CD147分子和基质金属蛋白酶在类风湿关节炎滑膜中的表达

目的 探讨类风湿关节炎(RA)滑膜组织CD147的表达及其与基质金属蛋白酶(MMP)-01和MMP-2表达的相关性。方法 采用免疫组织化学SP(streptavidin／peroxidase)染色方法检测11例RA患者受损关节软骨-血管翳接合部（CPJ)滑膜组织中CD147和MMP-1及MMP-2的表达，并与3例骨关节炎（OA)患者滑膜组织CD147和MMP-1及MMP-2的检测相对照。结果 3例OA滑膜组织CD147和MMP-1及MMP-2的表达均为阴性，而11例RA滑膜组织中均有CD147和MMP-1及MMP-2的表达。其中，表达CD147的细胞为单核-巨噬细胞、淋巴细胞和滑膜成纤维样细胞，表达MMP-1及MMP-2的细胞为滑膜成纤维样细胞。统计学分析表明RA滑膜细胞CD147的表达和MMP-1及MMP-2的表达间存在显著相关性。结论 CD147在RA滑膜组织中表达增高，可能是导敏RA受损关节软骨、骨基质降解的重要因素之一。     

Glycation cross-links inhibit matrix metalloproteinase-2 activation in vascular smooth muscle cells cultured on collagen lattice

Aims/hypothesis. Extracellular matrix glycation has been proposed to contribute to the arterial stiffness observed in aging and diabetes. We examined whether matrix protein glycation regulates the proleolytic process through the manipulation of matrix metalloproteinases (MMPs) activation, using collagen fibrils model. Methods. Vascular smooth muscle cells were cultured on control or glycated collagen fibrils. Matrix metalloproteinase-2 activation and the production of tissue inhibitors of metalloproteinase (TIMPs) were measured in the conditioned medium by using gelatin zymography and immunoblotting. Membrane type 1 matrix metalloproteinase (MT1-MMP) expression was also measured in cell lysates. Results. When smooth muscle cells were cultured on collagen fibrils, pro-MMP-2 processing to active form was observed in the conditioned medium in coincidence with the increased MT1-MMP expression and the suppressed TIMP-2 production. Culturing smooth muscle cells on glycated collagen fibrils inhibited MMP-2 activation and attenuated MT1-MMP expression without the alteration of TIMP-2 production compared with control fibrils, indicating the possible mechanism of the suppression of MT1-MMP expression for the inhibition of MMP-2 activation on glycated collagen fibrils. Inclusion of aminoguanidine, an inhibitor of cross-linking formation, during collagen glycation restored the MMP-2 activation, suggesting the role of cross-links on the inhibition of MMP-2 activation. Conclusion/interpretation. These observations suggest that glycation-induced cross-linking formation in interstitial collagen contributes to arterial stiffness in aging and diabetes through the manipulation of matrix metalloproteinase activation along with the reduction of the susceptibility to proteolytic enzymes. [Diabetologia (2001) 44: 433–436] Received: 16 October 2000 and in revised form: 18 December 2000     

Involvement of membrane-type matrix metalloproteinases (MT-MMPs) in capillary tube formation by human endometrial microvascular endothelial cells: role of MT3-MMP

In the endometrium, angiogenesis is a physiological process, whereas in most adult tissues neovascularization is initiated only during tissue repair or pathological conditions. Pericellular proteolysis plays an important role in angiogenesis being required for endothelial cell migration, invasion, and tube formation. We studied the expression of proteases by human endometrial microvascular endothelial cells (hEMVECs) and their involvement in the formation of capillary tubes and compared these requirements with those of foreskin MVECs (hFMVECs). Inhibition of urokinase and matrix metalloproteinase (MMP) both reduced tube formation in a fibrin or fibrin/collagen matrix. hEMVECs expressed various MMP mRNAs and proteins; in particular MMP-1, MMP-2, and membrane-type (MT)1-, MT3-, and MT4-MMPs. MT3- and MT4-MMP mRNA expressions were significantly higher in hEMVECs than in hFMVECs. Other MT-MMP mRNAs and MMP-9 were hardly detectable. Immunohistochemistry confirmed the presence of MT3-MMP in endothelial cells of endometrial tissue. Overexpression of tissue inhibitor of MMP (TIMP)-1 or TIMP-3 by adenoviral transduction of hEMVECs reduced tube formation to the same extent, whereas only TIMP-3 was able to inhibit tube formation by hFMVECs. Tube formation by hEMVECs was partly inhibited by the presence of anti-MT3-MMP IgG. Thus, in contrast to tube formation by hFMVECs, which largely depends on MT1-MMP, capillary-like tube formation by hEMVECs is, at least in part, regulated by MT3-MMP.     

