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The success of endovascular repair of abdominal aortic aneurysms remains limited due to the development of endoleaks. Sac embolization has been proposed to manage endoleaks, but current embolizing materials are associated with frequent recurrence. An injectable agent that combines vascular occlusion and sclerosing properties has demonstrated promise for the treatment of endoleaks. Moreover, the inhibition of aneurysmal wall degradation via matrix metalloproteinases (MMPs) may further prevent aneurysm progression. Thus, an embolization agent that promotes occlusion, MMP inhibition and endothelial ablation was hypothesized to provide a multi-faceted approach for endoleak treatment. In this study, an injectable, occlusive chitosan (CH) hydrogel containing doxycycline (DOX)-a sclerosant and MMP inhibitor-was developed. Several CH-DOX hydrogel formulations were characterized for their mechanical and sclerosing properties, injectability, DOX release rate, and MMP inhibition. An optimized formulation was assessed for its short-term ability to occlude blood vessels in vivo. All formulations were injectable and gelled rapidly at body temperature. Only hydrogels prepared with 0.075M sodium bicarbonate and 0.08M phosphate buffer as the gelling agent presented sufficient mechanical properties to immediately impede physiological flow. DOX release from this gel was in a two-stage pattern: a burst release followed by a slow continuous release. Released DOX was bioactive and able to inhibit MMP-2 activity in human glioblastoma cells. Preliminary in vivo testing in pig renal arteries showed immediate and delayed embolization success of 96% and 86%, respectively. Altogether, CH-DOX hydrogels appear to be promising new multifunctional embolic agents for the treatment of endoleaks., Statement of Significance: An injectable embolizing chitosan hydrogel releasing doxycycline (DOX) was developed as the first multi-faceted approach for the occlusion of blood vessels. It combines occlusive properties with DOX sclerosing and MMP inhibition properties, respectively known to prevent recanalization process and to counteract the underlying pathophysiology of vessel wall degradation and aneurysm progression. After drug release, the biocompatible scaffold can be invaded by cells and slowly degrade. Local DOX delivery requires lower drug amount and decreases risks of side effects compared to systemic administration. This new gel could be used for the prevention or treatment of endoleaks after endovascular aneurysm repair, but also for the embolization of other blood vessels such as venous or vascular malformations., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs., (Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.)

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

1. The potential of the matrix metalloproteinase (MMP) inhibitor ABT-518 to affect pre-adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using mouse models of adipogenesis and obesity. 2. Differentiation of 3T3-F442A pre-adipocytes into mature adipocytes was enhanced in a dose-dependent manner by the addition of ABT-518 (0-100 μmol/L). This was associated with increased expression of the adipogenic markers adipocyte fatty acid-binding protein 2 (AP2), peroxisome proliferator-activated receptor γ and adiponectin. 3. Feeding 5-week-old male wild-type mice with a high-fat diet, with or without ABT-518 (to achieve a dose of 100 mg/kg per day), for 16 weeks resulted in a significant reduction in bodyweight throughout the experimental period. Magnetic resonance spectroscopy revealed that the lipid : water ratio was significantly lower in ABT-518-treated mice. The total weight of isolated subcutaneous or gonadal fat depots did not differ significantly following ABT-518 treatment, but adipocyte and blood vessel size were significantly reduced in the gonadal fat. 4. Administration of ABT-518-2 (100 mg/kg per day for 10 weeks) to 5-week-old male wild-type mice with established obesity maintained on a high-fat diet had no effect on total bodyweight at the end of the experiment, but was associated with reduced blood vessel size in the fat depots. 5. Thus, the MMP inhibitor ABT-518 stimulates differentiation of 3T3-F442A pre-adipocytes in vitro. It mildly reduces bodyweight gain in a murine model of diet-induced obesity, but does not affect established obesity., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

Investigation of collagenase and gelatinase inhibitory natural components afforded two isoflavonoids. Two isoflavonoids, tectorigenin-7-O-β-D-glucoside (1) and luteolin-7-O-β-D-glucuronopyranoside (2), were isolated from ethyl acetate fraction of Viola patrinii fermentation extracts (VPFE). Of these, compounds 1 and 2 exhibited collagenase inhibitory activity (IC(50)) at a concentration of less than 1.5 μM, and compound 2 showed gelatinases A and B inhibitory activity (IC(50)) at 0.3 μM and 0.8 μM, respectively.

