Localization and expression of cartilage oligomeric matrix protein by human rheumatoid and osteoarthritic synovium and cartilage.

Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.     

人关节软骨脱细胞基质的制备

目的制备人关节软骨脱细胞基质。方法切取人关节软骨,对之冷冻干燥后,采取化学去污剂TritonX100及DNA酶和RNA酶等试剂制备脱细胞的人关节软骨。做HE、番红0及软骨蛋白聚糖(aggrecan)免疫组化染色等方法进行检测。结果HE染色、番红0染色均显示细胞陷窝内己无细胞结构番红花0染色阳性;软骨蛋白聚糖免疫组化染色阳性。结论人关节软骨冻干后,经去污剂酶等处理方法可脱去软骨的细胞成分,并且保留了软骨细胞外基质,成功制备了人关节软骨脱细胞基质。     

Increased degradation and alteered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage

We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67–94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominately localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrices. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrices, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis. (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oligomeric matrix protein in the different disease processes.     

S-100 protein in human articular cartilage

We studied the immunohistochemical distribution of the alpha and beta subunits of S-100 protein in human articular cartilage by the Avidin-Biotin Complex method using monoclonal antibodies against each subunit. Immunostaining for both subunits was detected in chondrocytes in superficial. intermediate, and deep zones of normal articular cartilage. In the matrix, only the superficial zone was stained positively. In arthrotic joints, we detected intense immunostaining in clustered chondrocytes in the hypercellular area. and weak or no immunostaining in isolated chondrocytes in the hypocellular area of articular cartilage.     

S-100 protein in human articular cartilage

We studied the immunohistochemical distribution of the alpha and beta subunits of S-100 protein in human articular cartilage by the Avidin-Biotin Complex method using monoclonal antibodies against each subunit. Immunostaining for both subunits was detected in chondrocytes in superficial, intermediate, and deep zones of normal articular cartilage. In the matrix, only the superficial zone was stained positively. In arthrotic joints, we detected intense immunostaining in clustered chondrocytes in the hypercellular area, and weak or no immunostaining in isolated chondrocytes in the hypocellular area of articular cartilage.     

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.     

Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotype.

Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies.     

Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.     

Isolation of RNA from small human articular cartilage specimens allows quantification of mRNA expression levels in local articular cartilage defects.

Human adult cartilage is an inherently difficult tissue from which to isolate RNA. The RNA isolation techniques described so far have generally only been successfully applied to the isolation of RNA from larger amounts of cartilage. However, it is important to be able to analyse focal cartilage lesions in order to understand the local processes in the cartilage degeneration process. Therefore, we have developed a protocol for isolating RNA directly from as little as 10 mg wet weight of cartilage followed by quantitative PCR analysis. We were able to analyse the expression levels of several genes in parallel including aggrecan and type II collagen.     

The characterization of the tidemark in human articular cartilage

The tidemark has been characterized in fourteen human diarthrodial joints using histochemical and biochemical techniques. It stained selectively for lipid as well as for the enzymes alkaline phosphatase and ATPase. Preliminary biochemical data has shown a high concentration of calcium phospholipid phosphate complexes.     

尿草酸钙晶体基质蛋白的分离及理化特征

为了进一步了解晶体基质蛋白（ＣＭＰ）的理化特性和其在结石形成中的作用，利用ＤＥＡＥＳｅｐｈａｄｅｘＡ５０、ＳｅｐｈａｄｅｘＧ２００对草酸钙结晶中的基质蛋白进行了分离，并用ＳＤＳＰＡＧＥ、双向电泳和Ｗｅｓｔｅｒｎｂｌｏｔ进行检测。结果表明：ＣＭＰ是草酸钙过饱和方法制备的晶体基质中的主要成分，其分子量为３１０００，等电点为５．０～５．５；Ｗｅｓｔｅｒｎｂｌｏｔ证明与抗人凝血酶原抗体有交叉反应。结果认为ＣＭＰ是活化的人凝血酶原片段     

Impact of knee joint loading on fragmentation of serum cartilage oligomeric matrix protein

