To study the relative contribution of various matrix degrading enzymes in the pathogenesis of arthritis, changes in the levels of various matrix metalloprtoteinases (MMPs) during the progression of collagen induced arthritis was studied in experimental animals. Arthritis was induced in male wistar rats by injecting an emulsion containing collagen type II and Freund’s complete adjuvant. The duration of the experiment was 35 days. Synovial effusate was collected at regular intervals after induction. At the end of the experiment serum and cartilage were collected and analysed. Synovial fluid of osteoarthritic patients was also analyzed. Levels of MMP-2, MMP-3, MMP-9 and MT1-MMP were found to be high in synovial effusate and cartilage of experimental animals. In synovial effusate of arthritic animals the expression of MMP-3 was found to be high during the early stages while increase in MMP-2 and MMP-9 occurred at later stages. Synovial fluid of osteoarthritic patients also showed elevated levels of MMP-2, MMP-3 and MMP-9. Our results indicated that sequential action of MMPs such as MMP-3, MMP-2 and MMP-9 can cause degradation of articular cartilage extracellular matrix.
Matrix metalloproteinases; Osteoarthritis; Synovial effusate; Synovial fluid
Hypoxia is a feature of the inflamed synovium in rheumatoid arthritis (RA). Intra-articular injection of hyaluronan (HA) may be considered a potential way to treat RA. However, the exact molecular mechanism of HA on decreased cellular responses to hypoxic environment is unclear. The present study has been designed to use the adjuvant-induced arthritis model to examine the effects of HA on the changes of immunohistochemical expressions of hypoxia-inducible factor-1alpha (HIF-1alpha), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-3 (MMP3) in the synovial tissues at the early phase of arthritic inflammation.
Monoarthritis was induced in adult male Sprague-Dawley (250-300 g) via intraarticular injection of complete Freund's adjuvant (CFA) into the tibiotarsal joint. The CFA-induction arthritis animals were divided into three groups: treatment (intraarticular injection of HA), placebo (intraarticular injection of saline) and controls (no treatments). Functional evaluations of edema and pain behavior, histology, and HIF-1alpha, iNOS, and MMP3 immunohistochemistry were performed before, after the first injection, three injections, and on the follow-up injection of the treatments.
Intra-articular injection of HA also significantly suppressed the mechanical allodynia (p < 0.001) and overexpressions of HIF-1alpha (p < 0.001), iNOS (p = 0.004) and MMP3 (p < 0.001) immunoreactivity in synovium.
This study demonstrated that early intervention of HA is an effective protection against accumulation of inflammation-induced HIF-1alpha, iNOS, and MMP3 to limit erosive damage in CFA-induced model of arthritis.
Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (Ad-MMP-9), affects breast cancer sensitivity to radiation.
In the present study, we used antisense MMP-9 adenoviral construct (Ad-MMP-9) to downregulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro prior to irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were performed with orthotopic breast tumors and the radiosensitivity evaluated both in vitro and in vivo.
Ad-MMP-9 infection resulted in downregulation of radiation-induced levels of hypoxia-inducible factor 1 alpha (HIF1α) and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9 in combination with radiation decreased levels of the transcription factors NF-κB and AP 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas-L apoptotic cascade, which resulted in the cleavage of PARP-1 and caspases 10, 3 and 7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model.
In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.
