Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts

OBJECTIVE: Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. METHODS: Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. RESULTS: MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). CONCLUSION: The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.     

Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo. METHODS: MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA. RESULTS: Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA. CONCLUSION: Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.     

Elevation of activated protein C in synovial joints in rheumatoid arthritis and its correlation with matrix metalloproteinase 2

OBJECTIVE: To investigate the involvement of the anticoagulant serine protease activated protein C (APC) in tissue remodeling in rheumatoid arthritis (RA). METHODS: PC/APC, matrix metalloproteinase 2 (MMP-2), and MMP-9 were detected in synovial fluid by Western blotting, and their antigen levels were quantified by enzyme-linked immunosorbent assay in patients with osteoarthritis (OA) or RA. Enzymatic activity of MMP-2 was assayed using a specific fluorogenic substrate. We developed an improved assay to measure APC activity in synovial fluid utilizing a chromogenic substrate following immunoprecipitation with a specific PC/APC antibody. PC/APC and MMP-2 were localized by immunohistochemistry in RA, OA, and normal synovial tissues. RESULTS: Synovial fluid analysis demonstrated that APC is present in both RA and OA synovial fluid, with APC activity being markedly higher in RA (mean +/- SEM 462 +/- 112 ng/ml versus 136 +/- 42 ng/ml; P < 0.02). A correlation (r(2) = 0.61) was found between APC and MMP-2 activity levels in RA patients, but not in OA patients. Immunohistochemical studies of synovial sections showed colocalization of APC and MMP-2 in endothelial and synovial lining cells. Additionally, APC and MMP-2 coimmunoprecipitated with an anti-PC/APC antibody. CONCLUSION: Our results show, for the first time, that APC and MMP-2 are coordinately up-regulated and tightly bound in RA synovial fluid and colocalized in synovia. Their association suggests that APC may modulate MMP-2 activity in RA.     

Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1beta (IL-1beta)-induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene alpha (GROalpha/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROalpha produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-kappaB. RESULTS: EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 microM or 20 microM significantly inhibited IL-1beta-induced ENA-78, RANTES, and GROalpha, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 microM caused a complete block of IL-1beta-induced production of RANTES, ENA-78, and GROalpha, and reduced production of MCP-1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1beta-induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCdelta and inhibited the activation and nuclear translocation of NF-kappaB in IL-1beta-treated RA synovial fibroblasts. CONCLUSION: These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.     

p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion

OBJECTIVE: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF. METHODS: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies. RESULTS: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process. CONCLUSIONS: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.     

膜型基质金属蛋白酶-1与肿瘤的侵袭和转移的研究进展

基质金属蛋白酶在肿瘤侵袭和转移中起着非常重要作用,而膜型基质金属蛋白酶是一类基质金属蛋白酶家族的新成员,其中膜型基质金属蛋白酶-1是研究最深、意义极为重要的一种,它直接或间接降解细胞外基质中的多种成分,通过调节多种细胞效应分子,影响肿瘤细胞的浸润、转移以及血管生成等过程,在肿瘤的浸润和转移的过程中有重要作用.     

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.     

Early joint erosions and serum levels of matrix metalloproteinase 1, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 in rheumatoid arthritis

OBJECTIVE: To further evaluate the roles of matrix metalloproteinase 1 (MMP-1), MMP-3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) in the pathogenesis of joint inflammation and articular erosions in early inflammatory arthritis. METHODS: Untreated patients with joint symptoms for <2 years were evaluated at presentation and followed up prospectively for 18 months. Swollen joint count and serum levels of C-reactive protein (CRP) were determined every 6 months. Serum levels of MMP-1, MMP-3, and TIMP-1 were measured by double-antibody sandwich enzyme-linked immunosorbent assay at the same time intervals. The number of joint erosions in serial radiographs of the hands and feet was also recorded. Analysis of synovial fluid levels of MMPs and TIMP-1 at presentation was completed in some patients. RESULTS: Of 175 patients evaluated at baseline, 85 had rheumatoid arthritis (RA), 39 had seronegative spondylarthropathy, 38 had undifferentiated arthritis, and 13 had self-limiting arthritis. Of 164 patients with available radiographs of the hands and feet at presentation, 33 (20.1%) had joint erosions. Baseline levels of MMP-1, MMP-3, and TIMP-1 were significantly higher (P = 0.0001, P = 0.013, and P = 0.0001, respectively) and ratios of TIMP-1:MMP-1 and TIMP-1:MMP-3 were significantly lower (P = 0.0001 and P = 0.013, respectively) in RA versus non-RA patients. In RA patients, serum levels of CRP correlated with MMP-3 and TIMP-1 levels, but not with MMP-1 levels. The number of erosions at presentation correlated with baseline levels of both MMP-1 and MMP-3, but not with levels of TIMP-1. One hundred one patients were followed up for the next 18 months. The number of patients with erosions and the number of erosions per patient increased significantly during this period. Area under the curve (AUC) measurements of MMP-1 and TIMP-1 levels, but not of MMP-3 levels, yielded significantly higher values in RA than in non-RA patients. In RA patients, only the AUC level of MMP-3 correlated with the AUC CRP level (r = 0.67, P = 0.0001), while only the AUC level of MMP-1 correlated with the number of new joint erosions (r = 0.28, P = 0.034). CONCLUSION: These data suggest an uncoupling of the pathophysiologic mechanisms associated with joint inflammation and articular erosion. Treatments that inhibit the production and activity of MMP-1 may preferentially limit the formation of new joint erosions and improve the long-term functional outcome of some patients with inflammatory arthritis.     

