming for the first time that SNUPN and NUDT11 were associated with early diagnosis and prediction of OA.
Conclusions
The regulators of m7G RNA methylation play a crucial role in the advancement of OA and hold promise in terms of prognosis. However, the clinical effects of these biomarkers on OA need further study.
... Synovial inflammation exists in all stages of OA, even in the early stage [10]. Inflammatory regulators and matrix degradation enzymes produced by OA synoviocytes contribute to the progression of OA [11,12]. For example, the production of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and chemokines causes synovial histological changes, and recruits mononuclear cells (such as monocytes and macrophages) and lymphocytes (activated T cells and B cells) [13,14]. ...
Background: Osteoarthritis (OA) is a degenerative arthritis with high levels of clinical heterogeneity. Aberrant metabolism such as shifting from oxidative phosphorylation to glycolysis is a response to changes in the in-flammatory microenvironment of OA. Therefore, there is a pressing need to identify novel glycolysis regulators during OA progression. Methods: We systematically studied glycolysis patterns mediated by 141 glycolysis regulators in 74 human sy-novial samples and discussed the characteristics of the immune microenvironment modified by glycolysis. The random forest (RF) method was applied to screen candidate hub glycolysis regulators in OA. RT-qPCR was performed to validate these key regulators. Then distinct glycolysis patterns were identified, and systematic correlation between these glycolysis patterns and immune cell infiltration was analyzed. The glycolysis score was constructed to quantify glycolysis patterns together with immune infiltration of individual OA patient. Results: 56 glycolysis-related differentially expressed genes (DEGs) were identified between OA and non-OA samples. STC1, VEGFA, KDELR3, DDIT4 and PGAM1 were selected as candidate genes to predict the probability of OA. Two glycolysis patterns in OA were identified. Glycolysis cluster A with higher glycolysis score was related to an inflamed phenotype. Conclusions: Taken together, our results established a glycolysis-based genetic signature for OA, guided in-depth studies on the metabolic mechanism of OA, and facilitated to explore new clinical treatment strategies.
... Synovial inflammation has been found to be present in the OA joints and associated with radiographic and pain progression [5,42]. Scientists discovered that OA synoviocytes would produce some inflammatory regulators and matrix degradation enzymes contributing to the progression of OA [8,9]. Additionally resrearchers have also discovered cuproptosis and senescence related gene signature in OA synovial tissue have contributed to OA progression [42,43]. ...
Background:
Accumulating evidence has shown that aberrant N7-methylguanosine (m7G) RNA methylation played an important role in the occurrence and development of cancer. However, knowledge of m7G modifications in inflammatory diseases is limited. Osteoarthritis (OA) is the most common arthritic disease with poor prognosis. Our research aimed to identify m7G-related hub biomarkers and investigate m7G regulator expression pattern in immune landscape of OA patients.
Methods:
Gene expression profiles and their clinical information were obtained from the Gene Expression Omnibus (GEO) database, and differential analysis of 14 m7G-related regulators between elective OA and normal samples was performed. M7G-related hub genes for OA were mined based on single-sample gene set enrichment analysis (ssGSEA) and the random forest (RF) algorithm, and qRT-PCR was performed to confirm the abnormal expression of hub genes. Enrichment, protein-protein interaction (PPI), transcription factor (TF)-gene interaction and microRNA (miRNA)-gene coregulatory analysis based on m7G hub genes were performed. Then we predicted several candidate drugs related to m7G hub genes using DSigDB database. Moreover, we comprehensively evaluated m7G methylation patterns in OA samples and systematically correlated these modification patterns with the characteristics of immune cell infiltration. The m7G score was generated to quantify m7G methylation patterns for individual OA patients by the application of principal component analysis (PCA) algorithm.
Results:
We constructed an OA predictive model based on 4 m7G hub genes (SNUPN, METTL1, EIF4E2 and CYFIP1). Two m7G methylation patterns in OA were discovered to show distinct biological characteristics, and an m7G score were generated. M7G cluster A and a higher m7G score were found to be related to an inflamed phenotype.
Conclusions:
Our study was the first to comprehensively investigate the m7G methylation dysregulations in immune landscape during the progression of OA. These 4 m7G gene-related signatures can be used as novel OA biomarkers to predict the occurrence of OA. Evaluating the m7G methylation patterns of OA individuals will contribute to enhancing our cognition of immune infiltration characterization and guiding more effective immunotherapy strategies.
... 25,30 TNF-α together with IL-1 stimulate synovial fibroblasts to release MMP-1, 3, and 13, but also simulate the expression of intracellular MMP-8. 31 In OA, especially advanced stages, MMP-9 was found to be produced by synovial fibroblasts. 32 Overall, the effects of TNFα are synergistic to IL-1β, as both initiate similar inflammatory pathways in OA. 24 In the present study, we found that TNF-α and IL-1 expressions were significantly higher in patients diagnosed with cartilage lesions. ...
Objective:
The aim of this study was to evaluate if a similar catabolic and inflammatory gene pattern exists between the synovium, hyaline cartilage, and blood of patients with the knee joint tissues and if one precedes the other.
Design:
A total of fifty-eight patients (34 females and 24 males) with a mean age of 44.7 years (range, 18-75) underwent elective knee arthroscopy due to previously diagnosed pathology. Full blood samples were collected preoperatively from synovium and cartilage samples intraoperatively. Real time PCR with spectrophotometric analysis was performed. Following genes taking part in ECM (extracellular matrix) remodeling were selected for analysis: MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, MMP-14, ADAMTS-4 (Agg1) and ADAMTS-5 (Agg2) proteases, TIMP-1, and TIMP-2 - their inhibitors - and IL-1 and TNF-α cytokines.
Results:
Analysis revealed a strong and significant correlation between gene expression in synovial and systemic blood cells (p <0.05 for all studied genes) with ADAMTS-4, ADAMTS-5, IL-1, TNF-α and TIMP-2 expression most positively correlated with an R>0.8 for each. An analysis between chondrocytes and systemic blood gene expression shown no significant correlation for all genes. Bivariate correlation of International Cartilage Repair Society grading and genes expression revealed significant associations with synovial MMP-1, MMP-2, MMP-8, MMP-9, IL-1, TNF-α and TIMP-2.
Conclusion:
We suggest that the synovial tissue is the first responder for knee joint stress factors in correlation with the response of blood cells. The chondrocyte's genetic response must be further investigated to elucidate the genetic program of synovial joints, as an organ, during OA development and progression.
... Synovial tissue in ammation, which is known to contribute to OA development, exists in all stages of OA, even in the early stages [8]. In ammatory regulators and matrix degradation enzymes produced by OA synoviocytes contribute to the progression of OA [9,10]. For example, the production of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and chemokines causes synovial histological changes in the OA synovium, accompanied by in ltrated mononuclear cells (such as monocytes and macrophages) and lymphocytes (activated T cells and B cells) [11,12]. ...
Background:Osteoarthritis (OA) is a degenerative joint disease characterized by low-grade inflammation and high levels of clinical heterogeneity. Aberrant metabolism such as shifting from oxidative phosphorylation to glycolysis is a response to changes in the inflammatory micro-environment and may play a key role in cartilage degeneration and OA progression. Therefore, there is a pressing need to identify glycolysis regulators in the diagnosis of OA, determination of individualized risk, discovery of therapeutic targets, and improve understanding of pathogenesis.
Methods: We systematically studied glycolysis patterns mediated by 141 glycolysis regulators in 74 samples and discussed the characteristics of the immune microenvironment modified by glycolysis. The random forest was applied to screen candidate glycolysis regulators to predict the occurrence of OA. RT-qPCR was performed to validate these glycolysis regulators. Then two distinct glycolysis patterns were identified and systematic correlation between these glycolysis patterns and immune cell infiltration was analyzed. The glycolysis score was constructed to quantify glycolysis patterns together with immune infiltration of individual OA patient.
Results: 56 differentially expressed genes (DEGs) of glycolysis were identified between OA and normal samples. STC1, VEGFA, KDELR3, DDIT4 and PGAM1 were selected as candidate genes to predict the risk of OA using the random forest (RF) method. Two glycolysis patterns in OA were identified and glycolysis scoring system was constructed to show distinct individual immune characteristics. Glycolysis cluster A and higher glycolysis score was revealed to be related to an inflamed phenotype.
Conclusions: Taken together, these results established a genetic signature for OA based on glycolysis, which has reference significance for the in-depth study of the metabolic mechanism of OA and the exploration of new clinical treatment strategies.
... The NF-κB signaling pathway promotes the transcription of genes encoding cytokines like TNF-α and IL-1β and then induces the production of IL-6, IL-8, and cyclooxygenase-2 (COX-2). After that, the NF-κB signaling pathway stimulates the production of matrix metalloproteinases (MMPs) in synovial cells, monocytes, or chondrocytes, and ultimately mediates critical events in in ammatory response [31]. In addition to regulating the expression of TNF-α and IL-1β, NF-κB is activated by these cytokines as well. ...
Background. The aim of this study was to explore the relationship between coagulation/fibrinolysis function and serum lipid levels in people with advanced knee osteoarthritis (KOA).
Methods. From January 1st 2013 to January 1st 2018, a total of 206 patients underwent total or unicompartmental knee arthroplasty in Changzhou No.1 People’s Hospital were enrolled consecutively in this study. Baseline characteristics and preoperative laboratory indicators of patients were collected. The association between coagulation/fibrinolysis function and serum lipid levels was analyzed using Spearman correlation analysis and multiple linear regression.
Results. Spearman correlation analysis revealed that coagulation/fibrinolysis function was associated with serum lipid levels, especially prothrombin time was moderately negatively correlated with Apolipoprotein-A1 (r=-0.4585, P<0.05). The multiple linear regression analyses were performed with serum lipid levels as the independent variables and coagulation/fibrinolysis function parameters as the dependent variables after adjusting for sex, age, body mass index, hypertension, diabetes mellitus and coronary heart disease. We found that the regression analysis was reliable (its imitation degree was ideal, adjusted R^2 = 0.3108) and predictive (P<0.0001) when the dependent variable was prothrombin time. The significant contributors to prothrombin time were triglyceride (P<0.001), high density lipoprotein cholesterol (P=0.003), Apolipoprotein-A1 (P=0.017) and Lipoprotein (a) (P=0.015).
Conclusions. The results of this study showed that coagulation/fibrinolysis function was correlated with serum lipid levels in patients with advanced KOA, although the correlation may be small. It may be hypothesized that strategies for lipid control may help improve coagulation/fibrinolytic dysfunction in patients with KOA.
Trial registration. This clinical study was retrospectively registered in a publicly available registry because this study was a retrospective study began in 2018. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University in Jiangsu Province of China (No.CZYY2021187).
... Synovial fibroblasts produce and secrete proteins into the synovial fluid for joint lubrication and impact on the joint integrity. Also, inflammation of the synovial membrane is observed in more than half of the patients with OA (26) and is known to contribute to cartilage destruction by secreting cartilage catabolic factors, such as MMP1/3/13 (27). Thus, we examined the possibility that reductions in ERAD function in synovial fibroblasts may also trigger cartilage destruction. ...
Chondrocytes secrete massive extracellular matrix (ECM) molecules that are produced, folded, and modified in the endoplasmic reticulum (ER). Thus, the ER-associated degradation (ERAD) complex—which removes misfolded and unfolded proteins to maintain proteostasis in the ER— plays an indispensable role in building and maintaining cartilage. Here, we examined the necessity of the ERAD complex in chondrocytes for cartilage formation and maintenance. We show that ERAD gene expression is exponentially increased during chondrogenesis, and disruption of ERAD function causes severe chondrodysplasia in developing embryos and loss of adult articular cartilage. ERAD complex malfunction also causes abnormal accumulation of cartilage ECM molecules and subsequent chondrodysplasia. ERAD gene expression is decreased in damaged cartilage from patients with osteoarthritis (OA), and disruption of ERAD function in articular cartilage leads to cartilage destruction in a mouse OA model.
... Interestingly, we identify a similar pattern in infected fibroblasts with upregulation and/or secretion of IL-6 and RANTES, but not matrixmetalloproteases (MMPs), as described before [4]. MMP expression and secretion by synovial fibroblasts can, similar to IL-6 secretion, be induced through external stimulation with IL-1 and TNF and by activated immune cells [54]. A paracrine stimulation of MMP expression by infiltrating immune cells has not been addressed in this model, but is likely to contribute to the direct induction of rheumatoid arthritis-like symptoms. ...
