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.     

The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles

Rheumatoid arthritis is a chronic inflammatory disease characterized by destruction of cartilage and bone that is mediated by synovial fibroblasts. To determine the mechanisms by which these cells are activated to produce matrix metalloproteinases (MMPs), the effects of microparticles were investigated. Microparticles are small membrane-bound vesicles whose release from immune cells is increased during activation and apoptosis. Because microparticles occur abundantly in the synovial fluid in rheumatoid arthritis, they could represent novel stimulatory agents. Microparticles derived from T cells and monocytes strongly induced the synthesis of MMP-1, MMP-3, MMP-9, and MMP-13 in fibroblasts. The induction was time-dependent, with effects primarily observed after 36 h; under these conditions, MMP-2, MMP-14, and tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and TIMP-3 were not induced. Microparticles also increased the synthesis of inflammatory mediators including IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and MCP-2. In Ikappa-B-transfected synovial fibroblasts, MMPs were less inducible by microparticles compared with wild-type fibroblasts. Blocking of TNFalpha and IL-1beta with antibodies against TNFalpha and with IL-1 receptor antagonist did not abrogate stimulation by microparticles. These data provide evidence for a novel mechanism by which vesicles derived from activated or apoptotic immune cells can promote the destructive activity of synovial fibroblasts in rheumatoid arthritis.     

Urokinase induces matrix metalloproteinase-9/gelatinase B expression in THP-1 monocytes via ERK1/2 and cytosolic phospholipase A2 activation and eicosanoid production

OBJECTIVE: Urokinase-type plasminogen activator (uPA) regulates cell migration and invasion by pericellular proteolysis and signal transduction events. We characterized the mechanisms by which uPA regulates matrix metalloproteinase-9 (MMP9) function in THP-1 monocytes. METHODS AND RESULTS: In THP-1 monocytes, MMP9 production induced by urokinase was completely inhibited by the ERK1/2 inhibitor, PD98059, but not by the p38 mitogen-activated protein kinase inhibitor, SB202190. A dominant negative MEK1 adenovirus also blocked MMP9 expression. The effect of urokinase was completely suppressed by genistein and by herbimycin A indicating that tyrosine kinase(s) are required for MMP9 production. Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, did not decrease MMP9 expression suggesting that PKC activation is not required. Key roles for cytosolic phospholipase A2 (PLA2) and eicosanoid production were shown by complete inhibition with methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA2), and indomethacin (a cyclooxygenase inhibitor), with no effect of monoalide, a secretory PLA2 inhibitor. uPA stimulated phosphorylation of cytosolic PLA2. CONCLUSIONS: Induction of MMP9 by uPA in THP-1 monocytes is via a pathway involving MEK1-ERK1/2-mediated activation of cytosolic PLA2 and eicosanoid generation. These data suggest important roles for eicosanoids in monocyte migration induced by uPA and MMP9.     

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.     

葛根素对兔动脉粥样硬化基质金属蛋白酶-9及其组织抑制物-1表达的影响

目的：观察葛根素对动脉粥样硬化兔髂动脉分泌和表达基质金属蛋白酶-9（MMP-9）及其组织抑制物-1（TIMP-1）的影响。方法：20只家兔分为正常对照组（正常饮食，6只）、病理对照组（球囊和高脂饮食，8只）和葛根素组（球囊、高脂饮食和葛根素，8只）。球囊损伤后4周处死，取一侧病变髂动脉做病理切片，应用免疫组化法测定MMP-9和TIMP-1的蛋白表达；取另一侧病变髂动脉抽提总RNA应用半定量逆转录多聚酶链式反应（RT—PCR）测定MMP-9和TIMP-1 mRNA的表达。结果：兔动脉粥样斑块MMP-9 mRNA（mRNA／GAP—DH mRNA）表达：正常对照组、病理对照组、葛根素组的分别为0．81±0．17，1．52±0．24，1.03±0．19，病理对照组、葛根素组的较正常对照组显著增加（P〈0．05），而葛根素组的较病理对照组显著下降（P〈0．05），上述三组的TIMP—1 mRNA的表达依次为1．44±0．14，2．63±0．16，2．67±0．12，病理对照组与葛根素组的较正常对照组显著增加（P〈0．05），但病理对照组与葛根素组间无显著差异（P〉0．05）。免疫组化检测显示葛根素抑制MMP-9蛋白质表达（P〈0．05），但对TIMP-1蛋白质的表达无影响。结论：葛根素可能是通过调节兔动脉粥样斑块分泌MMP-9途径发挥稳定动脉粥样硬化斑块的作用。     

A matrix metalloproteinase inhibitor, ONO-4817, suppresses the development of aortic intimal hyperplasia in experimental hyperlipidemic rabbit

Inhibition of matrix metalloproteinases (MMPs) would be expected to suppress atherosclerotic neointimal proliferation and thus limit atheromatous plaque progression, but this has not yet been demonstrated morphologically in atherosclerotic intimal hyperplasia induced by cholesterol loading in experimental animals. We therefore investigated whether a broad-spectrum MMP inhibitor (MMPi), ONO-4817, could inhibit the development of intimal hyperplasia in male hyperlipidemic rabbits (n = 6) fed laboratory chow supplemented with 1% cholesterol for 2 months followed by a 1% cholesterol diet plus 100 mg/kg ONO-4817 for another month (Chol + ONO group). Control animals (n = 6) received no ONO-4817. When the aortas were studied both histologically and immunohistochemically, intimal hyperplasia was inhibited in Chol + ONO rabbits. The distribution of macrophages and MMP-12 in the hyperplastic tissue of the Chol + ONO rabbits was limited to the luminal side of the lesions. No such limitation in the distribution of macrophages and MMP-12 was observed in the control group. The distribution of smooth muscle cells in the hyperplastic tissue was not different between the Chol + ONO and control groups. However, the distribution of MMP-2 and MMP-12 was limited to the luminal side of lesions in the Chol + ONO group. This is the first reported evidence that an MMPi can suppress the development of intimal hyperplasia in hyperlipidemic rabbits.     