Matrix metalloproteinase (MMP)-2 and MMP-9 have been associated with the ability of tumor cells to metastasize because of their capacity to degrade type IV collagen, the main component of basement membrane, and to their elevated expression in malignant tumors. (S)-methyl 6-(benzyloxycarbonylamino)-2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido) piperidin-1-yl) acetamido) hexanoate (CH1104I) is a galloyl cyclic-imide derivative designed to fit and extend into the S1' active pocket of MMP-2 and MMP-9. We aimed to evaluate the efficacy of CH1104I as a candidate compound for antiinvasion and antimetastasis of tumor cells. CH1104I significantly blocked gelatinase activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that the compound (7-210 micromol/l) inhibited the activity of MMP-2 and MMP-9 produced by human ovarian carcinoma SKOV3 cells. Inhibition of MMP-2 and MMP-9 expression was also observed using the assays of immunocytochemical staining and western blot analysis. The results showed that CH1104I suppressed the expression of zymogens and active MMP-2 and MMP-9. The effects of CH1104I on the invasion and migration of SKOV3 cells were then measured. Both the trans-well motility assay and wound scratch assay indicated that CH1104I was very effective for the antiinvasion and antimigration of SKOV3 cells. Furthermore, the Lewis lung carcinoma model was used to evaluate the efficacy of CH1104I in vivo. A significant inhibition of pulmonary metastasis of carcinoma cells was observed in CH1104I-administrated mice (25-100 mg/kg). These results suggest that CH1104I is a potential MMP-2 and MMP-9 inhibitor that may effectively suppress tumor invasion and metastasis.

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30mg/(kgday) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215+/-8mmHg versus 167+/-13mmHg in 2K-1C rats and 2K-1C+doxy rats, respectively; P<0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline.

(4-Phenoxyphenylsulfonyl)methylthiirane (inhibitor 1) is a highly selective inhibitor of gelatinases (matrix metalloproteinases 2 and 9), which is showing considerable promise in animal models for cancer and stroke. Despite demonstrated potent, selective, and effective inhibition of gelatinases both in vitro and in vivo, the compound is rapidly metabolized, implying that the likely activity in vivo is due to a metabolite rather than the compound itself. To this end, metabolism of inhibitor 1 was investigated in in vitro systems. Four metabolites were identified by LC/MS-MS and the structures of three of them were further validated by comparison with authentic synthetic samples. One metabolite, 4-(4-thiiranylmethanesulfonylphenoxy)phenol (compound 21), was generated by hydroxylation of the terminal phenyl group of 1. This compound was investigated in kinetics of inhibition of several matrix metalloproteinases. This metabolite was a more potent slow-binding inhibitor of gelatinases (matrix metalloproteinase-2 and matrix metalloproteinase-9) than the parent compound 1, but it also served as a slow-binding inhibitor of matrix metalloproteinase-14, the upstream activator of matrix metalloproteinase-2.

Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, are implicated in tumor progression. We describe herein the development of a resin-immobilized, broad-spectrum synthetic MMP inhibitor for selective binding of the active forms of MMPs from different experimental samples. We confirmed the activity-based binding of MMPs to the inhibitor-tethered resin with purified human recombinant MMP-2, -9, and -14, samples of cultured cells, and tissue extracts. Our results show that only the free active MMPs, and not the zymogens or MMP/TIMP (enzyme-protein inhibitor) complexes, bound specifically to the resin. In our comparison of benign and carcinoma tissue extracts, we detected active MMP-2 and MMP-14 forms only in the cancerous tissue samples, indicating that a pool of the tumor MMPs is free of endogenous inhibitors (TIMPs), and is thus likely to contribute to proteolytic events that precipitate tumor metastasis.