... Interestingly, we identify a similar pattern in infected fibroblasts with upregulation and/or secretion of IL-6 and RANTES, but not matrix-metalloproteases (MMPs), as described before [4]. MMP expression and secretion by synovial fibroblasts can, similar to IL-6 secretion, be induced through external stimulation with IL-1 and TNF and by activated immune cells [54]. A paracrine stimulation of MMP expression by infiltrating immune cells has not been addressed in this model, but is likely to contribute to the direct induction of rheumatoid arthritis-like symptoms. ...
Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analyzed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.
... Transcription(by cytokines) [6,7,8,9,10,11] It is performed by cytokines. Cytokines are the chemical messengers that affect the surrounding or the distant cells by up-regulating or down-regulating the protein synthesis by these cells. ...
Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn(++)-endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. This review article discusses in detail the types of MMPS, its biological actions and role in periodontal disease. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed.
... Additionally, MMP production is strongly dependent on macrophage NF-kB activity, even when it is produced by fibroblasts [42,43]. Fibroblasts produce MMPs after being stimulated by cytokines, namely TNFa and IL-1 [44]. In line with these observations, in our study, we showed that in advanced OA, fibroblasts produce a higher percentage of total MMP-9 compared to early OA, but still less than the macrophages. ...
Synovitis of the knee synovium is proven to be a precursor of knee osteoarthritis (OA), leading to a radiologically advanced stage of the disease. This study was conducted to elucidate the expression pattern of different inflammatory factors—NF-kB, iNOS, and MMP-9 in a subpopulation of synovial cells. Thirty synovial membrane intra-operative biopsies of patients (ten controls, ten with early OA, and ten with advanced OA, according to the Kellgren–Lawrence radiological score) were immunohistochemically stained for NF-kB, iNOS, and MMP9, and for different cell markers for macrophages, fibroblasts, leukocytes, lymphocytes, blood vessel endothelial cells, and blood vessel smooth muscle cells. The total number of CD68+/NF-kB+ cells/mm2 in the intima of early OA patients (median = 2359) was significantly higher compared to the total number of vimentin+/Nf-kB+ cells/mm2 (median = 1321) and LCA+/NF-kB+ cells/mm2 (median = 64) (p < 0.001 and p < 0.0001, respectively). The total number of LCA+/NF-kB+ cells/mm2 in the subintima of advanced OA patients (median = 2123) was significantly higher compared to the total number of vimentin+/NF-kB+ cells/mm2 (median = 14) and CD68+/NF-kB+ cells/mm2 (median = 29) (p < 0.0001). The total number of CD68+/iNOS+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/iNOS+ cells/mm2 and LCA+/iNOS+ cells/mm2 (p < 0.0001 and p < 0.001, respectively). The total number of CD68+/MMP-9+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/MMP-9+ cells/mm2 and CD5+/MMP-9+ cells/mm2 (p < 0.0001). Macrophages may have a leading role in OA progression through the NF-kB production of inflammatory factors (iNOS and MMP-9) in the intima, except in advanced OA, where leukocytes could have a dominant role through NF-kB production in subintima. The blocking of macrophageal and leukocyte NF-kB expression is a possible therapeutic target as a disease modifying drug.
... Studies of the knee OA have shown correlation of synovial inflammation and progression of disease [29] and correlation of synovial inflammation and sensation of pain expressed in a visual-analogue scale as well [30]. Macrophages and FLS from the synovium also secrete MMPs [31]. Given the important proinflammatory role of the synovial macrophages, FLS and T cells [32] in the pathogenesis of OA, in this paper we decided to investigate their co-localization with BCL-2, MMP-9 and iNOS in order to achieve a better insight into the inflammatory changes of the synovial membrane in HOA. ...
Hip osteoarthritis (HOA) is characterized by degradation of the cartilage and synovitis. However, the pathohistological effects of synovial tissue inflammation on HOA are not clear. The aim of this study was to evaluate the expression of iNOS, BCL-2 and MMP-9 markers in different synovial cell populations. A total of 32 patients were evaluated retrospectively. Age, sex, height, weight, body mass index were recorded and lymphocyte, fibrocytes and macrophages were analysed in tissue sections. Osteoarthritis cartilage histopathology assessment system (OARSI), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Krenn score, Harris Hip Score (HHS) and Kellgren–Lawrence (K-L) grading of the hip joints were performed. Total hip arthroplasty was performed on 32 patients and controls. Patients were divided into two groups according to their disease severity. The tissues were immunohistochemically analysed. K-L grade and Krenn score differ between all three groups, but also between moderate and severe OA. Synovial lining cell layer, resident cells in stroma and especially inflammatory infiltration were increasing with severity of OA. iNOS expression in both intima and subintima was positively correlated with Krenn score in moderate and severe osteoarthritis (OA) groups. Expression of BCL-2 in intima of severe OA patients was positively correlated with Krenn score. In conclusion, iNOS, BCL-2 and MMP-9 are involved in the regulation of HOA. Our study indicates a relationship between the pathohistological features, the synovial inflammation and the cartilage condition at the time of hip replacement due to OA or femoral neck fracture.
... 12 Furthermore, synovial inflammation facilitates the production of pro-inflammatory and pain neurotransmitters such as nerve growth factor and bradykinin which are potential therapeutic targets for OA treatment. 13 Inflammatory regulators and matrix degradation enzymes produced by OA synoviocytes contribute to the progression of OA. 6,14 It has been reported that synovial inflammation plays an initiator role in OA by releasing pro-inflammatory mediators and cartilage destructive factors that induce cartilage damage, which in turn magnifies the synovial inflammation, forming a vicious cycle. 15 Fibroblast-like synoviocytes (FLS) constitute the predominant cellular component of the joint synovium. ...
Osteoarthritis (OA), the most ubiquitous degenerative disease affecting the entire joint, is characterized by cartilage degradation and synovial inflammation. Although the pathogenesis of OA remains poorly understood, synovial inflammation is known to play an important role in OA development. However, studies on OA pathophysiology have focused more on cartilage degeneration and osteophytes, rather than on the inflamed and thickened synovium. Fibroblast‐like synoviocytes (FLS) produce a series of pro‐inflammatory regulators, such as inflammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2). These regulators are positively associated with the clinical symptoms of OA, such as inflammatory pain, joint swelling and disease development. A better understanding of the inflammatory immune response in OA‐FLS could provide a novel approach to comprehensive treatment strategies for OA. Here, we have summarized recently published literatures referring to epigenetic modifications, activated signalling pathways and inflammation‐associated factors that are involved in OA‐FLS‐mediated inflammation. In addition, the current related clinical trials and future perspectives were also summarized.
... Several pathways are involved in the pathogenesis of OA. Mechanical damage can cause a localized inflammatory response of the joint, marked by increased of pro-inflammatory mediators such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nitrite oxide (NO) and prostaglandin E2 (PGE2) in the joint space [46][47][48][49][50]. This inflammatory response further exaggerates cartilage tissue damage via oxidative stress and damage, thus forming a vicious self-destructive cycle. ...
Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2–2 mg mL−1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 μg i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.
... Increased levels of MMP-13 have been detected in cartilage from OA patients, where it is associated with a greater destruction [104][105][106], as well as in SF from RA patients, correlating with uPA levels [107]. MMP-13 is also present in SF and synovial fluid from OA and RA patients [108][109][110][111], where cytokines such as IL-1β and other inflammatory mediators are involved in the induction of its expression [80,92]. ...
The extracellular matrix (ECM) is a complex and specialized three-dimensional macromolecular network, present in nearly all tissues, that also interacts with cell surface receptors on joint resident cells. Changes in the composition and physical properties of the ECM lead to the development of many diseases, including osteoarthritis (OA). OA is a chronic degenerative rheumatic disease characterized by a progressive loss of synovial joint function as a consequence of the degradation of articular cartilage, also associated with alterations in the synovial membrane and subchondral bone. During OA, ECM-degrading enzymes, including urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), cleave ECM components, such as fibronectin (Fn), generating fibronectin fragments (Fn-fs) with catabolic properties. In turn, Fn-fs promote activation of these proteinases, establishing a degradative and inflammatory feedback loop. Thus, the aim of this review is to update the contribution of ECM-degrading proteinases to the physiopathology of OA as well as their modulation by Fn-fs.
... Ohara et al. reported that one of the reasons for flexor tendon rupture with DRUJ OA after Galeazzi fracture-dislocation was capsule perforation [14]. In the past reports, it has been reported that matrix metalloproteinase-(MMP-) 1, MMP-3, MMP-8, and MMP-13 are expressed in synovial fibroblasts of OA [14,[19][20][21]. MMP-1, MMP-8, and MMP-13 also reportedly degrade type I collagen that is abundant in tendons [13,22]. ...
Spontaneous flexor tendon rupture is rare, occurring most commonly in the little finger or flexor pollicis longus. To the best of our knowledge, there have been no reports of spontaneous flexor tendon rupture due to primary distal radioulnar joint (DRUJ) osteoarthritis (OA). We present a case of spontaneous flexor tendon rupture in the index finger due to primary DRUJ OA in a 71-year-old female farmer. Surgical exploration confirmed that, at the wrist joint level, the flexor digitorum profundus of the index finger had undergone degeneration and complete rupture. The flexor digitorum superficialis of the index finger was elongated and thinned. A bony spur toward the volar side was covered with synovial fluid from a pinhole-sized perforation of the capsule. The combination of direct friction from the DRUJ spur and the matrix metalloproteinases in the synovial fluid from the perforation of the DRUJ capsule may have caused the spontaneous flexor tendon rupture. Palmar-side symptoms associated with DRUJ OA should be carefully examined because of the risk of spontaneous flexor tendon rupture.
... MMPs are involved in the etiology of many diseases and medical conditions, such as metastasis, rheumatoid arthritis, osteoarthritis, periodontal diseases, inflammatory diseases, atherosclerosis, diabetes, Parkinson's disease pathology, and skin aging. During aging, increased MMP-1, MMP-8, and MMP-13 from the collagenase family break down collagen and elastin, leading to degenerative changes of the extracellular matrix, and accelerate wrinkle formation, and sagging and aging of the skin (Bailey, 2001;Gosline et al., 2002;Heim et al., 2006;Nema et al., 2013) MMP-1 and MMP-8 are also known to cause osteoarthritis (Fuchs et al., 2004). The physiological activity process of MMPs is controlled by endogenous tissue inhibitor metalloproteinases (TIMPs) (Öncel, 2012). ...
Phytomics or metabolomics is analysis of large-scale primary and secondary metabolites of plant extracts and provides very meaningful data to monitor or evaluate cellular function or systems biology. The activity of plant extracts depends on the synergistic/antagonistic effect of different metabolites rather than single active metabolites. Matrix metalloproteinases (MMPs) have an active role in the formation of many diseases. To our knowledge, there is no study on the correlation between the phytomics and MMP inhibitory activity of Achillea millefolium, Achillea filipendulina (Asteraceae), Mentha piperita, and Salvia officinalis (Lamiaceae), (AAMS). Therefore, this study aimed to correlate the metabolomics profiling of AAMS extracts to identify the metabolites responsible for the MMP inhibitory activity based on phytomics data. The AAMS extracts showed a significant MMP inhibitory effect (57.73–92.73%) at different concentrations (25–500 μg/mL). In order to identify the metabolites responsible for such activities in the extract, the metabolomic profiling of the plants was investigated using gas chromatography-mass spectrometry (GC-MS). After deconvolution and aligning of the chromatograms, 284 metabolites were detected, of which 149 were annotated using retention index libraries. Multivariate analyses results indicated that A. millefolium and A. filipendulina showed similar metabolomic profiles, while M. piperita and S. officinalis differed both from each other and from Achillea species. The correlation analysis was applied to evaluate the correlation between metabolomic levels and MMP inhibitory activities, and 96 metabolites had a negative correlation (r ≤ −0.70) and 55 had a highly positive correlation (r ≥ 0.70) with MMP inhibitory activity. This is the first study which revealed that phytomics, plant metabolomics, can be used for activity evaluation and a single metabolite may not be responsible for a specific activity. In conclusion, phytomics can be a more useful tool for the evaluation of the activities than investigating a single metabolite. This new perspective can also provide a better understanding of plant metabolomics and can be easily employed for future research on plant activity.