Ad-MMP-9; Radiation; Orthotopic breast tumor; Hypoxia; Tumor growth
This study was aimed at examining the effect of an ointment containing essential oils (EO) on the severity of adjuvant arthritis (AA), an experimental model of human rheumatoid arthritis (RA), in Lewis rats and to define the underlying mechanisms. At the onset of AA, rats received topical application twice daily of ointment containing 20% EO or placebo ointment. The synovial fluid (SF) and synovium-infiltrating cells (SIC) of rats were tested for pro-inflammatory cytokines TNF-α and IL-1β. The hind paws and skin were examined histologically. The activity/level of matrix metalloproteinases (MMPs) and anti-mycobacterial heat-shock protein 65 (Bhsp65) antibodies was tested. Arthritic rats treated with ointment containing EO developed less severe clinical arthritis compared to the controls, and this activity was attributable to EO and not the carrier oil. The levels of TNF-α and IL-1β, and the activity of MMPs in SF and SIC-lysate were significantly (p<0.05) reduced in EO-treated arthritic rats compared to the controls. However, the levels of anti-Bhsp65 antibodies were unaffected by treatment. Thus, topical dermal delivery of EO-containing ointment downmodulates the severity of AA in Lewis rats by inhibiting defined mediators of inflammation. Such ointments should be tested in patients with RA and other arthritic conditions.
Essential oils; Arthritis; Cytokines; Topical application; MMPs; Inflammation
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion. Numerous substrates and binding partners have been identified for MT1-MMP, and its role in collagenolysis appears crucial for tumor invasion. However, development of MT1-MMP inhibitors must consider the substantial functions of MT1-MMP in normal physiology and disease prevention. The present review examines the plethora of MT1-MMP activities, how these activities relate to cancer initiation and progression, and how they can be monitored in real time. Examination of MT1-MMP activities and cell surface behaviors can set the stage for the development of unique, selective MT1-MMP inhibitors.
MT1-MMP; MMP-14; matrix metalloproteinases; CD44; collagen; metastasis
Inflammation represents an early and key event in the development of both the cutaneous psoriasis and psoriatic arthritis. Compelling evidences indicate that the production of TNF-α plays a central role in psoriasis by sustaining the inflammatory process in the skin as well as in the joints. Among the multiple effects produced by TNF-α on keratinocytes, the induction of matrix metalloproteinase-9 (MMP-9), a collagenase implicated in joint inflammatory arthritis which acts as an angiogenesis promoting factor, might represent a key mechanism in the pathogenesis of the disease. Aims of the present study were to investigate a) the role of MMP-9 in the development of psoriasis by assessing the presence of MMP-9 in lesional skin and in sera of psoriatic patients; b) the association of MMP-9 with the activity of the disease; c) the relationship between MMP-9 and TNF-α production.
Eleven psoriatic patients, clinically presenting joint symptoms associated to the cutaneous disease, were included in a therapeutic protocol based on the administration of anti-TNF-α monoclonal antibody (Infliximab). Sera and skin biopsies were collected before treatment and after 6 weeks of therapy. Tissues were kept in short term cultures and production soluble mediators such as TNF-α, MMP-9, MMP-2, VEGF and E-Selectin, which include angiogenic molecules associated to the development of plaque psoriasis, were measured in the culture supernatants by immunoenzymatic assays (ng/ml or pg/ml per mg of tissue). MMP-9 concentrations were also measured in the sera. The cutaneous activity of disease was evaluated by the Psoriasis Area and Severity Index (PASI).
Clinical and laboratory assessment indicated that all but one patients had a significant improvement of the PASI score after three months of therapy. The clinical amelioration was associated to a significant decrease of MMP-9 (P = 0.017), TNF-α (P = 0.005) and E-selectin (P = 0.018) levels, spontaneously released by lesional biopsies before and after therapy. In addition, significant correlations were found between the PASI measurements and TNF-α (r2 = 0.33, P = 0.005), MMP-9 (r2 = 0.25, P = 0.017), E-selectin (r2 = 0.24, P = 0.018) production. MMP-9 levels were significantly correlated with those of TNF-α (r2 = 0.30, P = 0.008). A significant decrease of MMP-9 in the sera, associated to the clinical improvement was also found.
Our findings show the existence of a direct relationship between MMP-9 and TNF-α production strongly suggesting that MMP-9 may play a key role in the skin inflammatory process in psoriasis.
We previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope.
By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex.
These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.