BAT1 promoter polymorphism is associated with rheumatoid arthritis susceptibility

OBJECTIVE: To analyze whether the polymorphisms -22 (G/C) and -348 (C/T) of the BAT1 gene are associated with susceptibility to rheumatoid arthritis (RA). METHODS: One hundred fifty-six patients with RA and 154 controls were genotyped for HLA-DRB1 and the polymorphisms -22 and -348 of the BAT1 gene. RESULTS: HLA-DRB1*04 alleles were associated with RA susceptibility (33.9% vs 20.1%; pc = 0.04). Among these, HLA-DRB1*0401 (13.4% vs 5.1%; pc = 0.04) and HLA-DRB1*0404 (5.7% vs 1.2%; pc = 0.2) were increased in patients with RA. Additionally, carriage of BAT1 -348T polymorphism was strongly associated with RA (23.7% vs 12.1%; pc = 0.0002). Significantly, BAT1 -348T was in linkage disequilibrium with HLA-DRB1*0404 and HLA-DRB1*0405. However, BAT1 -348 T was associated independently with HLA-DRB1 shared-epitope alleles (42.6% vs 18.9%; p = 0.001). CONCLUSION: The BAT1 -348T polymorphism is associated with RA susceptibility independently of HLA-DRB1. The role of BAT1 in the regulation of tumor necrosis factor-a suggests that BAT1 may regulate the inflammatory response observed in patients with RA.     

Serum matrix metalloproteinase activity relating to cartilage destruction in rheumatoid arthritis

Aims: The aim of this study was to determine which matrix metalloproteinases (MMPs) are involved with C‐reactive protein (CRP) and which stage of rheumatoid arthritis (RA) correlated with MMP‐9 or MMP‐13 level. Methods: In the present clinical trial, we analysed MMP‐9 activity and MMP‐13 activity in 53 RA patients. We examined the presence of MMP‐9 and MMP‐13 in the synovium tissue and articular cartilage in RA patients by immunohistochemistry. In addition, we compared these factors and clinical Steinbrocker staging. Results: We found that MMP‐9 in blood serum correlates significantly with CRP. MMP‐13 also correlates with CRP but the coefficiency with CRP is much higher in MMP‐9 (r = 0.6694) than in MMP‐13 (r = 0.4037). MMP‐9 and MMP‐13 did not correlated with rheumatoid factor (RF). MMP‐9 level is increased in stages II and III in RA. On the other hand, MMP‐13 is increased in stages III and IV. Our results indicated that early stage RA shows high MMP‐9 release in serum while late stage RA shows high MMP‐13 release. Conclusion: MMP‐9 activity may correlate with synovium proliferation with vascularization, and serum MMP‐13 activity may correlate with the grade of joint destruction in rheumatoid arthritis.     