... OA development depends on a balance of extracellular matrix (ECM) production and degradation. Proteolytic enzymes such as ADAMTS as well as matrix metalloproteinases (MMPs) are found to be up-regulated in OA joints (1)(2)(3)(4)(5) and are directly involved in OA pathophysiology (6,7). Therefore, inhibition of these metalloproteinases is a potential route for the design of pharmaceutical agents to retard, suppress, and even halt the progression of OA. ...
Objective
Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or load‐induced etiology. In addition, we do not know the long‐term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP‐3), called [‐1A]TIMP‐3, on spontaneous OA development and bone formation in STR/Ort mice.
Methods
Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [‐1A]TIMP‐3, either ubiquitously or conditionally in chondrocytes. [‐1A]TIMP‐3 has an extra alanine at the N‐terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA‐related measures in all mice at age 40 weeks.
Results
Mice expressing high levels of [‐1A]TIMP‐3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [‐1A]TIMP‐3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [‐1A]TIMP‐3 in vitro showed elevated rates of mineralization.
Conclusion
The results provide evidence that [‐1A]TIMP‐3–mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.
... Knee OA, the most common form of arthritis, is characterized as sustained synovial inflammation and progressive cartilage degradation, in which OA fibroblast-like synoviocytes (OA-FLS) play a pivotal role [22,23]. OA-FLS promotes those pathological changes of synovium and cartilage through the secretion of inflammatory or catabolic factors, including TNF-α, interleukin (IL)-1β, and matrix degrading metalloproteinases (MMPs) [24][25][26]. ...
Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal and non-neuronal cells. Recently, emerging evidences suggested the crucial role of TRPA1 in the disease progression of osteoarthritis (OA). Therefore, we aimed to investigate whether TRPA1 mediate lipopolysaccharide (LPS)-induced inflammatory responses in primary human OA fibroblast-like synoviocytes (OA-FLS). The expression of TRPA1 in LPS-treated OA-FLS was assessed by polymerase chain reaction (PCR) and western blot (WB), and the functionality of TRPA1 channel by Ca²⁺ influx measurements. Meanwhile, production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, matrix metalloproteinase (MMP)-1, and MMP-3 in LPS-treated cells was measured by immunoassay. Histological observation after inhibition of TRPA1 was also performed in rats with LPS-induced inflammatory arthritis. After being induced by LPS, the gene and protein expression of TRPA1 was increased in the time-dependent or dose-dependent manner. Meanwhile, Ca²⁺ influx mediated by TRPA1 in human OA-FLS was also enhanced. In addition, pharmacological inhibition and gene silencing of TRPA1 downregulated the production of IL-1β, TNF-α, IL-6, MMP-1, and MMP-3 in LPS-treated FLS. Finally, synovial inflammation and cartilage degeneration were also reduced by the TRPA1 antagonist. We found the LPS caused the increased functional expression of TRPA1, the activation of which involved in LPS-reduced inflammatory responses in primary human OA-FLS, and the inhibition of TRPA1 produces protective effect in LPS-induced arthritis.
... Numerous studies have reported the involvement of inflammation in the progression of OA. Mechanical damage can cause a localized inflammatory response of the joint, marked by increased pro-inflammatory mediators, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), nitrite oxide (NO) and prostaglandin E 2 (PGE 2 ) in the joint space [11][12][13][14][15]. This inflammatory response further exaggerates cartilage tissue damage via oxidative stress and damage, thus forming a vicious self-destructive cycle. ...
Osteoarthritis is a major cause of morbidity among the elderly worldwide. It is a disease characterized by localized inflammation of the joint and destruction of cartilage, leading to loss of function. Impaired chondrocyte repair mechanisms, due to inflammation, oxidative stress and autophagy, play important roles in the pathogenesis of osteoarthritis. Olive and its derivatives, which possess anti-inflammatory, antioxidant and autophagy-enhancing activities, are suitable candidates for therapeutic interventions for osteoarthritis. This review aimed to summarize the current evidence on the effects of olive and its derivatives, on osteoarthritis and chondrocytes. The literature on animal and human studies has demonstrated a beneficial effect of olive and its derivatives on the progression of osteoarthritis. In vitro studies have suggested that the augmentation of autophagy (though sirtuin-1) and suppression of inflammation by olive polyphenols could contribute to the chondroprotective effects of olive polyphenols. More research and well-planned clinical trials are required to justify the use of olive-based treatment in osteoarthritis.
... Cartilage breakdown will amplify synovial inflammation, thus creating a vicious cycle. Previous studies have demonstrated that OA synovial cells are vital in maintaining arthritic pathologies by producing matrix degradation enzymes and inflammatory mediators [1,2]. In the light of the role of synovium in the pathogenesis and the symptoms of OA, synovium-targeted therapy may potentially halt the progression of structural destruction and mitigate the symptoms of the disease [3]. ...
Background
Osteoarthritis (OA) is manifested by synovial inflammation and cartilage destruction that is directly linked to synovitis, joint swelling and pain. In the light of the role of synovium in the pathogenesis and the symptoms of OA, synovium-targeted therapy is a promising strategy to mitigate the symptoms and progression of OA. Transforming growth factor beta 1 (TGF-β1), a secreted homodimeric protein, possesses unique and potent anti-inflammatory and immune-regulatory properties in many cell types. Heme oxygenase 1 (HO-1) is an inducible anti-inflammatory and stress responsive enzyme that has been proven to prevent injuries caused by many diseases. Despite the similar anti-inflammatory profile and their involvement in the pathogenesis of arthritic diseases, no studies have as yet explored the possibility of any association between the expression of TGF-β1 and HO-1.
Methodology/Principal findings
TGF-β1-induced HO-1 expression was examined by HO-1 promoter assay, qPCR, and Western blotting. The siRNAs and enzyme inhibitors were utilized to determine the intermediate involved in the signal transduction pathway. We showed that TGF-β1 stimulated the synthesis of HO-1 in a concentration- and time-dependent manner, which can be mitigated by blockade of the phospholipase (PLC)γ/protein kinase C alpha (PKC)α pathway. We also showed that the expression of miRNA-519b, which blocks HO-1 transcription, is inhibited by TGF-β1, and the suppression of miRNA 519b could be reversed via blockade of the PLCγ/PKCα pathway.
Conclusions/Significance
TGF-β1 stimulated the expression of HO-1 via activating the PLCγ/PKCα pathway and suppressing the downstream expression of miRNA-519b. These results may shed light on the pathogenesis and treatment of OA.
... Even though the siRNA was mostly absorbed into the synovium tissue and only into superficial chondrocytes, the inhibitory effect on cartilage degeneration was identified in vivo. While much research has focused on MMP13 or ADAMTS5 expression in OA cartilage (Aigner et al. 2003;Fosang et al. 2008), synovial fibroblasts is also considered as an important source of them in OA (Fosang et al. 2008;Fuchs et al. 2004). This supports previous studies suggesting that chemical mediators released from synoviocytes can affect cartilage degeneration (Clockaerts et al. 2010;Hardy et al. 2013). ...
Matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) are thought to play critical roles in cartilage degradation at the early phase of osteoarthritis (OA). The aim of this study is to examine the effect of chemically modified Mmp13 or Adamts5 small interfering RNA (siRNA), alone or in combination, in a mouse OA model. OA pathology was surgically induced in 9-week-old male C57/BL6 mice (n = 64) via destabilization of the medial meniscus (DMM). We used chemically modified siRNA (Accell siRNAs®) for Mmp13 and Adamts5, as well as a non-targeting control and evaluated their combined and individual effects after injection in the DMM model. The control group (n = 16) was injected with non-targeting siRNA and the normal group (n = 16) did not undergo any surgical induction or intra-articular injection. Histological assessment of the articular cartilage was conducted at 4 and 8 weeks post-DMM surgery to evaluate OA progression. Significant improvement in the histological score was observed at 8 weeks after DMM in all three siRNA-treated groups compared to the control siRNA-injected group. The score of the combined group was significantly lower than that of the Adamts5 siRNA-only group. No significant differences were noted between the Mmp13 siRNA-only group and the combined group. Combined intra-articular injection of Mmp13 and Adamts5 siRNA resulted in almost the same inhibitory effects as Mmp13 siRNA alone on cartilage degradation at the early phase of OA.
... Previous studies have indicated that non-invasively localized synovitis commonly exist in the joint of OA patient, particularly the synovium adjacent articular cartilage 22 . It's generally agreed that synovitis was not only the secondary pathological changes of OA, but also accelerated the progress of OA 23 . During synovitis, immune cells diffusing into the synovial fluid (SF) and synovium produce inflammatory mediators that are able to stimulate cartilage degradation by synoviocytes and chondrocytes and that cause the hyperplasia of the synovial tissue. ...
Objective
Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Osteoarthritis (OA), mainly resulting from the regression of cartilage, chronic inflammation of the synovium and the subchondral bone remodeling. Although the inflammatory response of AOPPs on fibroblast-like synoviocytes (FLSs) were reported, the effect of AOPPs on cartilage and synovial in vivo remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the articular cartilage and synovial in a rabbit model of OA.MethodsOA model were created by anterior cruciate ligament transection and medial meniscus resection (ACLT + MMx). Forty-eight male New Zealand rabbits were randomly divided into 3 groups: sham-operated group, AOPPs/ACLT + MMx group, and phosphate buffered saline (PBS)/ACLT + MMx group. In sham-operated group, the anterior cruciate ligament was just exposed without transection, and then the incision was sutured. Then intra-articular injection of AOPPs or PBS was performed in the other two groups. Through four weeks and eight weeks of treatment, rabbits in each group were sacrificed. Both hind legs were removed. India ink staining and Safranin O and fast green staining were used to evaluate the macroscopic and microscopic cartilage morphology. The protein expression of matrix metalloproteinases (MMP)-3, MMP-13 in synovium was measured by Western blot.ResultThe India ink score and Mankin score of AOPPs/ACLT + MMx group were both higher than the other two groups at the two time points. Western blot have revealed that intra-articular injection of AOPPs upregulated the protein expression of MMP-3 and MMP-13 in synovium.ConclusionAOPPs participated in the occurrence and development of OA by upregulating the protein expression of MMP-3 and MMP-13 in synovium.
... A growing body of evidence indicates the importance of activated FLS in OA pathogenesis. FLS promotes cartilage degradation through the secretion of inflammatory/catabolic mediators and other soluble factors [6][7][8]. Therefore, cultured FLS is a good in vitro model for OA pathogenesis and drug target discovery [6,9]. ...
Our previous work has revealed that expression of follistatin-like protein 1 (FSTL1) is elevated in the synovial tissues from osteoarthritis (OA) patients. The aim of this study was to elucidate the underlying molecular mechanisms by which FSTL1 plays a role in the pathogenesis of OA.
Cultured fibroblast-like synoviocytes (FLSs) from synovial tissues of OA patients were stimulated with human recombinant FSTL1, and then the expression of inflammatory cytokines in FLS and their concentrations in the cell supernatants were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immuno sorbent assay (ELISA), respectively. Nuclear factor kappa B (NF-κB) activation was examined by western blot and chromatin immunoprecipitation (ChIP) assay at p65 binding site. Finally, the proliferation of FLSs and the expression level of the proliferation-related tumor suppressors (p53 and p21) were determined by MTS assay kit and western blot in the presence or absence of FSTL1, respectively.
FSTL1 remarkably promoted expression levels of several inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6)) in vitro. Western blot analysis showed that FSTL1 activated the inflammatory-related NF-κB signaling pathway, as validated by ChIP assay detecting p65-binding level on cytokine promoter region. Moreover, FSTL1 promoted the proliferation of OA FLS by down-regulating the expression of p53 and p21. Interestingly, the concentration of synovial fluid IL-6 was remarkably elevated in OA patients, and was correlated with synovial fluid and serum FSTL1 levels.
These findings show that FSTL1 functions as an important proinflammatory factor in the pathogenesis of OA by activating the canonical NF-κB pathway and enhancing synoviocytes proliferation, suggesting that FSTL1 may be a promising target for the treatment of OA.
... Besides altering chondrocyte activity, activation of RAGE was also reported to affect synoviocyte activity and thus contribute to the pathogenesis of OA. There was a study proposing that the fibroblast-like synoviocyte (FLS) appeared to play an outstanding role in the pathogenesis of OA [117]. Another study showed that inflammation and cartilage degradation were inhibited in mice immunized and challenged with collagen type II (CII) when the mice were treated with sRAGE [118]. ...