The matrix metalloproteinase (MMP) family is believed to play a role in the ovulatory process because MMP inhibitors block oocyte release. However, little is known about the mechanisms by which the MMPs affect ovulation. The present study investigated the degradomic actions of the gelatinases, MMP2 and MMP9, by identifying gelatinolytic targets in periovulatory granulosa cells. Granulosa cells were collected from immature rats 48 h after equine chorionic gonadotropin treatment and were cultured with human chorionic gonadotropin (hCG) in the absence or presence of a specific MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid) for an additional 24 h. The conditioned media was analyzed for gelatinolytic activity, progesterone, and peptide profiles. Gelatinolytic activity and progesterone were induced in response to hCG; however, there was no difference in progesterone between cells treated with or without the inhibitor. Peptide fragments of proteins altered in the presence of the gelatinase inhibitor were identified by two-dimensional gel electrophoresis and mass spectrometry. Protein disulfide isomerase A3 (PDIA3), which plays a role in protein folding, was identified as a peptide that decreased in the presence of inhibitor while the serine protease hepsin, was found to increase with inhibitor treatment. Subsequent experiments established that PDIA3 and hepsin were targets of MMP2/9 action by cleavage with MMP2 and Western blot analysis, respectively. Additionally, hepsin was identified as a gelatinolytic target in ovarian cancer cells. In the present study, proteomics has identified proteins that may be involved in novel ways in the complex cascades that are mediated by gelatinolytic MMPs during the periovulatory period.
Gelatinases from rat granulosa cells degrade hepsin and protein disulfide isomerase A3.
corpus luteum; hepsin; matrix metalloproteinase; ovulation; protein disulfide isomerase A3
The matrix metalloproteinases (MMPs) have been implicated in the aggressive course of non-small cell lung cancer (NSCLC). However, there are a large number of MMP subtypes with diverse proteolytic substrates and different induction pathways. This study tested the hypothesis that a differential MMP profile would exist between NSCLC and normal lung and that MMP patterns would differ between NSCLC histologic type.
NSCLC samples and remote normal samples were obtained from patients with stage I or II NSCLC with either squamous cell (n=22) or adenocarcinoma (n=19) histology. Absolute concentrations for each of the MMP subclasses; collagenases (MMP-1, 8, -13), gelatinases (MMP-2,-9), lysins (MMP-2, -7) and elastase (MMP-12) were determined by a calibrated and validated multiplex suspension array.
Overall, MMP levels were significantly increased in NSCLC compared to normal. For example, MMP-1 and MMP-7 increased by approximately 10 fold in NSCLC (p<0.05). Moreover, a different MMP portfolio was observed between NSCLC histologic types. For example MMP-1,-8,-9 and -12 increased by over 4-fold in squamous cell versus adenocarcinoma (p<0.05). In those patients who recurred within 3 years of resection, 3-fold higher levels of MMP-8 and -9 were observed (p<0.05).
Increased levels of a number of MMP types occur with NSCLC, but the MMP profile was distinctly different between histologic types and in those patients with recurrence. These different MMP profiles may be important in the mechanistic basis for the natural history of different NSCLC types, as well as identifying potential prognostic and therapeutic targets.
matrix metalloproteinases; lung cancer; multiplex; recurrence
Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the joints that can lead to deformities and disability. The prolonged use of conventionally used drugs is associated with severe adverse reactions. Therefore, safer and less expensive therapeutic products are continually being sought. Huo-Luo-Xiao-Ling dan (HLXL), a traditional Chinese herbal mixture, and its modified versions possess anti-arthritic activity. In this paper, we examined the influence of modified HLXL on two of the key mediators of arthritic inflammation and tissue damage, namely, chemokines and matrix-metalloproteinases (MMPs) in the rat adjuvant-induced arthritis (AA) model of RA. We treated arthritic Lewis rats with HLXL (2.3 g/kg) by daily gavage beginning at the onset of AA. The control rats received the vehicle. At the peak phase of AA, rats were sacrificed and their draining lymph node cells (LNC) and spleen adherent cells (SAC) were tested. The HLXL-treated rats showed a significant reduction in the levels of chemokines (RANTES, MCP-1, MIP-1α, and GRO/KC), MMPs (MMP 2 and 9), as well as cytokines (IL-6 and IL-17) that induce them, compared to the control vehicle-treated rats. Thus, HLXL controls arthritis in part by suppressing the mediators of immune pathology, and it might offer a promising alternative/adjunct treatment for RA.
Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC-SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.
arthritis, experimental; reactive oxygen species; rheumatoid arthritis; superoxide dismutase; synovial membrane
Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9−/− mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9−/− exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9−/− and wild-type mice. We examined whether decreased inflammation in MMP-9−/− mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.
BACKGROUND/AIMS—Matrix metalloproteinases (MMPs) accumulate in the tears of patients with active peripheral ulcerative keratitis (PUK) but it is unknown whether these enzymes have a central role in disease progression. The aims of the present investigation were to determine the source of these enzymes and to ascertain whether their accumulation in tears is a phenomenon specific to PUK or a general feature of other anterior segment diseases.
METHODS—The experimental samples were obtained from the culture media of conjunctival and corneal epithelial cells, from fractionated blood plasma and leucocytes of healthy subjects and patients with rheumatoid arthritis, and from the tears of healthy subjects and patients with a variety of anterior segment diseases. The MMPs of all samples were visualised by zymography and tear samples were assayed using nitrophenol acetate and an MMP-9 susceptible quenched fluorescent peptide as substrate.
RESULTS—The major MMPs that accumulate in the tears of patients with rheumatoid arthritis with active ocular disease are MMP-9 and a species of Mr 116 000. By comparing the zymographic activity profiles of the gelatinases present in the samples obtained, it was deduced that the main source of these MMPs was granulocytes. Their accumulation in tears was not unique to patients with PUK; detectable amounts of the enzymes also occurred in the tears of patients with keratoconus with associated atopic disease, patients undergoing treatment for herpetic eye disease, and patients with systemic and non-systemic dry eye disease.
CONCLUSION—The MMPs that accumulate in tears are mainly derived from granulocytes. This may be effected by autoimmune diseases that involve ocular tissue or by ocular diseases that induce an inflammatory response.
Matrix metalloproteinase (MMP)-1, MMP-8 and MMP-13 are interstitial collagenases that degrade type II collagen in cartilage; this is a committed step in the progression of rheumatoid arthritis and osteoarthritis. Of these enzymes, the expression of MMP-1 and MMP-13 is substantially increased in response to IL-1 and tumor necrosis factor-α, and elevated levels of these collagenases are observed in arthritic tissues. Therefore, cytokine-mediated MMP-1 and MMP-13 gene regulation is an important issue in arthritis research. In this review, we discuss current models of MMP-1 and MMP-13 transcriptional regulation, with a focus on signaling intermediates and transcription factors that may be future targets for the development of new arthritis drugs.
arthritis; matrix metalloproteinases; mitogen-activated protein kinases; nuclear factor κB; transcription
matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.
Expression of the metalloproteinase Mmp10 mRNA is stimulated by hCG prior to follicular rupture in both the human and the rat ovary, indicating involvement in ovulation and subsequent luteinization.
extracellular matrix; granulosa cells; matrix metalloproteinase; ovulation; ovulatory cycle; proteinases; theca cells
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.