实验性肝纤维化逆转过程中基质金属蛋白酶表达的动态研究

探讨基质金属蛋白酶在实验性肝纤维化逆转过程中的表达及意义。建立大鼠实验性四氯化碳中毒性肝纤维化模型,采用核酸原位杂交和免疫组化法检测实验性肝纤维化自然逆转过程中MMP-13、MMP-2、MT—MMP2的来源细胞、时序和量的变化。MMP-13、MMP-2、MT—MMP2在间质细胞和部分肝细胞表达,MMP-13先维持在一定水平,然后逐渐减弱;MMP-2、MT—MMP2先增强后减弱。肝脏间质细胞是基质金属蛋白酶的主要来源细胞,MMP-13、MMP-2、MT—MMP2表达与肝纤维化程度相关,在肝纤维化逆转过程中起重要作用。     

Expression of membrane type 1 matrix metalloproteinase, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 in human cartilaginous tumors with special emphasis on mesenchymal and dedifferentiated chondrosarcoma

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as an activator of the proenzyme of matrix metalloproteinase 2 (MMP-2: gelatinase A), and has also been shown to play a crucial role in tumor invasion by activating proMMP2 in both lung and gastric carcinoma. The tissue inhibitor of metalloproteinase 2 (TIMP-2) plus the MT1-MMP complex also plays an important role in the activation of proMMP-2. In this study, the expressions of MT1-MMP, MMP-2 and TIMP-2 were evaluated in 10 enchondromas, 34 conventional chondrosarcomas, 5 clear-cell chondrosarcomas, 7 mesenchymal chondrosarcomas and 8 dedifferentiated chondrosarcomas. The expressions were immunohistochemically visualized on paraffin sections and the levels of expression were assessed semiquantitatively. The extent of staining was assessed by the extent score in order to determine the overall level of expression. The extent scores of MT1-MMP, MMP-2 and TIMP-2 in grade 2 chondrosarcoma were significantly higher than those in either enchondroma or grade 1 chondrosarcoma (P < 0.05). In conventional chondrosarcoma, significant correlations were found between the extent scores of MT1-MMP and MMP-2 (P < 0.001), MT1-MMP and TIMP-2 (P < 0.01), and MMP-2 and TIMP-2 (P < 0.01). The undifferentiated small round tumor cells of mesenchymal chondrosarcoma showed lower positive rates and extent scores for MT1-MMP (2/7, 0.7 ± 0.5) and MMP-2 (3/7, 0.7 ± 0.4) than for cartilaginous components of mesenchymal chondrosarcoma [MT1-MMP (4/7, 1.3 ± 0.5) and MMP-2 (7/7, 1.9 ± 0.3)] or conventional chondrosarcoma. In dedifferentiated chondrosarcoma, the extent scores of MT1-MMP, MMP-2 and TIMP-2 in low-grade cartilaginous components were not significantly different from those in conventional chondrosarcoma; however, the high-grade anaplastic components showed high extent scores for MT1-MMP, MMP-2 and TIMP-2, compared with the low-grade cartilaginous components of dedifferentiated chondrosarcoma or conventional chondrosarcoma. According to our results, the expression of MT1-MMP as well as that of MMP-2 or TIMP-2 demonstrated a significant correlation with the tumor grade in human cartilaginous tumors. Furthermore, the expressions of MT1-MMP, MMP-2 and TIMP-2 were also found to play a crucial role in invasion in the high-grade components of dedifferentiated chondrosarcoma. Received: 17 February 1999 / Accepted: 21 April 1999     

Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis

Objective. To determine the expression of matrix metalloproteinase 9/gelatinase B (MMP-9) in synovial fluid (SF), plasma, and synovial tissue from individuals with rheumatoid arthritis (RA), inflammatory arthritis (IA), and osteoarthritis (OA), using specific monoclonal antibody reagents. Methods. Gelatinolytic activity in the SF and plasma of patients with RA, IA, and OA was assessed by gelatin zymography. A mouse monoclonal antiserum, 277.13, which selectively recognizes soluble latent forms of human MMP-9, was used to quantitate MMP-9 levels in patient synovial effusions, plasma, and synovial tissue with a capture sandwich enzyme-linked immunosorbent assay (ELISA). Fifty-one SF samples (31 RA, 9 OA, 11 IA) were analyzed. Immunolocalization of MMP-9 in RA, OA, and normal synovium was investigated using MMP-9–specific antisera. Results. MMP-9 antigen levels in synovial effusions were elevated 67-fold in RA samples compared with OA samples. In addition, although MMP-9 antigen levels in IA synovial effusions were 2.7-fold less than the values in RA samples, they were elevated 34-fold over the values in OA samples. These data indicate an association between increased MMP-9 levels and inflammatory arthritis. A predominant 92-kd gelatinolytic activity (specifically inhibited by EDTA) was evident in RA and IA samples, but no activity was observed in OA samples. Among 86 plasma samples (17 RA, 9 IA, 60 normal controls) analyzed for MMP-9 antigen levels by immunocapture ELISA, MMP-9 antigen levels were elevated 7-fold in RA plasma compared with normal plasma. RA synovial tissue extracts demonstrated elevated levels of MMP-9 antigen compared with OA synovial tissue. MMP-9 immunolocalization studies demonstrated expression in infiltrating leukocytes (neutrophils and macrophages), endothelial cells, and synovial fibroblasts in RA synovium. Conclusion. Latent MMP-9 and/or MMP-9–tissue inhibitor of metalloproteinases 1 (TIMP-1) complexes are elevated in RA and IA SF compared with OA SF. In addition, MMP-9 is increased in RA plasma versus normal control plasma. Synovial tissue levels of MMP-9 antigen are also elevated in RA versus OA. The tissue distribution of MMP-9 within RA synovium is localized to sites of inflammation comprising surface synovial lining cells, endothelium, and leukocytes. Taken together, these observations suggest that connective tissue turnover occurs as a result of excessive MMP activity over TIMP action in the invading pannus, periarticular tissue, or SF. Further studies such as those used in the present investigation will help elucidate the role of a number of different enzymes and inhibitors in the destructive arthropathies.     

1,25-二羟维生素D3对人成骨细胞基质金属蛋白酶及其抑制因子的作用

目的　研究 1, 25 二羟维生素D3 [ 1α, 25 (OH)2D3 ]对人成骨细胞基质金属蛋白酶(MMP) 1、MMP 2、膜型基质金属蛋白酶 1 (MT1 MMP)、基质金属蛋白酶抑制因子 1 (TIMP 1 )的影响,探讨 1α, 25(OH)2D3 调节骨代谢作用机制。方法　人成骨细胞用 1α, 25 (OH)2D3 干预。Western杂交检测MT1 MMP蛋白质表达。MMP 1、MMP 2、TIMP 1的分泌及MMP 2的活性用ELISA检测。Northern杂交检测维生素D受体、MT1 MMPmRNA表达。结果　 1α, 25 (OH)2D3 对人成骨细胞MMP 1、MMP 2、TIMP 1表达无影响, 10-10 ~ 10-8 mol/L 1α, 25 (OH)2D3 干预诱导成骨细胞MT1 MMP表达呈剂量依赖性 (P值均 <0 05);促进MMP 2激活呈剂量依赖性 [MMP 2活性分别为(42 3 ± 8 6)、(64 4 ±11 4)、(93 5 ±9 9)μg/L, P值均<0 05]。结论　由于MT1 MMP在骨吸收过程中起着关键作用, 1α, 25(OH)2D3 可通过诱导成骨细胞MT1 MMP表达刺激骨吸收。     

Increased level of heme oxygenase-1 in rheumatoid arthritis synovial fluid

Abstract

We investigated the expression and localization of heme oxygenase-1 (HO-1) in synovial fluid and synovial tissue, and examined the stimulation of HO-1 production in rheumatoid synovial fibroblasts (RASFs). Synovial fluid samples were obtained from knee joints of 20 rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients, and concentration of HO-1 and matrix metalloproteinase-3 (MMP-3) were measured by enzyme-linked immunosorbent assay (ELISA). Synovial tissues obtained from RA or OA patients during total knee arthroplasty (TKA) were used for immunohistochemical analysis of HO-1. HO-1 production by RASFs in response to various cytokines was examined by ELISA. HO-1 levels in synovial fluid were higher in the RA group than in the OA group with significant difference (P < 0.001), and correlated with serum C-reactive protein (CRP) level (r = 0.80, P < 0.01) in the RA group. Higher levels of HO-1 were seen in the RA-L group (Larsen grade III–V) than in the RA-E (Larsen grade 0-II) group (P < 0.001). There was weak correlation between the levels of HO-1 protein and MMP-3 in synovial fluid in the RA group (r = 0.31, P < 0.01), while no positive correlation was observed in OA. Positive immunoreaction for HO-1 was observed in cells of synovial tissue including synovial fibroblasts and cells in synovial pannus. HO-1 protein levels in cultured media of RASFs were increased by stimulation by interleukin-1β at 6 h and tumor necrosis factor-alpha at 12 h, but suppressed by interferon-gamma at 12 and 24 h. These results indicated that HO-1 expression in synovial tissue might be stimulated by inflammatory cytokines. The correlation of HO-1 concentration in synovial fluid with serum CRP and MMP-3 in joint fluid indicated that HO-1 might be useful as a marker of joint inflammation in RA patients.     