The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily, capable of binding a broad repertoire of ligands. RAGE-ligands interaction induces a series of signal transduction cascades and lead to the activation of transcription factor NF-κB as well as increased expression of cytokines, chemokines, and adhesion molecules. These effects endow RAGE with the role in the signal transduction from pathogen substrates to cell activation during the onset and perpetuation of inflammation. RAGE signaling and downstream pathways have been implicated in a wide spectrum of inflammatory-related pathologic conditions such as arteriosclerosis, Alzheimer's disease, arthritis, acute respiratory failure, and sepsis. Despite the significant progress in other RAGE studies, the functional importance of the receptor in clinical situations and inflammatory diseases still remains to be fully realized. In this review, we will summarize current understandings and lines of evidence on the molecular mechanisms through which RAGE signaling contributes to the pathogenesis of the aforementioned inflammation-associated conditions.
... Several lines of evidence support the involvement of synoviocytes in OA cartilage degradation 3 . Hypoxia is recognized as an important event in the perpetuation of joint destruction in OA 4 . ...
Objective:
Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions.
Methods:
Human cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-β (IL-1β), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. ·NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2(·-) production, NOX subunit expression and nitrosylation were also assessed.
Results:
iNOS expression and nitrite (but not peroxynitrite) production were ~0.20 to ~0.12 nmol min(-1) mg proteins(-1) (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2(·-) production was restored at normoxic levels (0.21 nmol min(-1) mg of proteins(-1)).
Conclusions:
Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia.
... Weiterhin konnte gezeigt werden, dass die Arthrosedisposition sich nicht nur auf spezielle Gelenke beschränkt, sondern praktisch den gesamten Bewegungsapparat einschließlich der Wirbelsäule betrifft. Wahrscheinlich ist die unterschiedliche Manifestation an einzelnen Gelenken und Wirbelsäulenabschnitten sowohl auf die schwache Expressivität und Penetranz des Krankheitsbildes als auch auf multifaktorielle lokale, systemische, endo-und exogene Faktoren zurückzuführen, die an einigen Gelenken stärker manifestationsfördernd wirken als an anderen [2,7,20,23]. ...
Background
The definition of generalized osteoarthritis in the literature is just as controversial as the discussion about correlations between GOA and Heberden’s nodes (HN). Therefore, both questions were investigated in patients with proven heredity in a genealogical study of 931 family members.
Material and methods
In 106 patients with HN and 109 control subjects, 70 joints and spinal segments were investigated with respect to characteristic functional parameters. In addition, 44 joints and spinal segments were investigated radiologically.
Results
GOA affects both the small and large joints as well as the spine. This phenomenon is the more pronounced the more finger joints are affected by Heberden’s and Bouchard’s nodes.
Conclusions
GOA affects the entire musculoskeletal system. The varying manifestation in individual joints and spinal segments is probably attributable to multifactorial local and systemic factors. In an earlier study, a genetic disposition with a maximum HA prevalence of 30% was identified in the study population. Since HA is considered a genetic marker for GOA, it can be assumed that the same is true of GOA prevalence.
... Figure 5A shows that MMP activity released into the medium was significantly decreased by cell treatment with either 100 or 200 mM CORM-2. In particular, MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) are strongly induced by IL-1b in this cellular system and play a relevant role in OA synoviocytes [14,15]. Our results indicated that the observed reduction in MMP activity could be dependent on inhibitory effects of CORM-2 on MMP-1 and MMP-3 secretion into the medium ( Figure 5B and 5C) and mRNA expression in OA synoviocytes ( Figure 6A and 6B). ...
Osteoarthritis (OA) is the most widespread degenerative joint disease. Inflamed synovial cells contribute to the release of inflammatory and catabolic mediators during OA leading to destruction of articular tissues. We have shown previously that CO-releasing molecules exert anti-inflammatory effects in animal models and OA chondrocytes. We have studied the ability of CORM-2 to modify the migration of human OA synoviocytes and the production of chemokines and other mediators sustaining inflammatory and catabolic processes in the OA joint.
OA synoviocytes were stimulated with interleukin(IL)-1β in the absence or presence of CORM-2. Migration assay was performed using transwell chambers. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. CORM-2 reduced the proliferation and migration of OA synoviocytes, the expression of IL-8, CCL2, CCL20, matrix metalloproteinase(MMP)-1 and MMP-3, and the production of oxidative stress. We found that CORM-2 reduced the phosphorylation of extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2 and to a lesser extent p38. Our results also showed that CORM-2 significantly decreased the activation of nuclear factor-κB and activator protein-1 regulating the transcription of chemokines and MMPs in OA synoviocytes.
A number of synoviocyte functions relevant in OA synovitis and articular degradation can be down-regulated by CORM-2. These results support the interest of this class of agents for the development of novel therapeutic strategies in inflammatory and degenerative conditions.
... As demonstrated by the use of neutralizing antibodies, the main inducer of MMP-2 secretion present in these supernatants was TNF-␣. The preponderant role of TNF-␣ as an MMP inducer in FLS agrees with the findings of several previous studies (12,32,33,53). It has been shown that TNF-␣ is upregulated in the joints of mice with S. aureus arthritis and that the local secretion of TNF-␣ in the joint cavity gives rise to an increased severity of arthritis (58). ...
Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated.
Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the
production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment
and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte
chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture
supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion.
The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced
by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted
in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate.
These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during
brucellar arthritis through the production of MMPs and proinflammatory mediators.
... With respect to MMP production, numerous cytokines have been shown to upregulate the mRNA expression of aggrecan-degrading MMPs in articular cartilage. In particular, a strong induction of MMP3 mRNA has been observed within a few hours in human chondrocytes treated with IL-1β/OSM (Barksby et al., 2006; Cawston et al., 1998; Koshy et al., 2002), and MMP protein synthesis has also been shown to be upregulated by these cytokines (Arner and Tortorella, 1995; Bonassar et al., 1996; Fuchs et al., 2004; Sanchez et al., 2004; Tetlow et al., 2001). In addition, the present data show that MMP2 and MMP9 are readily synthesized and present in the culture media throughout the 19-day treatment of bovine cartilage explants with IL-1β and that the addition of APMA to these cultures is able to promote aggrecan degradation within the IGD through MMP action. ...
Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu(373-374)Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as "aggrecanase" (ADAMTS) cleavage sites, while cleavage between Ser(341-342)Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu(2047-2048)Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.
... Synovial inflammation has been demonstrated in tissue samples of OA patients and may be related to disease progression [17,29,30]. Several lines of evidence support the involvement of synoviocytes in OA cartilage degradation through the production of inflammatory and catabolic mediators [31] . In this regard, pro-inflammatory cytokines such as IL-1b play a role in driving synovitis during OA and influencing the production of cytokines and MMPs [32]. ...
High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).
HMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-κB activation by transient transfection with a NF-κB-luciferase plasmid.
In the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1β, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-κB activation in the presence of IL-1β.
Our results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1β to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.
Remitting seronegative symmetrical synovitis and pitting edema (RS3PE) is an uncommon condition that is characterized as an acute polysynovitis associated with pitting edema that tends to affect individuals who are over 50 years of age. This condition was first described by McCarty et al. in 1985 and although now usually thought of as a separate entity was originally thought to represent a subset of late onset rheumatoid arthritis (LORA). The controversy over the last 30 years as to whether RS3PE is a separate entity or forms a part of a clinical spectrum that includes LORA and polymyalgia rheumatica (PMR) is due to the similarities in clinical and demographic characteristics that are shared by these conditions. Additionally, RS3PE is also considered a paraneoplastic condition as it is often associated with malignancy. In this chapter, we will discuss the epidemiological and clinical characteristics of RS3PE, associated conditions including malignancy, diagnosis and management, as well as current data on mechanism of disease pathogenesis including areas of ongoing research and future directions.
Tissue engineering offers promising perspectives in the therapy of osteoarthritis. In the context of cell-based therapy, chondrogenic progenitor cells (CPCs) may be used to regenerate defects in cartilage tissue. An in-depth characterization of the secretome of CPCs is a prerequisite to this approach. In this study, a method was developed for the qualitative and quantitative analysis of the secretome of undifferentiated and differentiated CPCs. Secreted proteins from cells grown in two-dimensional as well as three-dimensional alginate cultures were extracted and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Quantitation was achieved by internal standardization using stable isotope-labeled amino acids in cell culture (SILAC). Qualitative analysis of CPC secretomes revealed ECM-components, signal proteins and growth factors most of which were also found in healthy cartilage. A quantitative comparison revealed significantly upregulated proteins with regenerative potential during differentiation, while proteins involved in catabolic metabolism were significantly downregulated. The development of methods for qualitative and quantitative analysis of the secretome of CPCs by mass spectrometry provides a foundation for the investigation of progenitor or stem cells from other sources.
Gastrodia elata (GE), which belongs to the Orchidaceae family, was found to possess anti-inflammatory activity. However, the effect of GE on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) remains largely unknown. Thus, the aim of this study was to investigate the effects of GE on tumor necrosis factor-α (TNF-α)-induced inflammatory response in RA-FLS and the underlying molecular mechanism was also explored. Our results demonstrated that GE significantly attenuated TNF-α-induced IL-6 and IL-8 production in RA-FLS. GE also inhibited TNF-α-induced MMP-3 and MMP-13 expression in RA-FLS. Furthermore, pretreatment with GE significantly attenuated TNF-α-induced the expression of p-p65 and IκBα degradation in RA-FLS. In conclusion, this study demonstrated for the first time that GE attenuated inflammatory response by inhibiting the NF-κB pathway signaling in RA-FLS. Thus, GE might have a therapeutic potential towards the treatment of RA.
Objective: To explore the effects of angelica sinensis polysaccharides (ASP) on cartilage of osteoarthritis in rats. Methods: Fifty-six rats were divided into seven groups with eight in each as follow: normal control group, 0.50 g/L ASP control group, model control group, 0.10, 0.25, 0.50 g/L ASP group, and positive control group. Animal models of osteoarthritis were established by intra-articular injection of 4% papain in the knees of the rats in the latter five groups. The animals in APS group began receiving intra-articular ASP injection of different concentrations every three days. The normal and model groups received same amount of saline instead of APS. The positive control groups received 100 mg/kg glucosamine sulfate by gavage everyday. Five weeks latter, all animals were sacrificed for histological examination on synovium and articular cartilage. The transmission electron microscopy were also adopted to observe the changes in articular cartilage. Results: In 0.25 and 0.50 g/L ASP group, Mankin's scores and synovial membrane scores significantly decreased as compared with those in model control group (both P<C0.01), but Mankin's scores were higher (P<0.01) and synovial membrane scores had no difference (P> 0.05) versus that respectively in positive control group. The 0.10 g/L ASP showed similar effects to 0.25 and 0.50 g/L ASP, but those effects were weaker (all P<0.01). The transmission electron microscopy showed ASP could decrease the damage of cartilage. The 0.50 g/L ASP control group showed no difference with nomal control group in every index (all P>0.05). Conclusion: Intra-articular injection of ASP promotes the repairing process of degenerated cartilage of osteoarthritis in the rat models.
This study investigated the effect of MMP-13 gene knock down on cartilage degradation by injecting small interfering RNA (siRNA) into knee joints in a mouse model of osteoarthritis (OA). OA was induced in male C57BL/6 mice by destabilization of medial meniscus (DMM) surgery. Change of Mmp13 expression over time was determined by qPCR analysis from 3 days to 6 weeks after surgery. Mmp13 and control chemically modified siRNA were injected into the knee joint 1 week after surgery and expression levels were assessed in synovium by qPCR 48 h later. Cartilage degradation was histologically assessed 8 weeks after DMM surgery according to OARSI recommendations. Mmp13 expression levels were elevated 1 week after surgery and peaked at 77 fold at 2 weeks compared to expression at 3 days. A 55% decrease of Mmp13 levels in cartilage was observed 48 h after injection of Mmp13 siRNA (p = 0.05). Significant reduction in the histological score at 8 weeks after surgery was observed in the Mmp13 siRNA-treated group compared to the control siRNA group (p < 0.001). Intra-articular injection of Mmp13 siRNA at the early phase of OA development resulted in effective knock down of Mmp13 expression and delay in cartilage degradation in vivo. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Introduction
Chondroitin sulfate (CS) and hyaluronic acid (HA) are used in the symptomatic treatment of osteoarthritis (OA). Cholagenase-1 (MMP-1) and stromelysin-1 (MMP-3), are responsible for degradation of the extracellular matrix in OA. Few studies have determined the in vitro effect of CS and HA on MMP-1 synthesis and that of HA on MMP-3 expression in human OA chondrocyte culture. In the literature reviewed, there were no studies evaluating the effect of CS on MMP-3.