Many proteinases, including gelatinase B/MMP-9, fulfill crucial regulatory or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. Denatured collagen type II provides one of the best gelatinase B substrates, and the characteristics of its cleavage were employed to define the requirements of a novel optimal substrate probe. A synthetic fluorescent derivative was used for the development of a new high-throughput technology for the selection of inhibitors on the principles of sensitivity of confocal fluorescence detection, resolution capacity of capillary electrophoresis, and multichannel power of DNA sequencers. Combinatorial chemical synthesis of a library of peptide-based inhibitors, library deconvolution, high-throughput screening, isolation, and mass spectrometric techniques enabled us to identify a novel single-peptide gelatinase B inhibitor. A notable finding is that the in vitro-selected inhibitor mimics many of the characteristics of the evolution-selected MMP propeptide sequence.

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 degrees C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family.

Background: Chemically modified tetracyclines (CMTs), devoid of antimicrobial activity, inhibit pathologically elevated collagenase activity both in vivo and in vitro. In the current study, doxycycline and 5 different CMTs were tested to prevent matrix metalloproteinase (MMP)-dependent periodontal tissue breakdown in an animal model of periodontitis., Methods: Adult male rats received intragingival injections with either 10 microl of physiologic saline or Escherichia coli endotoxin (1 mg/ml) every other day for 6 days and were distributed into 8 treatment groups (12 rats/group): saline (S), endotoxin alone (E), E + CMT-1, E + CMT-3, E + CMT-4, E + CMT-7, E + CMT-8, and doxycycline. All animals were treated daily with 1 ml of 2% carboxymethyl cellulose (CMC) alone or containing one of the above-mentioned CMTs (2 mg/day) orally. The gingival tissues were removed, extracted, and assayed for gelatinase (GLSE). Some rat maxillary jaws from each treatment group were fixed in buffered formalin and processed for histology and immunohistochemistry for the cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6, and MMP-2 and MMP-9., Results: Endotoxin injection induced elevated GLSE activity (functional assay and osteoclast-mediated bone resorption), the former identified as predominantly MMP-9 (92 kDa GLSE) by gelatin zymography. All 6 tetracyclines (2 mg/day) inhibited periodontal breakdown in the following order of efficacy: CMT-8 > CMT- 1 > CMT-3 > doxycycline > CMT-4 > CMT-7. Immunohistochemistry was positive for TNF, IL-1, and IL-6 in the inflammatory cells from untreated endotoxin rat tissues, whereas treatment with CMTs decreased the number of immuno-positive stained cells for cytokines and MMPs. The in vivo efficacy of these drugs varied with CMT structure and was significantly correlated with bone resorption: r2 = -0.77, P<0.01; gelatinase inhibitory activity: r2 = -0.84, P <0.01; and serum drug concentrations., Conclusion: Since both conventional (antimicrobial) and non-antimicrobial tetracyclines inhibited periodontal bone resorption induced by endotoxin injection, MMP-mediated bone loss in this model can be prevented by inhibition of MMPs.

Periodontal disease is characterized by excessive host collagenase resulting in loss of gingival and periodontal ligament collagen and adjacent alveolar bone. Intragingival endotoxin injection induces a model of periodontal disease characterized by rapid bone loss with biochemical features similar to that of naturally occurring adult periodontitis. CH1766, a peptide with a zinc binding moeity which fits into the active site of the enzyme, and CH6631, a hydroxamic acid derivative with aryl-substituted sulphonamide residues, are inhibitors of matrix metalloproteinases (MMPIs) with differing inhibitory profiles as characterized by in vitro assays. In this study, endotoxin was injected into the gingivae of rats which were then treated orally with either 3 mg/kg or 30 mg/kg of one of the two inhibitory compounds. The gingival tissues were assessed for collagenase and gelatinase activity, plus three different pro-inflammatory cytokines. In addition, alveolar bone height in defleshed jaws was studied by computerized morphometric analysis and scanning electron microscopy. Both drugs reduced active and/or total MMP activity, in many cases to normal, and also partially normalized cytokine levels as well. A dose-response effect was seen with regard to amelioration of lipopolysaccharide-induced alveolar bone loss with both drugs. Other than studies with tetracyclines, this is the first report of beneficial effects of MMPIs in a model of periodontal disease, strongly suggesting that this class of agents could bring therapeutic benefit to patients with this disorder, and that periodontal disease can be used as a model to demonstrate in vivo efficacy of this class of drugs.