Herein, we for the first time report a novel activatable photoacoustic (PA) imaging nano-probe for in vivo detection of cancer-related matrix metalloproteinases (MMPs). A black hole quencher 3 (BHQ3) which absorbs red light is conjugated to near-infrared (NIR)-absorbing copper sulfide (CuS) nanoparticles via a MMP-cleavable peptide linker. The obtained CuS-peptide-BHQ3 (CPQ) nano-probe exhibits two distinctive absorption peaks at 630 nm and 930 nm. Inside the tumor microenviorment where MMPs present, the MMP-sensitive peptide would be cleaved, releasing BHQ3 from the CuS nanoparticles, the former of which as a small molecule is then rapidly cleared out from the tumor, whereas the latter of which as large nanoparticles would retain inside the tumor for a much longer period of time. As the result, the PA signal at 680 nm which is contributed by BHQ3 would be quickly diminished while that at 930 nm would be largely retained. The PA signal ratio of 680 nm / 930 nm could thus serve as an in vivo indicator of MMPs activity inside the tumor. Our work presents a novel strategy of in vivo sensing of MMPs based on PA imaging, which should offer remarkably improved detection depth compared with traditional optical imaging techniques.
Peptide; Photoacoustic imaging; Enzyme cleavage; Copper sulfide; MMPs detection.
Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-α) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1β, TNF-α and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.
Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.
Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.
PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.
Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
Rheumatoid arthritis is an autoimmune disease in which joint inflammation leads to progressive cartilage and bone erosion. Matrix metalloproteinases (MMPs) implicated in homeostasis of the extracellular matrix play a central role in cartilage degradation. However, the role of specific MMPs in arthritis pathogenesis is largely unknown. The aim of the present study was to investigate the role of Mmp-8 (collagenase-2) in an arthritis model.
Arthritis was induced in Mmp8-deficient and wildtype mice by K/BxN serum transfer. Arthritis severity was measured by a clinical index and ankle sections were scored for synovial inflammation, cartilage damage and bone erosion. cDNA microarray analysis, real-time PCR and western blot were performed to identify differential changes in gene expression between mice lacking Mmp8 and controls.
Mmp8 deficiency increased the severity of arthritis, although the incidence of disease was similar in control and deficient mice. Increased clinical score was associated with exacerbated synovial inflammation and bone erosion. We also found that the absence of Mmp8 led to increased expression of IL-1β, pentraxin-3 (PTX3) and prokineticin receptor 2 (PROKR2) in arthritic mice joints.
Lack of Mmp-8 is accompanied by exacerbated synovial inflammation and bone erosion in the K/BxN serum-transfer arthritis model, indicating that this Mmp has a protective role in arthritis.
Herein we demonstrate for the first time that a fluorogenic probe can be used as an in vivo imaging agent for visualizing activities of membrane-tethered, membrane-type matrix metalloproteinases (MT-MMPs). An MT-MMP fluorogenic probe that consisted of an MT1-MMP (MMP-14) substrate and near-infrared (NIR) dye-quencher pair exhibited rapid, efficient boosts in fluorescence upon cleavage by MT1-MMP in tumor-bearing mice. In particular, unlike similar fluorogenic probes designed to target extracellular, soluble-type MMPs (EC-MMPs)--which can be cleared from the blood stream after activation--the fluorescence signals activated by MT1-MMP enable clear visualization of MT1-MMP-positive tumors in animal models for up to 24 hours. The results indicate that a simple form of a fluorogenic probe that is less effective in EC-MMP imaging is an effective probe for imaging MT-MMP activities in vivo. These findings can be widely applied to designing probes and to applications targeting various membrane-anchored proteases in vivo.
activatable probe; fluorogenic probe; membrane-type matrix metalloproteinase; optical imaging; protease
Allergic rhinitis (AR) is a chronic upper respiratory tract disease that inflames the mucous membranes of the nose and occurs when circulating inflammatory cells including eosinophils and basophils migrate to and accumulate in the inflammation area by passing through the interstitium and capillary walls. To pass through these barriers, the inflammatory cells degrade extracellular matrix proteins. Matrix metalloproteinases (MMPs) released by inflammatory cells mediate the degradation of these proteins. MMPs have synthetic inhibitors and doxycycline, a tetracycline antibiotic, inhibits MMPs. This study investigated the efficiency of intranasal doxycycline in decreasing the symptoms and inflammatory cell infiltration in an animal model of AR.