The aim of the study was to examine the effect of mechanical knee joint loading on the fragmentation pattern of serum cartilage oligomeric matrix protein (COMP). Ten healthy men ran with knee orthoses that were passive or active (+30.9 N·m external flexion moments) on a treadmill (30 minute; v = 2.2 m/s). Lower-limb mechanics, serum COMP levels, and fragmentation patterns (baseline; 0, 0.5, 1, 2 hours postrunning) were analyzed. Running with active orthoses enhanced knee flexion moments, ankle dorsiflexion, and knee flexion angles (P < .05). There was an increase in serum COMP (+25%; pre: 8.9 ± 2.4 U/l; post: 10.7 ± 1.9 U/l, P = .001), COMP pentamer/tetramer (+88%; 1.88 ± 0.81, P = .007), trimer (+209%; 3.09 ± 2.65, P = .005), and monomer (+78%; 1.78 ± 0.85, P = .007) after running with passive orthoses and in serum COMP (+41%; pre: 8.5 ± 2.7 U/l; post: 11.3 ± 2.1 U/l, P < .001), COMP pentamer/tetramer (+57%; 1.57 ± 0.39, P = .007), trimer (+86%; 1.86 ± 0.47, P = .005), and monomer (+19%; 1.19 ± 0.34, P = .114) after running with active orthoses. Increased fragmentation might indicate COMP release from cartilage while running. Interestingly, 0.5 h up to 2 hours after running with passive orthoses, trimer (0.5 hour: 2.73 ± 3.40, P = .029; 2 hours: 2.33 ± 2.88, P = .037), and monomer (0.5 hour: 2.23 ± 2.33, P = .007; 1 hour: 2.55 ± 1.96, P = .012; 2 hours: 2.65 ± 2.50, P = .009) increased while after running with active orthoses, pentamer/tetramer (1 hour: 0.79 ± 0.28, P = .029), and trimer (1 hour: 0.63 ± 0.14, P = .005; 2 hours: 0.68 ± 0.34, P = .047) decreased. It seems that COMP degradation and clearance vary depending on joint loading characteristics.     

血清COMP在骨关节炎早期诊断中的作用

目的:运用血清COMP含量检测从有临床症状而未出现影像学改变的人群中筛选出骨关节炎(os-teoarthritis,OA)亚临床患者,从而开展OA的早发现、早治疗。方法:收集2007年8月至2009年9月入院诊断为膝骨关节炎患者或骨关节炎高危人群组115例(OA组),体检健康对照人群35例(健康组)。OA组男55例,女60例;年龄39～76岁,平均(55±13.32)岁;体重指数15.1～29.8;病程6～60个月。健康组男16例,女19例;年龄36～77岁,平均(53±12.53)岁;体重指数14.8～29.2。按照国际骨关节炎学会制定的诊断分类标准以及Kellgren和Lawrence衡量标准,确诊OA并分为Ⅰ-Ⅳ级,采用口服塞来昔布胶囊治疗,检测血清COMP含量并分析与OA级别的相关性。OA亚临床组患者随访调查24～38个月,平均33.4个月,检测随访前后的血清COMP含量。结果:健康组血清COMP含量受年龄影响较大(t=2.50,P=0.02),但健康组和OA组均不受性别(t=0.98,P=0.34;t=0.18,P=0.86),体重指数(t=0.56,P=0.92;t=0.17,P=0.85)和吸烟史(t=1.89,P=0.08;t=0.70,P=0.49)影响。随着OA分级的升高,血清COMP含量逐渐升高(F=15.56,P=0.001)。即使针对未出现影像学改变的OA亚临床患者,也可以在该患者确诊的2年前检测出显著的血清COMP含量升高,并能有效地将其与其他相关疾病的亚临床患者区分(t=2.55,P=0.03)。结论:血清COMP可作为潜在的生物学标记物为膝关节OA的亚临床和早期患者提供新的诊断指标。     

Enhanced concentration of COMP (cartilage oligomeric matrix protein) in osteochondral fractures from racing Thoroughbreds.