Objectives
To analyze the effect of CS and HA (500-730 kDa) on MMP-3 and MMP-1 synthesis induced by interleukin-1β (IL-1β) in OA chondrocytes.
Material and methods
Chondrocytes were incubated for 48 hours with IL-1β (2.5 ng/ml) in the presence or absence of different HA concentrations (Hyalgan®, Bioibérica Farma) (10, 50, 100, 150, 200 and 1000 μg/ml). Functional evaluation of chondrocytes was performed by enzyme-immunoanalysis of MMP-1 and MMP-3 levels.
Results
CS and HA inhibited IL-1β-induced MMP-3 synthesis, without significantly modifying MMP-1. CS and HA reduced levels of MMP-3 expression at all the studied concentrations, with no statistically significant differences among these concentrations.
Conclusions
The results of this study show for the first time that CS inhibits MMP-3 synthesis in OA cartilage. and corroborates the few existing data on the ability of HA to inhibit this enzyme.
The myocardial interstitium is highly organized and orchestrated, whereby small disruptions in composition, spatial relationships, or content lead to altered myocardial systolic and/or diastolic performance. These changes in extracellular matrix structure and function are important in the progression to heart failure in pressure overload hypertrophy, dilated cardiomyopathy, and ischemic heart disease. The myocardial interstitium is not a passive entity, but rather a complex and dynamic microenvironment that represents an important structural and signaling system within the myocardium.
Differing extents of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiologic context of tendon degeneration and the extent of tendon retraction are unclear. Tendon integrity depends on the extracellular matrix, which is regulated by matrix metalloproteinases (MMP). It is unknown which enzymes play a role in tendon degeneration. The hypotheses are that (1) the expression of MMPs 1, 3, and 9 is altered in the torn rotator cuff when compared with healthy tendon samples; and (2) that there is a relationship between MMP expression and the extent of tendon retraction in the torn cuff.
Rotator cuff tendon samples of 33 patients with full-thickness rotator cuff tears (Bateman grade III) were harvested during reconstructive surgery. Samples were dehydrated and paraffin-embedded. Immunohistologic determination of MMP 1, 3, and 9 expression was performed by staining sample slices with MMP antibody. The extent of tendon retraction was determined intraoperatively according to Patte's classification and patients were assigned to 4 groups (control group, and by tendon retraction grade Patte I-III). The control group consisted of six healthy tendon samples.
Expression of MMPs 1 and 9 was significantly higher in torn cuff samples than in healthy tendons whereas MMP 3 expression was significantly decreased (P < 0.05). MMP 9 expression significantly increased with rising extent of tendon retraction in the torn cuff (P < 0.05). No significant association was found between expression of MMPs 1 and 3 and the rising extent of tendon retraction by Patte's classification.
Elevated expression of MMPs 1 and 9 as well as decreased MMP 3 expression can be detected in torn rotator cuff tendon tissue. There is a significant association between the extent of tendon retraction and MMP 9 expression. The results of this study give evidence that early surgical treatment of small and partial-thickness rotator cuff tears is required.
The aim of this study was to evaluate a subset of matrix metalloproteinases (MMPs) and proinflammatory cytokines within the synovium, from traumatic knee disorders (TKDs) and nontraumatic osteoarthritis (OA).
MMP-1, MMP-3, interleukin 1beta, and tumor necrosis factor (TNF) alpha gene expression was quantified by use of TaqMan-based real-time polymerase chain reaction (Applied Biosystems, Foster City, CA) in synovial tissue samples obtained from 12 patients with OA and 32 with TKDs. In addition, the levels of serum inflammatory parameter C-reactive protein (CRP) were recorded. At arthroscopy, the amount of chondral damage was graded based on the Outerbridge classification.
Quantitative analysis showed no significant differences in the expression levels of MMPs, interleukin 1beta, or TNF-alpha messenger RNA between the synovial tissues of patients with OA and TKD, but CRP level was significantly increased in the OA group. A significant correlation was also seen regarding the gene expression levels between MMP-1 and -3, as well as between CRP and MMPs tested. Furthermore, significant relations between TNF-alpha and MMP-1 plus MMP-3 were observed in the OA synovial tissue. The level of TNF-alpha in the synovial tissue correlated with the time after injury as well as chondral damage in patients with TKD.
This study shows similar changes in the inflammatory patterns of synovial tissue of TKD and OA suggesting a likely disease progression. Moreover, the correlation between CRP and MMP expression levels indicates their essential role in joint degeneration in synovial tissue of primary OA patients.
TNF-alpha could provide a factor to quantify individual risk for the development of OA.
Curcumin, a major component of turmeric, has been shown to exhibit anti-oxidant and anti-inflammatory activities. The present study was performed to determine whether curcumin is efficacious against both collagen-induced arthritis (CIA) in mice and IL-1beta-induced activation in fibroblast-like synoviocytes (FLSs). DBA/1 mice were immunized with bovine type II collagen (CII) and treated with curcumin every other day for 2weeks after the initial immunization. For arthritis, we evaluated the incidence of disease and used an arthritis index based on paw thickness. In vitro proliferation of CII- or concanavalin A-induced splenic T cells was examined using IFN-gamma production. Pro-inflammatory cytokines TNF-alpha and IL-1beta were examined in the mouse ankle joint and serum IgG1 and IgG2a isotypes were analyzed. The expression levels of prostaglandin E(2) (PGE(2)), cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) in human FLSs were also determined. The results showed that compared with untreated CIA mice, curcumin-treated mice downregulated clinical arthritis score, the proliferation of splenic T cells, expression levels of TNF-alpha and IL-1beta in the ankle joint, and expression levels of IgG2a in serum. Additionally, by altering nuclear factor (NF)-kappaB transcription activity in FLSs, curcumin inhibited PGE(2) production, COX-2 expression, and MMP secretion. These results suggest that curcumin can effectively suppress inflammatory response by inhibiting pro-inflammatory mediators and regulating humoral and cellular immune responses.
Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.
Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.
Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.
The substrate specificity of human neutrophil collagenase was examined using both monomeric and fibrillar collagens. The neutrophil enzyme cleaved types I, II, and III collagens, but failed to attack types IV or V. Against monomeric collagen substrates at 25 degrees C, the neutrophil enzyme displayed values for the Michaelis constant (Km) of 0.6-1.8 X 10(-6) M, essentially indistinguishable from the substrate affinities that characterize human fibroblast collagenase. Catalytic rates, however, varied considerably; type I collagen was cleaved with a specificity (kappa cat/Km) some 20-fold greater than type III. Type II collagen was degraded with intermediate selectivity, approximately equal to 25% of the type I rate, but 450% that of type III. This specificity contrasted markedly with that of human fibroblast collagenase, which cleaved human type III collagen 15-fold faster than type I and greater than 500-fold more rapidly than type II. Interestingly, the 20-fold selectivity for type I over type III exhibited by neutrophil collagenase against monomeric collagens was largely abolished following the reconstitution of these substrates into insoluble fibrils, falling to a value of just 1.5-fold. The distinctive and opposite preference by the human fibroblast enzyme for monomeric type III collagen over type I (15-fold) was similarly reduced to less than 2-fold upon substrate aggregation. The transition from native soluble collagen monomers into insoluble fibrils appeared to be handled by both the human neutrophil and fibroblast collagenases with similar facility on type I substrates. By comparison, however, the neutrophil enzyme degraded type III collagen fibrils faster than would have been predicted from solution rates, while the fibroblast enzyme cleaved such fibrils much slower than expected from solution values. In exploring this phenomenon further, solvent deuterium isotope effects were measured. The deuterium studies suggest that neutrophil collagenase, acting on type III fibrils (kappa H2O/kappa D2O = 5.0), is less sensitive to factors which govern the availability of water at the relatively hydrophobic site of peptide bond hydrolysis in the collagen molecule than is fibroblast collagenase (kappa H2O/kappa D2O = 15.0).
Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.
We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/4C(short)) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/4C(short) neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C(short) from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process.
Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA.
Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with lambda DNA. Rheumatoid synovium was used as a positive control for MMP gene expression.
Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease.
The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.
Objective. To determine the involvement of interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 in the cartilage pathology of murine antigen-induced arthritis (AIA) and zymosan-induced arthritis (ZIA). Methods. Arthritis was induced by intraarticular injection of zymosan in naive mice or by subcutaneous injection of methylated bovine serum albumin in sensitized animals. Mini-osmotic pumps releasing human recombinant IL-1 receptor antagonist (IL-1ra) protein were implanted intraperitoneally 2 days before arthritis induction, and neutralizing antibodies directed against murine IL-1 alpha, IL-1 beta, TNF alpha, or IL-6 were administered 1 day before. Proteoglycan (PG) synthesis and degradation were assessed in patellar cartilage. Results. Murine IL-1 alpha and IL-1 beta injected intraarticularly at doses of 0.1-100 ng suppressed chondrocyte PG synthesis. The highest dose of TNF tested (100 ng) decreased PG synthesis marginally. In contrast, the maximum dose of IL-6 (1 lug) stimulated PG synthesis 2 days after injection. Treatment of AIA with neutralizing monoclonal antibodies against either TNF alpha or IL-6 did not reduce either the PG degradation or the suppression of its synthesis. However, treatment with anti-IL-1 (alpha + beta) polyclonal antibodies totally prevented PG suppression, although the initial breakdown of PG was unaffected. This effect was confirmed when IL-1ra was administered in high doses. Moreover, treatment of ZIA with anti-IL-1 (alpha + beta), but not with anti-TNF, resulted in normal PG synthesis, confirming the key role played by IL-1 in the inhibition of PG synthesis. Treatment of AIA with anti-IL-1 did not affect inflammation during the acute phase, but a significant reduction of ongoing inflammation was noted at day 7, and there was a marked reduction in the loss of cartilage PG. Conclusion. The suppression of PG synthesis in both ZIA and ALA in mice is due to the combined local action of IL-1 (alpha + beta), and neither IL-6 nor TNF is involved. Moreover, the normalization of PG synthesis brought about by blocking of IL-1 ameliorates the cartilage damage associated with AIA.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The phosphorylation of amino alcohols has been achieved using inorganic sodium cyclo-triphosphate hexahydrate, Na3P3O9·6H2O, in aqueous solution. The main phosphorylated products were imidotriphosphates of amino alcohols as evidenced by 1H, 13C, and 31P NMR spectra. In the phosphorylation of 3-amino-1-propanol (3A1P), 4-amino-1-butanol (4A1B), and 5-amino-1-pentanol (5A1P), only their amino groups were phosphorylated to give imidotriphosphates, with their maximum yields of more than 97%.
Levels of tissue inhibitor of metalloproteases (TIMP) and plasminogen activator (PA)/plasmin were measured and the distribution of PA was studied by immunohistochemical techniques in cartilage and synovium samples from dogs subjected to sectioning of the anterior cruciate ligament of their right knees and sham operation of their left knees (controls). Twenty-three animals were divided into 3 groups and killed at 2, 4, or 8 weeks after surgery. The levels of PA and plasmin were found to be significantly elevated in the osteoarthritic (OA) knee cartilage and synovium at all times after surgery, except for levels of PA in the OA cartilage at 2 weeks. There was a positive correlation between the levels of PA and plasmin in the synovial membrane (r = 0.64, P < 0.001). In OA knees, the presence of high levels of total and active collagenase was detected in cartilage and in synovium. The levels of these 2 forms of collagenase showed a positive correlation both in cartilage (r = 0.65, P < 0.001) and in synovium (r = 0.77, P < 0.001). The levels of TIMP in cartilage from OA and sham operated knees were similar. Although the TIMP level was increased in the OA synovium, it was found only in trace amounts in cartilage. Immunohistochemical studies revealed that both forms of PA, urokinase-type PA and tissue-type PA, and TIMP were present in OA tissues. In the synovium, they were found mainly in monocyte/macrophages, synovial lining cells, and blood vessel cells. In OA cartilage, PA was present only at the superficial level in chondrocytes and in cartilage matrix, whereas TIMP was present in chondrocyte lacunae throughout the full thickness of the cartilage. TIMP was also detected in the superficial level of cartilage from sham operated knees. The results of this study indicate that in OA tissues, there are conditions that favor the synthesis and activation of metalloproteases. PA and plasmin are likely to play an important role in the physiologic activation of metalloproteases, although they are probably not the only system involved in this process. The lack of increased TIMP levels in the OA cartilage, in the presence of increased metalloprotease activity, is also a possible contributing factor in the enzymatic degradation of this tissue.