EMMPRIN/CD147对类风湿关节炎患者滑膜成纤维细胞合成基质金属蛋白酶的刺激作用

目的 观察细胞外基质金属蛋白酶诱导剂（EMMPRIN／CD147）对类风湿关节炎（RA）患者滑膜成纤维细胞（FLS）合成基质金属蛋白酶（MMPs）的作用。方法 手术切除的RA患者的滑膜组织，分离成FLS，传代培养。明胶酶谱法测FLS的MMPs含量将FLS与高表达EMMPRIN／CD147的单核细胞株（THP）-1共培养，观察THP-1细胞对FLS MMPs表达量的影响，同时观察EMMPRIN／CD147拮抗肽激活蛋白（AP）-9对FLS MMPs表达的影响。结果 THP-1细胞表达高水平的CD147，而MMP-2、MMP-9的表达水平很低；分离的FLS表达低水平的CD147和一定水平的MMP-2、MMP-9。二者混合培养后，FLS表达MMP-2、MMP-9水平显著升高，随着THP-1细胞数的增加，MMP-2、MMP-9的合成量也增加THP-1对FLS合成MMP-2、MMP-9的刺激作用可被CD147拮抗肽AP-9所抑制。结论 CD147对RA患者FLS合成MMP-2、MMP-9有刺激作用，而这种刺激作用呵被CD147拮抗肽AP-9所抑制。     

Induction of membrane-type matrix metalloproteinase-1 stimulates angiogenic activities of bovine aortic endothelial cells

Matrix metalloproteinases (MMPs) have been reported to play critical roles in endothelial cell migration and matrix remodeling during the angiogenic process. Among these MMPs, membrane-type MMP-1 (MT1-MMP) is an important molecule that can trigger the invasion of tumor cells by activating MMP-2 on their plasma membrane. However, the precise involvement of MT1-MMP in the angiogenic process has not been determined. To investigate the roles of the MT1-MMP by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased expression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased in MT1-MMP transfectants. However, cotransfection with antisense MT1-MMP expression vector abolished the effects of MT1-MMP overexpression. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

TIMP-2、MMP-2和MT1-MMP在活化肝星状细胞中的表达及对细胞外基质合成分泌的影响

目的 研究金属蛋白酶组织抑制剂-2(TIMP-2)、基质金属蛋白酶-2(MMP-2)和膜型基质金属蛋白酶-1(MT1-MMP)在活化肝星状细胞(HSCs)中的表达,观察其对细胞外基质(ECM)合成分泌的影响.方法 原代分离培养大鼠HSCs活化后,分别给予40～160 pmol化学合成经修饰抗TIMP-2 siRNA进行干预,检测培养细胞上清液透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和羟脯氨酸(Hyp)的含量,采用荧光实时定量PCR法检测TIMP-2、MMP-2、MT1-MMP、MMP-13、COL Ⅰ和COL Ⅲ mRNA的表达,western印迹检测TIMP-2、MT1-MMP和MMP-13蛋白表达及明胶酶谱法检测MMP-2蛋白表达.结果 应用化学合成经修饰抗TIMP-2 siRNA后,TIMP-2、MMP-2、MT1-MMP、COL Ⅰ和COL Ⅲ的表达明显降低,而MMP-13的表达则明显增加,培养细胞上清液中HA、PCⅢ和Hyp的含量也明显减少.结论 TIMP-2通过MT1-MMP介导MMP-2的活化,抑制TIMP-2的表达,MT1-MMP和MMP-2的表达随之降低,而HSCs合成分泌ECM也相应减少.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE: Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS: Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [(3)H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS: Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS: Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.