Background: Tetracyclines, particularly doxycycline (Doxy), and their non-antimicrobial chemically-modified derivatives (CMTs) inhibit the activities of human matrix metalloproteinases (MMPs), and reduce the severity and progression of periodontal disease in animal models and humans. In this study, the effects of Doxy and CMT-1, -3, and -5 on proteolytic, serpinolytic, and progelatinase-B activation activities of potent periodontopathogens were studied., Methods: The effect of Doxy and CMTs (0.5 to 50 microM) on proteolytic activities were investigated by incubating bacteria with chromogenic substrates or human serum albumin. A collagenolytic fraction of Porphyromonas gingivalis was used to evaluate the effect of these substances on collagenolytic (type I collagen) and serpinolytic (alpha1-proteinase inhibitor) activities. Lastly, the effect of Doxy on progelatinase-B (pro-MMP-9) activation by purified proteinases from P. gingivalis and Treponema denticola was investigated by SDS-PAGE/Western immunoblotting., Results: Doxy and CMTs, except CMT-5 which lacks the structural elements required for cation chelation, inhibited Arg- and Lys-gingipain activities as well as collagenolytic activity of P. gingivalis. Doxy and CMTs did not markedly affect the chymotrypsin-like activity of T. denticola but inhibited its trypsin-like activity. In addition, degradation of human serum albumin by cells of P. gingivalis and T. denticola was strongly inhibited by Doxy and CMT-1. Doxy and CMT-1 also inhibited the inactivation of alpha1-proteinase inhibitor (serpinolytic activity) by a collagenolytic fraction of P. gingivalis. Lastly, Doxy prevented the latent to active conversion of human neutrophil progelatinase-B (pro-MMP-9) by Arg-gingipains A/B of P. gingivalis but not by the chymotrypsin-like proteinase of T. denticola., Conclusions: Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response.

Background: Chemically modified non-antimicrobial tetracyclines (CMTs) have been shown to inhibit pathologically elevated collagenase (and other matrix metalloproteinase, MMP) activity and bone resorption in vivo and in vitro., Methods: In the current study, suboptimal doses of CMT-8 (a non-antimicrobial chemically modified doxycycline) and a bisphosphonate (clodronate, an anti-bone resorption compound) were administered daily, either as a single agent or as a combination therapy, to rats with experimental periodontitis induced by repeated injection of bacterial endotoxin (LPS) into the gingiva. At the end of the 1-week protocol, the gingival tissues were dissected, extracted, and the extracts analyzed for MMPs (collagenases and gelatinases) and for elastase, and the defleshed jaws were morphometrically analyzed for alveolar bone loss., Results: LPS injection significantly (P<0.001) increased alveolar bone loss and increased collagenase (MMP-8), gelatinase (MMP-9), and elastase activities. Treatment of the LPS-injected rats with suboptimal CMT-8 alone or suboptimal clodronate alone produced slight reductions in the tissue-destructive proteinases and no significant reductions in alveolar bone loss. However, a combination of suboptimal CMT-8 and clodronate "normalized" the pathologically elevated levels of MMPs, elastase, and alveolar bone loss, indicating synergistic inhibition of tissue breakdown in this animal model of periodontitis., Conclusions: Combination of a CMT and a bisphosphonate may be a useful treatment to optimally suppress periodontal destruction and tooth loss and in other tissue-destructive inflammatory diseases such as arthritis.

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in pathologic oral processes such as periodontal tissue destruction, root caries, tumor invasion and temporomandibular joint disorders. The aim of this study was to test the effect of metal ions released from dental amalgam on the major gingival gelatinolytic MMPs. Gingival human explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers conditioned with dispersed phase or concentional phase dental amalgams. The major enzymes present in conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. The proteolytic activities of MMP-2 and MMP-9 were strongly inhibited by dispersed phase amalgams conditioned buffers. Inhibition of MMP-2 and MMP-9 activities was partly prevented by the addition of 1,10 phenanthroline, a divalent metal chelator, to the amalgam conditioned buffers. Dental amalgam conditioned buffer also inhibited the degradation of denatured type I collagen by purified MMP-2 on liquid phase assays. These findings suggest that the activity of oral tissue MMPs may be modulated by metal ions released from dental amalgam.