Objective
We investigated the response of human osteoarthritic (OA) chondrocytes, in terms of collagenase 3 production, to growth factors and cytokines involved in the anabolism and catabolism of articular cartilage, and explored the major signaling pathways leading to its up-regulation.Methods
Human OA chondrocytes were treated with the following factors: the proinflammatory cytokine interleukin-1β (IL-1β), the growth factors basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGFβ1), and TGFβ2, the protein kinase (PK) activator antagonists for PKC, PKA, and PKG pathways, and phospholipase A2 and tyrosine kinases, as well as the antiinflammatory cytokines IL-4, IL-10, and IL-13. Collagenase 3 expression and synthesis were determined. Comparison was made with collagenase 1.ResultsThe human OA chondrocyte population could be divided into 2 categories: the L chondrocytes, showing low collagenase 3 basal synthesis levels and high sensitivity to IL-1β stimulation; and the H chondrocytes, high collagenase 3 basal synthesis levels and low IL-1β inducibility. In L chondrocytes, all growth factors stimulated collagenase 3 production. In H chondrocytes, PTH, IGF-1, and TGFβ had little or no impact; bFGF slightly stimulated it and PDGF-BB showed the same pattern as in the L chondrocytes. The effects of all growth factors, except TGFβ, on collagenase 1 synthesis followed those of collagenase 3, albeit to a higher degree. Interestingly and unlike collagenase 3, the effects of TGFβ on collagenase 1 could not be related to the state of the cells, but rather, depended on the isoform. Indeed, TGFβ2 did not induce collagenase 1 synthesis, whereas TGFβ1 stimulated it. Among the PK activators tested, phorbol myristate acetate was the strongest inducer, suggesting a major involvement of the PKC pathway. IL-13 inhibited collagenase 3 production, IL-4 had little effect, and IL-10 had none.Conclusion
This study shows that collagenase 3 production in human OA chondrocytes depends on the physiologic state of the cell. TGFβ might be responsible for the change in cells from the L to the H state. Importantly, our in vitro data implicate TGFβ2 as a possible in vivo agent capable of specifically triggering collagenase 3 production over that of collagenase 1 in OA cartilage.
ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1α or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.
Objective. To examine the interaction between synovial fibroblasts and macrophages in the context of cartilage degradation.
Methods. An in vitro model of human cartilage degradation was used, in which purified populations of fibroblasts and macrophages were added to a radio-labeled cartilage disc. Cartilage destruction was measured by the percentage of radiolabel release.
Results. Fibroblasts, obtained from either rheumatoid arthritis (RA) or osteoarthritis synovial tissue, could mediate cartilage degradation if cocultured with the U937 macrophage cell line. Skin and RA bone marrow fibroblasts had no degradative effect on cartilage. Fibroblast—macrophage contact was not required for cartilage degradation. Cartilage degradation by synovial fibroblasts was inhibited by antibodies to tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6. Cartilage degradation was almost completely abrogated by a combination of antibodies to TNFα and IL-1β. Contact between fibroblasts and cartilage was shown to be essential. Antibodies to CD44, but not to intercellular adhesion molecule 1, markedly inhibited cartilage degradation.
Conclusion. TNFα, IL-1β, and IL-6 were involved in the activation of synovial fibroblasts to cause cartilage degradation. Cartilage degradation occurred only when fibroblasts were in contact with cartilage. CD44 was demonstrated to be involved in the fibroblast—cartilage interaction.
Objective. To show the eventual presence and extent of production of matrix metalloproteinase 13 (MMP-13, or collagenase 3) in rheumatoid synovial tissue samples and extracts, and to assess the inhibition characteristics of recombinant MMP-13.
Methods. Immunohistochemical avidin-biotin-peroxidase complex staining/morphometry was used to analyze MMP-13-positive cells in situ. Neutral salt extraction of synovial tissue, electrophoresis of the extract in different buffer systems, and Western blotting were also used. The inhibitory properties of doxycycline, clodronate, pamidronate, and D-penicillamine for recombinant enzyme were determined with a soluble type II collagen assay.
Results. MMP-13 was detected in fibroblast- and macrophage-like mononuclear cells in the synovial lining and stroma and in vascular endothelial cells. The overall expression of MMP-13 in these cells in the synovial stroma was high in rheumatoid arthritis (86 ± 12%) compared with osteoarthritis (17 ± 5%) patient samples (P = 0.0027). In a high-pH native electrophoresis gel, immunoreactivity to anti-MMP-1 and anti-MMP-13 were clearly separated, with anti-MMP-13-immunoreactive material migrating faster than anti-MMP-1-immuno-reactive material. Finally, in contrast to MMP-1 and MMP-8, MMP-13 was found to be relatively resistant to the inhibitory effects of doxycycline and clodronate in vitro.
Conclusion. Due to its localization in synovial tissue, its substrate profile, increased expression, and relative resistance to known MMP inhibitors, MMP-13 is suggested to play a major role in the pathogenesis of tissue destruction in rheumatoid arthritis.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Degradation of proteoglycans is an initial change in osteoarthritic cartilage. Matrix metalloproteinase-3 (MMP-3; stromelysin) capable of degrading cartilage proteoglycans and type IX collagen was immunolocalized in osteoarthritic and normal cartilage. Immunohistochemical studies showed MMP-3 in chondrocytes of the superficial and transition zones in approximately 90% of osteoarthritic cartilage (60 of 67 samples) and in 31% of those of the superficial zone in some normal cartilage (4 of 13 samples). MMP-3 staining correlated directly with the histological histochemical scores of Mankin and with proteoglycan depletion, up to a certain grade of severity. Chondrocytes in the deep radial zone, clusters, and osteophytes were immunostained only when proteoglycan depletion and fissures affected them. Culture media from osteoarthritic cartilage contained significantly higher levels of metalloproteinase activity that was identified as MMP-3 by immunoblotting and lower amounts of tissue inhibitor of metalloproteinases compared with those in the control samples. MMP-3 was also immunolocalized in the lining cells of most osteoarthritic synovium (20 of 23 specimens, 87%) with a direct correlation with scores of inflammatory cell infiltration in the synovium, but it was not detected in the normal synovium. Light and electron microscopic studies demonstrated that MMP-3 digests proteoglycan aggregates in human articular cartilage. Treatment of normal and osteoarthritic cartilage slices with tumor necrosis factor-alpha and/or interleukin-1 alpha increased the number of MMP-3-immunoreactive chondrocytes and the intensity of the staining. These data suggest that MMP-3 produced by the chondrocytes and synovial lining cells under stimulation with these cytokines may be important in proteoglycan degradation in human ostoearthritic cartilage.
Extracts of human articular cartilage have been examined for the presence of metalloproteinases that degrade proteoglycans and collagen of the extracellular matrix. Two enzymes (stromelysin and acid metalloproteinase) that degrade proteoglycan are elevated at least 150% in osteoarthritis (OA), whereas the tissue inhibitor of metalloproteinases (TIMP) shows only a small increase. We postulate an imbalance of enzyme over inhibitor that leads to net matrix destruction in OA. Stromelysin is shown to have an acid pH optimum of about 5.5. At this pH it can become spontaneously active and is less susceptible to TIMP inhibition than at pH 7.5. We postulate that the chondrocytes may secrete acid into the pericellular space, leading to localized enzyme activation and proteoglycan digestion.
Articular cartilage from arthritic joints of rats immunized with type II collagen is severely depleted of proteoglycans. Depletion begins within 48 hours after the onset of inflammation, prior to extensive pannus formation, and may represent a critical first step in cartilage destruction. We have immunolocalized stromelysin, an enzyme that is believed to play a major role in the pathologic degradation of proteoglycans, in the joints of rats with collagen-induced arthritis. Immunoperoxidase staining of frozen tissue sections demonstrated the presence of stromelysin in both the synovium and chondrocytes. In contrast, collagenase was localized primarily to the pannus-cartilage junction. Neither enzyme was detectable in joints from normal animals. To test the hypothesis that chondrocytes respond directly to inflammatory mediators by increasing the production of stromelysin, isolated chrondrocytes were incubated with various concentrations of interleukin-1. The culture media were also assayed for the presence of stromelysin by immunoreactivity on Western blots and by analysis of enzymatic activity on casein substrate gels. A 3-fold increase in a doublet of proteins synthesized in response to 10 units/ml of interleukin-1 was observed. These proteins also immunoreacted with the stromelysin antibody and degraded casein. Northern blotting results established that the increased levels of stromelysin were accompanied by increases in stromelysin-specific messenger RNA levels. These results suggest that stromelysin is responsible for proteoglycan degradation in early inflammatory arthritis, and that chondrocytes may play a direct role in the earliest stages of the degradation of their own matrices.
The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.
We investigated the nature of cytokines synthesized by human osteoarthritic (OA) synovium, particularly interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). We examined the capacity of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) to block the synthesis of metalloproteases (collagenase and stromelysin), IL-1 beta, and IL-6 in osteoarthritis (OA) synovium. Human OA synovium were incubated in the presence or absence of lipopolysaccharide (LPS) or increasing concentrations of rhIL-1ra. The determinations of IL-1 alpha, IL-1 beta, TNF alpha, IL-6, and IL-1ra in culture medium were carried out using specific ELISA. Although both IL-1 isoforms and TNF alpha could be produced by OA synovium, IL-1 beta was the predominant cytokine synthesized either in the presence or absence of LPS. Treatment of the OA synovium with an increasing concentration of rhIL-1ra (0-10 micrograms/ml) showed a dose dependent reduction of both metalloproteases and IL-6. Maximal inhibition was 70% for collagenase, 80% for stromelysin, and 76% for IL-6. LPS treated synovium also showed a consistent suppression of metalloproteases and IL-6, although a higher IL-1ra concentration was required. Conversely, IL-1 beta production was not inhibited by IL-1ra, irrespective of the concentration used and whether the membranes were LPS stimulated. These data showed that IL-1 appears to be the major autocrine cytokine involved in the stimulation of metalloproteases and IL-6 synthesis in OA synovium.
To determine the involvement of interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-6 in the cartilage pathology of murine antigen-induced arthritis (AIA) and zymosan-induced arthritis (ZIA).
Arthritis was induced by intraarticular injection of zymosan in naive mice or by subcutaneous injection of methylated bovine serum albumin in sensitized animals. Mini-osmotic pumps releasing human recombinant IL-1 receptor antagonist (IL-1ra) protein were implanted intraperitoneally 2 days before arthritis induction, and neutralizing antibodies directed against murine IL-1 alpha, IL-1 beta, TNF alpha, or IL-6 were administered 1 day before. Proteoglycan (PG) synthesis and degradation were assessed in patellar cartilage.
Murine IL-1 alpha and IL-1 beta injected intraarticularly at doses of 0.1-100 ng suppressed chondrocyte PG synthesis. The highest dose of TNF tested (100 ng) decreased PG synthesis marginally. In contrast, the maximum dose of IL-6 (1 microgram) stimulated PG synthesis 2 days after injection. Treatment of AIA with neutralizing monoclonal antibodies against either TNF alpha or IL-6 did not reduce either the PG degradation or the suppression of its synthesis. However, treatment with anti-IL-1 (alpha + beta) polyclonal antibodies totally prevented PG suppression, although the initial breakdown of PG was unaffected. This effect was confirmed when IL-1ra was administered in high doses. Moreover, treatment of ZIA with anti-IL-1 (alpha + beta), but not with anti-TNF, resulted in normal PG synthesis, confirming the key role played by IL-1 in the inhibition of PG synthesis. Treatment of AIA with anti-IL-1 did not affect inflammation during the acute phase, but a significant reduction of ongoing inflammation was noted at day 7, and there was a marked reduction in the loss of cartilage PG.