The aim of the present study was to correlate the levels of COMP and aggrecan as indicators of tissue damage, in synovial fluid (sf) from carpal joints of acutely lame racehorses, with macroscopical lesions of articular cartilage (OA), osteochondral fractures and ligament tears found at arthroscopy. Sixty-three lame horses [49 Standardbred trotters (STB) and 14 Thoroughbreds (TB)] in conventional training and racing that underwent arthroscopy of their middle carpal or radiocarpal joints were included in the study. Intact as well as fragmented COMP and aggrecan released into the synovial fluid were quantified by western blot analyses and ELISA. The expression of COMP in tissues was estimated by mRNA in situ hybridisation and protein immunolocalisation in cartilage and osteochondral fractures. The concentration of sf-COMP was higher in TB with an osteochondral fracture than in STB with osteochondral fractures and TB and STB with OA. The chondrocytes in middle and deep zones of the articular cartilage of the osteochondral fragments (from a TB) expressed COMP mRNA, in contrast to the cartilage on the opposite side of the fracture where no expression was detected. In the synovial fluid from a joint (TB) with osteochondral fractures only intact COMP was present, whereas, fragmented COMP was more prominent in synovial fluid from a joint with OA. The concentration of sf-aggrecan did not differ between the two breeds, or between different lesions. The increased concentration of sf-COMP in TB with osteochondral fractures, but not in synovial fluid from equine joints with OA, is a novel finding. The results from this study indicate that elevated sf-COMP concentration in the joints of Thoroughbreds may be a useful marker for carpal joint osteochondral fragments.     

骨关节炎患者滑液中COMP水平与病情严重程度的相关性研究

背景：软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）是软骨中非胶原蛋白的主要成分,软骨的损伤、修复和代谢变化均可能影响COMP的表达水平。目的：研究骨关节炎（osteoarthritis,OA）患者关节滑液中COMP水平与病变严重程度的相关性,探讨注射玻璃酸钠对关节滑液中COMP的影响。方法：52例膝关节0A患者接受关节内注射玻璃酸钠治疗,治疗前、治疗后6个月行X线片检查,按Kellgren放射学诊断标准评级,记录治疗前和治疗开始后5周、6个月时患者膝关节的WOMAC评分。采用ELISA方法测定在接受透明质酸钠治疗前、治疗开始后5周关节液中的COMP水平。19例因半月板或韧带损伤接受关节镜手术的患者作为正常对照组。结果：OA组患者滑液中COMP水平明显高于对照组,有统计学差异（P=0．036）。OA组患者滑液中COMP水平与OA严重程度（WOMAC评分）呈正相关（P=0．001）,与影像学Kellgren分级标准无相关性（P=0．12）。结论：滑液中COMP水平与OA病变严重程度呈正相关,测定OA患者血中COMP水平对进一步研究OA发病机理、早期发现并采取有效防治策略及监测防治效果具有极其重要的意义。     

Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts.

Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.     

Age-related biological characterization of mesenchymal progenitor cells in human articular cartilage

Adult articular cartilage has a low regeneration capacity due to lack of viable progenitor cells caused by limited blood supply to cartilage. However, recent studies have demonstrated the existence of chondroprogenitor cells in articular cartilage. A critical question is whether these mesenchymal progenitor cells are functionally viable for tissue renewal and cartilage repair to postpone cartilage degeneration. This study was designed to compare the number and function of mesenchymal progenitor cells in articular cartilage collected from human fetuses, healthy adults (aged 28-45 years), and elderly adults (aged 60-75 years) and cultured in vitro. We detected multipotent mesenchymal progenitor cells, defined as CD105+/CD166+ cells, in human articular cartilage of all ages. However, mesenchymal progenitor cells accounted for 94.69%±2.31%, 4.85%±2.62%, and 6.33%±3.05% of cells in articular cartilage obtained from fetuses, adults, and elderly patients, respectively (P<.001). Furthermore, fetal mesenchymal progenitor cells had the highest rates of proliferation measured by cell doubling times and chondrogenic differentiation as compared to those from adult and elderly patients. In contrast, alkaline phosphatase levels, which are indicative of osteogenic differentiation, did not show significant reduction with aging. However, spontaneous osteogenic differentiation was detected only in mesenchymal progenitor cells from elderly patients (with lower Markin scales). The lower chondrogenic and spontaneous osteogenic differentiation of mesenchymal progenitor cells derived from elderly patients may be associated with the development of primary osteoarthritis. These results suggest that measuring cartilage mesenchymal progenitor cells may not only identify underlying mechanisms but also offer new diagnostic and therapeutic potential for patients with osteoarthritis.