CD147基因沉默对兔外周血单核巨噬细胞基质金属蛋白酶表达的影响

目的筛选出高效抑制兔外周血单核巨噬细胞中CD147表达的短链RNA(shRNA)慢病毒载体,观察CD147基因沉默后对基质金属蛋白酶(MMP)的影响。方法兔源CD147 mRNA序列,设计并构建shRNA慢病毒载体,实验分为空白组,阴性组,加CD147 shRNA慢病毒干扰依次为A组、B组、C组、D组,将其分别转染兔外周血单核巨噬细胞,72 h后观察转染效果,用RT-PCR测CD147 mRNA表达,ELISA法测CD147 MMP-2及MMP-9蛋白表达。结果 A、B、C、D组CD147 mRNA及CD147、MMP-2和MMP-9蛋白较空白组显著减少(P<0.01),A组CD147 mRNA和蛋白降低最显著,较空白组分别减少57.7%和50.9%(P<0.01),MMP-2、MMP-9蛋白表达分别减少95.9%和45.4%。结论成功构建并筛选出能高效、且特异阻断CD147基因表达的shRNA慢病毒载体。CD147基因沉默后,MMP-2和MMP-9表达明显减少,可能成为防治动脉粥样硬化新的治疗靶点。     

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.     

Regulation of matrix metalloproteinase-9/gelatinase B expression and activation by ovarian steroids and LEFTY-A/endometrial bleeding-associated factor in the human endometrium

Various matrix metalloproteinases (MMPs) participate in the menstrual breakdown of the human endometrium. MMP-9/gelatinase B is proposed as a major factor because it degrades many extracellular matrix constituents, including in the vasculature. Although globally under ovarian steroids control, endometrial MMP-9 seems expressed differently than other MMPs, and conflicting publications prevent a clear understanding of its regulation. We therefore quantified MMP-9 expression in the cycling human endometrium, defined its localization, and analyzed its regulation by estradiol and progesterone and by LEFTY-A/endometrial bleeding-associated factor in explant cultures. In fresh tissues, a major increase in MMP-9 mRNA expression occurred at menstruation, after a larger increase in LEFTY-A mRNA. MMP-9 was immunodetected in all cell types throughout the cycle, especially in foci of stromal cells during menstruation. MMP-9 synthesis by these cells was confirmed in cultured explants. In proliferative explants, ovarian steroids slightly decreased MMP-9 mRNA. They had no consistent effect on MMP-9 release in culture medium but strongly inhibited proMMP-9 activation. Addition of recombinant LEFTY-A to explants induced MMP-9 in most samples, a response prevented by ovarian steroids. We propose that endometrial MMP-9 activity is overall controlled by the ovarian steroids and locally adjusted through a network of modulators, including LEFTY-A.     

Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells.

Members of the matrix metalloproteinase (MMP) family have been implicated in the metastasis of tumor cells, but no direct evidence linking any given member of the MMP family to metastatic behavior has been presented. Rat embryo cells transformed by the Ha-ras and v-myc oncogenes or by Ha-ras alone are metastatic in nude mice and release the 92-kDa gelatinase/collagenase (MMP-9), whereas those transformed by Ha-ras plus the adenovirus E1A gene are not metastatic and do not release MMP-9. Here we demonstrate that MMP-9 expression can be induced in these tumorigenic but nonmetastatic rat cells by transfection with an MMP-9 expression vector. Transfection of a MMP-9 expression vector, but not control DNAs, conferred metastatic capacity on the nonmetastatic cells. The majority of colonies isolated after continued passage either in vivo or in vitro had lost the MMP-9 expression vector. However, occasional cells were isolated from metastases which retained MMP-9 expression after passage. These cells retained metastatic capacity. In contrast, cells isolated after losing MMP-9 expression also lost the ability to metastasize. These results provide direct evidence that MMP-9 has a role in tumor metastasis.     

MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA-29b (miR-29b) down-regulation was significantly associated with poor recurrence-free survival of HCC patients. Therefore, the role of miR-29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain- and loss-of-function studies showed that miR-29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR-29b-expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase-2 (MMP-2) was a direct target of miR-29b. The blocking of MMP-2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR-29b, whereas introduction of MMP-2 antagonized the function of miR-29b. We further disclosed that miR-29b exerted its antiangiogenesis function, at least partly, by suppressing MMP-2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2-signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR-29b level was inversely correlated with MMP-2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. CONCLUSION: miR-29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR-29b represents a promising new strategy in anti-HCC therapy.     

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.