The suppression of PG synthesis in both ZIA and AIA in mice is due to the combined local action of IL-1 (alpha + beta), and neither IL-6 nor TNF is involved. Moreover, the normalization of PG synthesis brought about by blocking of IL-1 ameliorates the cartilage damage associated with AIA.
To assess the likely importance of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the arthritic process.
Synovial samples from seven joints with rheumatoid arthritis and three osteoarthritic joints were analysed by indirect immunofluorescence microscopy. Using specific human antisera, we documented the frequencies and distributions of collagenase, stromelysins 1 and 2, matrilysin, gelatinases A and B, TIMP-1, and TIMP-2.
Stromelysin 1 was found in all synovia, bound to extracellular matrix, within cells, or both, indicating stromelysin synthesis. Matrilysin was present in only one active inflammatory synovium, and focal synthesis of collagenase and gelatinase A was seen in four synovia. Stromelysin 2 and TIMP-2 were not observed, but TIMP-1 synthesis was seen in five synovia, and in two active synovia the distribution of TIMP-1 positive cells was more widespread than that of MMPs.
The presence of stromelysin 1 in all synovia clearly implicates this enzyme in joint damage. Collagenase, gelatinase A and matrilysin may also have a role in rheumatoid arthritis, but are not significant in osteoarthritis. However, marked regional variations were found in the synthesis of these MMPs, indicating not only that these diseases are episodic but that control of enzyme synthesis is focal. Only TIMP-1 may be considered an inhibitory factor.
We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lactoferrin and TIMP-1 of saliva (n = 10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment.
Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E373-A374 'aggrecanase' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.
In an attempt to identify the factor(s) involved in the modulation of the degradative pathway of articular cartilage, we previously reported a possible imbalance between the levels of biologically active forms of metalloproteases and tissue inhibitor of metalloprotease (TIMP) in osteoarthritis (OA) cartilage.
We extended our analysis on the protein level and the synthesis of stromelysin-1, collagenase, TIMP-1, and TIMP-2 in normal, OA, and RA cartilages, and provided information on the synthesis pattern of these proteins in respect to the action of interleukin-1 (IL-1). These protein concentrations were determined by specific sandwich EIA assays.
This study allowed us to establish that the concentration of stromelysin-1 and collagenase is elevated in both OA and rheumatoid arthritis (RA) cartilages when compared with normal, with significantly higher levels of collagenase found in OA (p < 0.0003) and RA (p < 0.0001), and of stromelysin-1 in RA (p < 0.02). In all cases, the level of stromelysin-1 significantly exceeded (a few 100-fold) the collagenase level. The cartilage TIMP-1 level was notably enhanced only in RA, whereas TIMP-2 was increased in both OA and RA cartilage. RA patients with active disease had a higher level of metalloproteases and TIMP than those patients with inactive disease. Moreover, patients taking steroids alone or in combination with methotrexate had a markedly lower metalloprotease level without any changes in the TIMP-1 level. In culture cartilage explants, the synthesis of stromelysin-1 was enhanced in RA cartilage, whereas the level of collagenase was increased both in OA and RA explants. When compared with normal patients, the TIMP-1 synthesis was essentially unchanged in arthritic explants, whereas the level of TIMP-2 was decreased in RA explants when compared to OA. IL-1 induced a statistically significant increased synthesis of metalloproteases with the highest level found in arthritic explants. IL-1 also significantly decreased the TIMP-1 synthesis in OA and RA explants, and the TIMP-2 synthesis in OA.
This study demonstrates that stromelysin-1 is the predominant metalloprotease synthesized in human articular cartilage and that both TIMP-1 and TIMP-2 are present in this tissue. The differential regulation of metalloprotease and TIMP syntheses by IL-1 suggests that this cytokine, during inflammatory conditions, may promote cartilage degradation by creating an imbalance between the level of these enzymes and their inhibitors.
To determine the relative expressions of matrix metalloprotease (MMP) genes pro-MMP1 and pro-MMP3 in the cartilage of rabbits with experimentally induced osteoarthritis (OA), and to assess the role of the chondrocyte in this process.
OA was induced in rabbits after partial medial meniscectomy. Rabbits were killed at 4 weeks or 8 weeks, and total cellular RNA was prepared from cartilage and probed by Northern blotting with pro-MMP 32P-labeled complementary DNA. Monolayer chondrocytes were used to assess MMP-inducing activity of chondrocyte factor(s).
Pro-MMP messenger RNAs (mRNAs) were up-regulated in experimental OA cartilage; pro-MMP3 mRNA expression exceeded that of pro-MMP1. Conditioned medium from OA-derived chondrocytes up-regulated pro-MMP mRNAs in normal chondrocytes.
Up-regulation of MMP genes in this OA model may contribute to cartilage degradation. Chondrocytes up-regulate MMP genes via an autocrine pathway.
Human neutrophil procollagenase was activated by incubation with recombinant active stromelysin. Activation was achieved by cleavage of the Gly78-Phe79 peptide bond at the end of the propeptide domain in a single-step activation mechanism. In addition, accelerated activation was achieved when N-terminally truncated, latent collagenase (with Phe49 as its N-terminal residue) was incubated with recombinant active stromelysin. Determination of the specific activity of recombinant-stromelysin-activated neutrophil collagenase with dinitrophenyl-octapeptide or type I collagen demonstrated the generation of high specific activity. The specific activity of stromelysin-activated enzyme was considerably higher than that of trypsin- or HgCl2-activated collagenase. Thus human neutrophil collagenase is superactivated, like the homologous fibroblast collagenase [Murphy, Cockett, Stephens, Smith and Docherty (1987) Biochem. J. 248, 265-268]. The occurrence of Phe79 at the N-terminus of the neutrophil collagenase seemed to be critical for superactivation, which is in agreement with data published by Suzuki, Enghild, Morodomi, Salvesen and Nagase [(1990) Biochemistry 29, 10261-10270] on fibroblast collagenase.
To investigate mechanisms of cartilage matrix destruction by a study of the effects of a specific inactivator of cathepsin B and an inhibitor of several matrix metalloproteinases (MMP) on cartilage proteoglycan release.
Cartilage explants were treated with either recombinant human interleukin-1 alpha (rHuIL-1 alpha) or retinoic acid in the presence or absence of the inhibitors, and proteoglycan release was quantitated. Tests for nonspecific effects of the inhibitors included reversibility, rates of protein synthesis and glycolysis, and effects on other rHuIL-1 alpha-mediated events.
The cathepsin B inactivator inhibited rHuIL-1 alpha-stimulated proteoglycan release at nanomolar concentrations, but failed to significantly inhibit retinoic acid-stimulated proteoglycan release. An inhibitor of MMP was inhibitory to both rHuIL-1 alpha-stimulated release and retinoic acid-stimulated release.
Cathepsin B is implicated in rHuIL-1 alpha-stimulated loss of cartilage proteoglycan. Its lack of involvement in retinoic acid-stimulated proteoglycan release suggests the existence of at least 2 pathways of cartilage proteoglycan breakdown, which may converge at the activation of a matrix prometalloproteinase.
To determine the concentrations of human stromelysin-1, collagenase, tissue inhibitor of metalloproteinases (TIMP), and proteoglycan fragments in knee synovial fluid in patients with injury to the meniscus or anterior cruciate ligament, posttraumatic osteoarthritis, primary osteoarthritis, or pyrophosphate arthritis.
Synovial fluid samples were collected from patients with knee disease diagnosed arthroscopically and radiologically. Concentrations of stromelysin-1, collagenase, and TIMP-1 were determined by sandwich immunoassay, using monoclonal and polyclonal antibodies. Fragments of cartilage proteoglycan containing the chondroitin sulfate-binding region were determined by immunoassay with a polyclonal antibody.
Average concentrations of metalloproteinases, TIMP, and proteoglycan fragments in joint fluid were significantly elevated in patients from all disease groups as compared with volunteers with healthy knees (reference group). Stromelysin concentrations in disease groups averaged 15-45 times that of the reference group. The molar ratios between stromelysin and collagenase varied between 10 and 150. The molar ratio between total stromelysin and free TIMP was 0.5 in the reference group and between 1.6 and 5.3 in the disease groups.
Stromelysin concentration in joint fluid is a parameter that distinguishes diseased joints from healthy joints, with a sensitivity of 84% and a specificity of 90%. The high concentrations of metalloproteinase relative to TIMP in joint fluid from patients with the conditions studied may be associated with cartilage matrix degradation in these arthritides.
The levels of metalloproteases and inhibitor expression in synovial membranes were measured by analyzing mRNA of collagenase, stromelysin, and tissue inhibitor of metalloproteinase (TIMP-1) in this tissue from 20 healthy persons and 20 patients with osteoarthritis (OA). Our results indicated that while most of the normal synovia expressed TIMP-1 mRNA at low levels, very few expressed metalloproteinase mRNA. In contrast 40% of patients with OA expressed a moderate level of collagenase compared to 55 and 80% cases with considerably elevated stromelysin and TIMP-1 mRNA, respectively. The mRNA expression of collagenase (p < 0.032), stromelysin (p < 0.0001) and TIMP-1 (p < 0.008) was significantly higher in OA than in normals. The greater abundance of stromelysin mRNA relative to collagenase indicates differential regulation of the 2 genes. We also demonstrated an association of IL-1 with metalloproteinase gene expression.
The loss of aggrecan from articular cartilage may lead to the development of osteoarthritis (OA). Degradation products of human aggrecan, generated in vivo by enzymatic cleavages, have been identified in synovial fluid of patients with rheumatoid arthritis and OA. One matrix metalloproteinase (MMP), stromelysin (MMP-3), and an unidentified proteinase called "aggrecanase" are believed to generate these products in pathologic conditions. Thus far, only one proteinase, neutrophil collagenase (MMP-8), has been shown in vitro to be capable of cleavage of the aggrecan molecule at the "aggrecanase" site. In this study, we compare the presence and distribution of MMP-3 and MMP-8 in cartilages from two different joints of normal human donors. We determined whether mRNA for MMP-8 is expressed in normal human articular cartilage from different joints. In addition, we compared differences in MMP-8 and MMP-3 gene expression between human ankle and knee cartilage after in vitro stimulation by interleukin (IL)-1 beta. These two joints were chosen because the incidence of symptomatic and radiographic OA varies between the different joints. The knee is the most frequently involved joint, whereas the ankle (talocrural) joint is relatively rarely affected. Message for MMP-8 was detected in untreated cartilage from normal knee joints, but not in untreated cartilage of normal ankle joints. Message for MMP-3 was detectable in most of the knee and ankle cartilages. Messenger RNA expression for both MMPs could be up-regulated by IL-1 beta. The highest doses of IL-1 beta appeared to be most effective in stimulation of mRNA for MMP-3, whereas MMP-8 expression was more sensitive to lower doses of IL-1 beta. The fact that ankle cartilage with a low incidence of OA does not express MMP-8, whereas knee cartilage with a high incidence of OA does not express MMP-8, whereas knee cartilage with a high incidence of OA does constitutively express MMP-8, suggests that MMP-8 might be one of the key enzymes in the pathogenesis of osteoarthritis. This is further supported by our finding that the earliest signs of cartilage degradation were very similar to those found in IL-1 beta-treated explants.
To analyze synovial membrane of patients with rheumatoid arthritis (RA) for the expression of unknown matrix metalloproteinases (MMP).
Degenerate oligonucleotides corresponding to highly conserved regions of the MMP gene family and the rapid amplification of cDNA ends (RACE) method have been used to search for new members of this gene family. MMP gene expression has been characterized by Northern blot analysis.
We cloned a MMP cDNA from the synovial membrane that is completely identical to the recently published collagenase 3 cDNA derived from a human breast cancer cDNA library (Freije, et al: J Biol Chem 1994;269:16766-73). Collagenase 3 is expressed in parallel with interstitial collagenase and stromelysin 1 in RA and osteoarthritis (OA). Collagenase 3 gene expression was not detected in several normal human tissues.
The expression of collagenase 3 in the synovial membrane in RA and OA suggests its involvement in articular tissue degradation.
Cytokines play a critical role in the pathogenesis of arthritis. The aim of this study was to compare the amounts of IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-alpha, TGF-beta 1 and IFN-gamma in the synovial fluid of 30 patients with rheumatoid arthritis (RA), 40 patients with osteoarthritis (OA) and 13 patients with seronegative spondylarthropathies (SpA).
Since some samples exhibited non-specific activities, all cytokines were measured by ELISA in the presence or absence of cytokine specific neutralizing antibodies.
