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BACKGROUND & AIMS - Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs.
METHODS - Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab.
RESULTS - MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders.
CONCLUSIONS - Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.
Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.
We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')₂ fragments, but not for full-length IgG or for closely-related F(ab')₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')₂ fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')₂ fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.
Bone Morphogenetic Proteins (BMPs) are secreted cytokines that are part of the Transforming Growth Factor β (TGFβ) superfamily. BMPs have been shown to be highly expressed in human breast cancers, and loss of BMP signaling in mammary carcinomas has been shown to accelerate metastases. Interestingly, other work has indicated that stimulation of dermal fibroblasts with BMP can enhance secretion of pro-tumorigenic factors. Furthermore, treatment of carcinoma-associated fibroblasts (CAFs) derived from a mouse prostate carcinoma with BMP4 was shown to stimulate angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts. A large number of secreted pro-inflammatory cytokines and matrix-metallo proteases (MMPs) were found to be upregulated in response to BMP4 treatment. Fibroblasts that were stimulated with BMP4 were found to enhance mammary carcinoma cell invasion, and these effects were inhibited by a BMP receptor kinase antagonist. Treatment with BMP in turn elevated pro-tumorigenic secreted factors such as IL-6 and MMP-3. These experiments demonstrate that BMP may stimulate tumor progression within the tumor microenvironment.
Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.
CONTEXT - Endometriosis is a common condition associated with infertility and pelvic pain in women. Recent in vitro studies have shown that statins decrease proliferation of endometrial stroma (ES) and inhibit angiogenesis.
OBJECTIVE - The aim was to evaluate effects of simvastatin on development of endometriosis in a nude mouse model.
METHODS - Proliferative phase human endometrial biopsies were obtained from healthy donors and established as organ cultures or used to isolate ES cells. To establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (E) for 24 h and subsequently injected into ovariectomized nude mice. Mice (n = 37) were treated with E (8 mg, SILASTIC capsule implants; made in author laboratory) alone or with E plus simvastatin (5 or 25 mg/kg x d) for 10 d beginning 1 d after tissue injection (from three donors). Mice were killed and examined for disease. Effects of simvastatin on matrix metalloproteinase-3 (MMP-3) were evaluated in cultures of ES cells.
PRIMARY OUTCOME - The number and size of endometriotic implants were measured.
RESULTS - Simvastatin induced a dose-dependent decrease of the number and size of endometrial implants in mice. At the highest dose of simvastatin, the number of endometrial implants decreased by 87%, and the volume by 98%. Simvastatin also induced a concentration-dependent decrease in MMP-3 in the absence and presence of inflammatory challenge (using IL-1alpha).
CONCLUSIONS - Simvastatin exerted a potent inhibitory effect on the development of endometriosis in the nude mouse. Mechanisms of action of simvastatin may include inhibition of MMP-3. The present findings may lead to the development of novel treatments of endometriosis involving statins.
Matrix metalloproteinase (MMP)-3 is induced by multiple cell types in the skin during processes involved in both normal and pathological tissue remodeling. We previously demonstrated that MMP3-null animals have an increased sensitivity to the development of squamous cell carcinoma, suggesting that overall, MMP3 has a protective role in squamous cell carcinoma. However, not all cellular responses affected by a loss of MMP3 are tumor-protective, and tumor expression of MMP3 is co-incident with an invasive tumor phenotype. Transgenic mice were generated with MMP3 targeted to keratinocytes to examine the biological role of tumor-produced MMP3. Overexpression of MMP3 reduced tumor multiplicity in response to chemically induced squamous cell carcinoma. Vascular density was increased with MMP3 overexpression; however, other cellular processes, including tumor growth and leukocyte infiltration, were unaffected. In accordance with the change in tumor multiplicity, SP-1 murine papilloma cell lines that were generated to stably express MMP3 lost the capacity to establish palpable tumors following orthotopic injection into immunocompromised mice. Analysis of epidermal biopsies taken at 1 to 2 weeks postinjection revealed that these MMP3-expressing Sp-1 lines had reduced levels of proliferation and pronounced differentiation. These same cells demonstrated an increased ability to differentiate in vitro, an effect that was inhibited by broad-spectrum MMP and selective MMP3 inhibition. These studies suggest that keratinocyte expression of MMP3 promotes cellular differentiation, impeding tumor establishment during tumorigenesis.
The extracellular matrix surrounding the neuromuscular junction is a highly specialized and dynamic structure. Matrix Metalloproteinases are enzymes that sculpt the extracellular matrix. Since synaptic activity is critical to the structure and function of this synapse, we investigated whether changes in synaptic activity levels could alter the activity of Matrix Metalloproteinases at the neuromuscular junction. In particular, we focused on Matrix Metalloproteinase 3 (MMP3), since antibodies to MMP3 recognize molecules at the frog neuromuscular junction, and MMP3 cleaves a number of synaptic basal lamina molecules, including agrin. Here we show that the fluorogenic compound (M2300) can be used to perform in vivo proteolytic imaging of the frog neuromuscular junction to directly measure the activity state of MMP3. Application of this compound reveals that active MMP3 is concentrated at the normal frog neuromuscular junction, and is tightly associated with the terminal Schwann cell. Blocking presynaptic activity via denervation, or TTX nerve blockade, results in a decreased level of active MMP3 at the neuromuscular junction. The loss of active MMP3 at the neuromuscular junction in denervated muscles can result from decreased activation of pro-MMP3, or it could result from increased inhibition of MMP3. These results support the hypothesis that changes in synaptic activity can alter the level of active MMP3 at the neuromuscular junction.
(c) 2007 Wiley Periodicals, Inc.
CONTEXT - Endometriosis, the growth of endometrial tissue outside the uterus, is principally an estrogen-dependent disease. In contrast, exposure to progesterone during pregnancy or therapeutically has been shown to provide benefit to some women with this disease. However, recent research suggests that the presence of endometriosis impairs the capacity of the eutopic endometrium to respond to endogenous progesterone.
OBJECTIVE - Reduced progesterone responsiveness results in an elevated endometrial expression of matrix metalloproteinases (MMPs) during the secretory phase of the menstrual cycle in women with endometriosis. Although cyclic MMP expression is critical for endometrial growth and remodeling, the failure of progesterone to down-regulate MMPs may impair nidation and promote the invasive establishment of endometriosis. In the current study we examined the ability of a newly developed progesterone receptor (PR) agonist, tanaproget (TNPR), to down-regulate endometrial MMP expression in vitro and regress experimental endometriosis in vivo.
SETTING - This study was performed at a university-based medical center.
PARTICIPANTS - Asymptomatic volunteers and patients with endometriosis were studied.
MAIN OUTCOME MEASURES - We examined the ability of TNPR to down-regulate endometrial MMP expression in vitro compared with that of natural progesterone and two currently marketed synthetic steroidal progestins. Using a human/mouse model of endometriosis, we also tested the in vivo ability of TNPR to regress ectopic lesions established by tissues with reduced progesterone sensitivity.
RESULTS - TNPR effectively down-regulated MMP expression in vitro and induced significant reduction of lesions in mice with disease established by tissues from endometriosis patients.
CONCLUSION - Given the positive preclinical pharmacological profile of TNPR that has recently been reported, additional development of this compound for the treatment of endometriosis is warranted.