AR was created in female Wistar rats by repeated intranasal challenge with ovalbumin by intraperitoneal injection. For 15 days, topical intranasal doxycycline was administered one hour before ovalbumin administration. Following intranasal administration, nasal symptoms were scored and the nasal mucosae of all rats were evaluated histopathologically. To investigate tissue changes, hematoxyline-eosin and Alcian blue/periodic acid Schiff stains were used. As well, cilia loss, goblet cell changes, vascular congestion, vascular proliferation, inflammatory cell infiltration, eosinophil infiltration and the degree of hypertrophy in chondrocytes were evaluated with light microscopy.
Typical symptoms of AR were decreased by intranasal doxycycline administration. These effects were stable after repeated intranasal ovalbumin administration. Histological evaluation of doxycycline treated rats did not reveal typical inflammatory changes associated with AR.
MMPs may have crucial functions in AR and topical intranasal doxycycline, which decreases inflammatory cell infiltration, may offer an alternative therapy for AR.
Doxycycline; Allergic rhinitis; Matrix metalloproteinase inhibitors
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 [3H]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.
The matrix metalloproteinases (MMPs) exhibit a broad array of activities, some catalytic and some non-catalytic in nature. An overall lack of selectivity has rendered small molecule, active site targeted MMP inhibitors problematic in execution. Inhibitors that favor few or individual members of the MMP family often take advantage of interactions outside the enzyme active site. We presently focus on peptide-based MMP inhibitors and probes that do not incorporate conventional Zn2+ binding groups. In some cases, these inhibitors and probes function by binding only secondary binding sites (exosites), while others bind both exosites and the active site. A myriad of MMP mediated-activities beyond selective catalysis can be inhibited by peptides, particularly cell adhesion, proliferation, motility, and invasion. Selective MMP binding peptides comprise highly customizable, unique imaging agents. Areas of needed improvement for MMP targeting peptides include binding affinity and stability.
matrix metalloproteinase; protease inhibitor; secondary binding site; cyclic peptide; phage display; triple-helix
AIM—Peripheral ulcerative keratitis (PUK) is an ocular manifestation of rheumatoid arthritis and other similar systemic diseases. The purpose of this inquiry was to investigate the involvement of matrix metalloproteinases (MMPs) in the induction and/or maintenance of PUK.
METHODS—Substrate gel electrophoresis was used to characterise the MMP activities secreted by primary cultures of keratocytes derived from normal and perforated pathological corneal specimens, and those present in tears of normal subjects and patients with PUK. Substrate specificity and the in vivo activity status of the secreted MMPs was assessed by SDS-polyacrylamide gel electrophoresis of standard collagens incubated in the presence or absence of the various enzyme preparations.
RESULTS—In addition to MMP-2 of Mr 66 000, cultured keratocytes derived from perforated corneas of patients with PUK abnormally produce the MMP-2 of apparent Mr 62 000. Other MMPs and in particular MMP-9 of Mr 92 000, also occur in the tears of these patients. Their visualisation on substrate polyacrylamide gels correlated with clinical manifestations of disease activity; during periods of disease quiescence they were barely detectable. The steroid prednisolone, frequently used in systemic therapy, had no effect on the in vitro activity of MMP-2, or on its production by cultured corneal keratocytes. Although the in vitro activity of MMP-2 was inhibited by both Cu2+ and Zn2+, Cu2+ apparently induced the keratocytes to produce activated enzyme and Zn2+ irreversibly inhibited their production of MMP-2.
CONCLUSION—Overexpression of corneal MMP-2 and tear film MMP-9 are characteristic features of patients with PUK and their activation may be a crucial facet of disease initiation or progression. Although effective in systemic therapy for PUK, prednisolone had no direct control over corneal MMP-2 production or activity. Zn2+ on the other hand inhibited both MMP-2 production and MMP-2 activity and may, therefore, be of therapeutic value if suitably formulated and used in conjunction with systemic steroid treatment.