Our data show that these cytokines can be detected in OA, RA and SpA. Compared to patients with OA, all cytokines were found in higher levels in patients with RA and SpA. Surprisingly, higher levels of IL-4, IL-10 and TGF-beta 1 were not associated with lower cytokine levels. In contrast, higher levels of IFN-gamma were associated with elevated monokine concentrations.
Our data indicate that the immunosuppressive effects of IL-4, IL-10 and TGF-beta 1 predicted from in vitro studies may not be active in vivo. Different monokine profiles could be observed in patients with IL-4 and/or IFN-gamma, indicating that the T cells involved in these diseases may have different immunoregulatory properties.
To investigate the in vivo effect of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) on the development of lesions and the expression of metalloproteases in the canine experimental osteoarthritis (OA) model.
The right anterior cruciate ligament was sectioned percutaneously in 3 groups of dogs. The control group (n = 5) received an intraarticular injection of sterile physiologic saline (1 ml) twice weekly for 4 weeks starting on the day of surgery. The remaining 2 groups received intraarticular injections of either 2 mg (n = 6) or 4 mg (n = 5) rHuIL-1Ra in 1 ml of physiologic saline according to the same schedule as the first group. All dogs were killed 4 weeks after surgery. The macroscopic appearance of femoral condyle osteophytes and the size and severity of cartilage lesions on femoral condyles and tibial plateaus were evaluated, as were the histologic features of cartilage and synovial membrane. Levels of collagenase-1 and stromelysin-1 messenger RNA expression in cartilage and synovium were determined by Northern blotting.
Recombinant human IL-1Ra exerted a dose-dependent protective effect on the development of osteophytes and cartilage lesions in vivo. Treatment with rHuIL-1Ra reduced the incidence (saline-treated group 70%, 2 mg rHuIL-1Ra-treated group 42%, 4 mg rHuIL-1Ra-treated group 20%) and size (saline-treated group 2.3 +/- 0.7 mm [mean +/- SEM], 2 mg rHuIL-1Ra-treated group 0.7 +/- 0.3 mm, 4 mg rHuIL-1Ra-treated group 0.5 +/- 0.3 mm) of femoral condyle osteophytes. In addition, a dose-dependent decrease in the size (saline-treated group 24.40 +/- 8.17 mm2, 2 mg rHuIL-1Ra-treated group 20.90 +/- 8.01 mm2, 4 mg rHuIL-1Ra-treated group 7.70 +/- 5.16 mm2) and the grade (0-4 scale; saline-treated group 1.20 +/- 0.29, 2 mg rHuIL-1Ra-treated group 1.00 +/- 0.26, 4 mg rHuIL-1Ra-treated group 0.30 +/- 0.21) of the tibial plateau cartilage lesions was found, with a significant difference (P < 0.04) reached only with 4 mg rHuIL-1Ra. Similarly, the histologic lesions in dogs treated with 4 mg rHuIL-1Ra (Mankin scale; mean +/- SEM 2.95 +/- 0.53) were significantly less severe (P < 0.002) compared with those in the saline-treated group (4.95 +/- 0.54). Importantly, rHuIL-1Ra treatment led to a significant reduction (P < 0.005) of collagenase-1 expression in OA cartilage.
This study demonstrated that intraarticular injections of rHuIL-1Ra can protect against the development of experimentally induced OA lesions. This effect could result, at least in part, from a reduction of collagenase-1 expression. However, other catabolic processes involved in the degradation of OA cartilage may also be affected.
To establish the presence of inflammation in and cytokine production by synovial membranes from patients with various stages of early osteoarthritis (OA), with knee pain, normal knee radiographs, and arthroscopic evidence of chondral damage.
Synovial membrane samples were obtained from the knees of 63 patients at the time of arthroscopy for unexplained knee pain or at the time of joint replacement surgery. Evaluations of synovial membrane variables including thickness of lining layer, vascularity, and inflammatory cell infiltrate were by a blinded observer. In a subset of 20 patients, production of interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) at the mRNA and protein levels was determined using in situ hybridization with biotin labeled ribo-probes and immunohistochemistry.
There was evidence of thickening of the lining layer, increased vascularity, and inflammatory cell infiltration in synovial membranes from patients with all grades of OA, with the most marked changes seen in synovial tissue from patients with advanced grades of OA. Similarly, production of IL-1 alpha, IL-1 beta, and TNF-alpha was present in synovial membranes from all patients with OA, irrespective of the degree of articular cartilage damage. There was a trend to decreased levels of IL-1ra in synovial membranes from patients with OA that did not attain statistical significance. Similarly, there was a decrease in the ratio of IL-1ra to IL-1 alpha and beta with increasing grades of OA.
Chronic inflammatory changes with production of proinflammatory cytokines are a feature of synovial membranes from patients with early OA, with the most severe changes seen in patients at the time of joint replacement surgery resembling those seen in rheumatoid arthritis. This low grade synovitis results in the production of cytokines that may contribute to the pathogenesis of OA.
To show the eventual presence and extent of production of matrix metalloproteinase 13 (MMP-13, or collagenase 3) in rheumatoid synovial tissue samples and extracts, and to assess the inhibition characteristics of recombinant MMP-13.
Immunohistochemical avidin-biotin-peroxidase complex staining/morphometry was used to analyze MMP-13-positive cells in situ. Neutral salt extraction of synovial tissue, electrophoresis of the extract in different buffer systems, and Western blotting were also used. The inhibitory properties of doxycycline, clodronate, pamidronate, and D-penicillamine for recombinant enzyme were determined with a soluble type II collagen assay.
MMP-13 was detected in fibroblast- and macrophage-like mononuclear cells in the synovial lining and stroma and in vascular endothelial cells. The overall expression of MMP-13 in these cells in the synovial stroma was high in rheumatoid arthritis (86 +/- 12%) compared with osteoarthritis (17 +/- 5%) patient samples (P = 0.0027). In a high-pH native electrophoresis gel, immunoreactivity to anti-MMP-1 and anti-MMP-13 were clearly separated, with anti-MMP-13-immunoreactive material migrating faster than anti-MMP-1-immunoreactive material. Finally, in contrast to MMP-1 and MMP-8, MMP-13 was found to be relatively resistant to the inhibitory effects of doxycycline and clodronate in vitro.
Due to its localization in synovial tissue, its substrate profile, increased expression, and relative resistance to known MMP inhibitors, MMP-13 is suggested to play a major role in the pathogenesis of tissue destruction in rheumatoid arthritis.
Four members of the tissue inhibitor of metalloproteinases (TIMP) family have been characterized so far, designated as TIMP-1, TIMP-2, TIMP-3, and TIMP-4. TIMP-1 and TIMP-2 are capable of inhibiting the activities of all known matrix metalloproteinases (MMPs) and as such play a key role in maintaining the balance between extracellular matrix (ECM) deposition and degradation in different physiological processes. Accelerated breakdown of ECM occurs in various pathological processes, including inflammation, chronic degenerative diseases and tumor invasion. TIMP-1 and TIMP-2 can inhibit tumor growth, invasion, and metastasis in experimental models which has been associated with their MMP inhibitory activity. Recent developments in TIMP research suggest that TIMP-1 and TIMP-2 are multifunctional proteins with diverse actions. Both inhibitors exhibit growth factor-like activity and can inhibit angiogenesis. Structure-function studies have separated the MMP inhibitory activity of TIMP-1 from its growth promoting effect. TIMP-1 has also been implicated in gonadal steroidogenesis and as a cellular elongation factor. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The most recently discovered TIMP, TIMP-4, may function in a tissue-specific fashion in extracellular matrix hemostasis. The main aim of this article is to review recent literature on TIMPs with special emphasis on their biological activities and the possibility that they may have paradoxical roles in tumor progression.
To assess the presence of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion.
Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis.
MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor alpha stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13.
These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.
Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-alpha (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 microM doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.
Molecular biology has provided various new insights into the mechanisms operative in the pathogenesis of rheumatoid arthritis. Reflecting the unique character of rheumatoid synovium, advances have been achieved addressing the molecular changes taking place at the area of interaction between the aggressively growing synovium and the articular cartilage and bone. Key issues in the review period addressing this interaction were hyperplasia of rheumatoid arthritis synovium, mechanisms of activation and cell cycle regulation of synovial fibroblasts, pathways of synovial attachment to cartilage and bone, and the regulation of matrix-degrading enzymes.
Matrix metalloproteinases (MMPs) are involved in connective tissue turnover under physiological and pathological conditions. MMP activity is regulated by the requirement for zymogen activation. This report describes a proMMP-3 activator produced by articular cartilage. The activator initiates a step-wise processing of proMMP-3 to generate an array of active species. Sequencing of activation intermediates demonstrated cleavage on the NH2-terminal side of certain basic residues in the MMP-3 propeptide. Metal ion chelators inhibited activator-dependent proteolysis, and activity was restored by low levels of ZnCl2. These catalytic properties suggest similarity to members of the insulinase superfamily of metalloendopeptidases with in vitro specificity for single arginine or paired basic processing sites in a variety of prohormones. Dibasic sites also exist in the propeptides of several MMPs including proMMP-3. This is the first report that cartilage produces a potent MMP proenzyme activator, opening the possibility of a novel intrinsic activation pathway for catabolic processes in this avascular tissue.
To study the expression of the cysteine proteinases cathepsin B and L and their most potent inhibitor cystatin C in the synovial membrane of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) on both the messenger RNA (mRNA) level and the protein level.
The expression of both cysteine proteinases and cystatin C was investigated in synovial tissue from 15 RA and 11 OA patients and compared with the expression of matrix metalloproteinase 1 (MMP-1; collagenase), MMP-3 (stromelysin), and tissue inhibitor of metalloproteinases 1 (TIMP-1). Expression of mRNA was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction. Protein expression was evaluated by immunohistochemistry. The results were correlated with the histologic evidence of inflammatory activity.
A significantly more pronounced expression of MMP mRNA was observed in RA synovium compared with OA. In contrast, the mRNA expression of cysteine proteinases, as well as TIMP-1 and cystatin C, did not differ between the patient groups. However, the protein expression of both MMP and cysteine proteinases was significantly more prominent in RA synovial lining compared with OA, whereas cystatin C and TIMP-1 protein were expressed equally.
The data indicate a more pronounced expression of MMP mRNA compared with cysteine proteinases in RA. The higher levels of cathepsin B and L proteins in RA synovial lining cells compared with OA are consistent with previous studies that assert a post-transcriptional up-regulation of these enzymes in RA.
The aim of this study was to define the relative regulation of matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinases-1 (TIMP-1), in chondrocytes and synovium in experimental osteoarthritis (EOA).
Partial-meniscectomized (PM) rabbits, surgical sham controls (SH), and normal non-surgical controls (N) were killed at times corresponding to early degenerative lesions (4 weeks) and increasingly progressive stages of EOA at 8 and 12 weeks post-PM. MMP-3 activity was measured in conditioned media from chondrocytes and synovium using a peptide cleavage assay with substance P (SP) as the substrate. TIMP-1 was quantitated using an enzyme-linked immunosorbent assay (ELISA).
Early degenerative lesions (4 weeks post-PM) were characterized by inflammatory responses in the synovium accompanied by a significant rise of MMP-3 activity in synovial cultures (P < 0.05). At 8 weeks there was no discernible inflammation, and MMP-3 activity in EOA synovial cultures was comparable to that in the controls; this was followed by a second increase in MMP-3 activity in EOA samples at 12 weeks. MMP-3 activity was significantly elevated in EOA chondrocyte cultures at 8 weeks post-PM relative to N controls, corresponding to the most destructive phase of EOA, but not in the early phase (4 weeks) or 'late' degenerative phase (12 weeks). Medium derived from chondrocytes contained little or no TIMP-1. Synovia secreted relatively higher amounts of TIMP-1, and this was elevated at 8 weeks post-PM relative to the SH controls. The majority (approximately 90%) of MMP-3 activity could be inhibited using recombinant TIMP-1 or a hydroxamate MMP inhibitor. Complete inhibition was achieved with EDTA or 1,10 phenanthroline.
Together, these data indicate that in EOA, MMP-3 is initially upregulated in the synovium which may play a pivotal role in the pathogenesis of cartilage lesions. In contrast, chondrocyte-derived MMP-3 is upregulated in the later phases of EOA, contributing further to progression of cartilage lesions.