Serum matrix metalloproteinase 3 in early rheumatoid arthritis is correlated with disease activity and radiological progression

OBJECTIVE: To analyze the clinical significance of serial measurements of serum matrix metalloproteinase 3 (MMP-3) levels in relation to markers of disease activity and radiological progression in early rheumatoid arthritis (RA). METHODS: In a 3 year prospective study of 33 patients with early RA (symptoms < 1 year at entry) monthly measurements of serum MMP-3 were transformed into time integrated values for 6 month periods for comparison with other markers of disease activity like swollen joint count (SJC), tender joint count (TJC), Ritchie articular index (RAI), the disease activity score (DAS), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and radiological progression, scored according to Sharp's method, in which erosions and joint space narrowing are scored separately and combined to a total Sharp score. RESULTS: Significant correlations were found between serum MMP-3 and SJC, ESR, and CRP during all periods and between 6 and 30 months with the DAS. There were no correlations between serum MMP-3 and TJC or the RAI. During the first 12 months serum MMP-3 was correlated only with the item joint space narrowing of the Sharp score. After 12 months of followup it was also correlated with the total Sharp score and after 18 months it was correlated with all 3 items of the Sharp score. There was a wide interindividual variation in the relation between serum MMP-3 and radiological progression but intraindividually this relation seemed to be rather constant. CONCLUSION: Time integrated values of serum MMP-3 are correlated with time integrated values of other markers of disease activity such as joint swelling, ESR, CRP, and the DAS. Of the radiological scores, as outcome measures, especially joint space narrowing correlated closely with cumulative serum MMP-3.     

Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein

BACKGROUND: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction. OBJECTIVE: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components. METHODS: Cell culture plates were coated with fibronectin, collagens I-VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37 degrees C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor alpha (TNF alpha) or interleukin 1 beta. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion. RESULTS: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNF alpha reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNF alpha. CONCLUSION: The increase of galectin-3 occurs after adhesion to COMP, and the alpha V beta 3 receptor (CD51/CD61) has a pivotal role in this process.     

Eotaxin-induced expression of membrane type 1 matrix metalloproteinase mRNA in human eosinophils

Eosinophil penetration across the basement membrane (BM) is thought to be dependent on the degradation of membrane components. In this process, matrix metalloproteinases (MMP) appear to be primarily responsible for degradation of the BM. Matrix metalloproteinases -2 and MMP-9 degrade type IV collagen, which is a major component of the BM. In the present study, the effects of eotaxin, a selective chemoattractant for eosinophils, on the expression of MMP mRNA were examined. Incubation with chemotactically active concentrations of eotaxin for 24 h enhanced the expression of mRNA for membrane-type 1 MMP (MT1-MMP), but not of mRNA for MMP-2 and MMP-9. An increase in the protein level of MT1-MMP was also detected in the cell lysate of eotaxin-treated eosinophils. These results suggest that up-regulation of MT1-MMP expression may be involved in the eotaxin-induced penetration of eosinophils.     

Reduction of serum matrix metalloproteinase 1 and matrix metalloproteinase 3 in rheumatoid arthritis patients following anti- tumour necrosis factor-alpha (cA2) therapy

Matrix metalloproteinase (MMP)-1 and MMP-3 levels were measured in serum samples from rheumatoid arthritis (RA) patients undergoing a double-blinded placebo-controlled trial with the chimaeric anti-tumour necrosis factor (TNF)-alpha antibody cA2. Both MMP-1 (P < 0.015), but to a larger extent MMP-3 (P < 0.001) levels were elevated in all RA patients prior to the commencement of the trial compared with normal control sera. Following cA2 therapy, MMP-1 and MMP-3 levels were assessed in the placebo, and 1 and 10 mg/kg cA2-treated groups at 7, 14, 21 and 28 days. In both the 1 and the 10 mg/kg cA2-treated groups, a significant decrease in serum MMP-3 levels at all time points was observed, reducing maximally to 41% of pre-infusion values at day 7. MMP-1 levels were also reduced, but less dramatically than MMP-3, to 85% of pre-infusion values after 14 days in the 10 mg/kg cA2 treated group. In a separate non-placebo-controlled study, we also evaluated the tissue inhibitor of metalloproteinase (TIMP)-1 levels in plasma following cA2 infusion. Pre-infusion TIMP-1 levels were above the normal control range, but were significantly reduced (P < 0.035) 14 days after infusion to 72% of pre-infusion values. This study confirms previous reports that MMP-3 levels are elevated and correlate with measures of inflammation in RA, and furthermore demonstrate that serum MMP-3 and MMP-1 levels are downmodulated following anti-TNF-alpha antibody therapy. Whilst serum MMP-3 levels correlated with C-reactive protein (CRP) both prior to and following anti-TNF-alpha antibody therapy, it remains to be demonstrated that serum MMP-3 and/or MMP-1 levels reflect the cartilage and bone resorptive processes which are evident in this disease.