Abdominal aortic aneurysms (AAAs) represent a chronic degenerative condition associated with a life-threatening risk of rupture. The evolution of AAAs is thought to involve the progressive degradation of aortic wall elastin and collagen, and increased local production of several matrix metallo-proteinases (MMPs) has been implicated in this process. We have previously shown that tetracycline derivatives and other MMP inhibitors suppress aneurysm development in experimental animal models of AAA. Doxycycline also reduces the expression of MMP-2 and MMP-9 by human vascular wall cell types and by AAA tissue explants in vitro. To determine whether this strategy might have a role in the clinical management of small AAA, we examined the effect of doxycycline on aortic wall MMP expression in vivo. Patients were treated with doxycycline (100 mg p.o. bid) for 7 days prior to elective AAA repair, and aneurysm tissues were obtained at the time of surgery (n = 5). Tissues obtained from an equal number of untreated patients with AAA were used for comparison. By reverse transcription-polymerase chain reaction and Southern blot analysis, MMP-2 and MMP-9 were both found to be abundantly expressed in the aneurysm wall. Preoperative treatment with doxycycline was associated with a 3-fold reduction in aortic wall expression of MMP-2 and a 4-fold reduction in MMP-9 (p < 0.05 compared to untreated AAA). These preliminary results suggest that even short-term treatment with doxycycline can suppress MMP expression within human AAA tissues. Given its pleiotropic effects as an MMP inhibitor, doxycycline may be particularly effective in suppressing aortic wall connective tissue degradation. While it remains to be determined whether MMP inhibition will have a clinically significant impact on aneurysm expansion, it is expected that this question can be resolved by a properly designed prospective randomized clinical trial.

Background: We have shown previously that the metalloproteinase inhibitor, BB-94, prolongs survival and attenuates MMP-2 activity in a murine model of pancreatic cancer. The purpose of this study was to determine the effect of BB-94 on the activity and activation of MMP-2 by PANC-1 cells in vitro., Materials and Methods: The poorly differentiated pancreatic cancer cell line PANC-1 was stimulated in vitro with the phorbol ester PMA (20 nM) and grown in the presence of increasing doses of BB-94 (0, 40, 200, and 400 ng/ml) for 24 h. Activation of MMP-2 was determined by gel zymography. In a separate experiment detailing the effects of BB-94 on MMP-2 activity, PANC cells were stimulated for 24 h with PMA and run out on four separate zymograms, each incubated in the previously noted concentrations of BB-94. Using densitometry, band intensity on all gels was determined and compared for each concentration of BB-94. The Matrigel assay was used to determine BB-94's effect on the invasive potential of PANC cells at the previously studied concentrations. The presence of MT-MMP (a putative component of MMP-2 activation) was confirmed using Western blot in each group., Results: BB-94 inhibited the conversion of latent to active MMP-2 in a dose-dependent fashion. BB-94 also inhibited the activity of MMP-2 when run out on gel zymograms incubated with increasing concentrations of BB-94. Decreased activity and activation of MMP-2 by BB-94 were manifested by a significant reduction in the invasive potential of PANC as determined by the Matrigel assay. MT-MMP was universally present in each study group., Conclusions: The previously described salutary effects of MMP blockade in mice implanted with pancreatic cancer can be explained in vitro by a dose-dependent diminution of MMP-2 activity and activation in PANC cells exposed to BB-94., (Copyright 1999 Academic Press.)

Three-dimensional models for the catalytic domain of gelatinases (MMP-9 and -2) have been constructed based on the X-ray crystal structure of MMP-3. Conformations of the loop segment which forms the bottom half of the S1' subsite but shows conformational diversity among the crystal structures of other MMPs have been explored by simulated annealing of each gelatinase model complexed with two highly potent "probe" inhibitors. Representative catalytic domain models have been selected for each gelatinase from the set of generated conformations based on shape complementarity of the loop to the probe inhibitors. The single model selected for MMP-9 was utilized to explain the structure-activity relationship of our novel sulfonamide inhibitors. Molecular dynamics (MD) simulations of the complex models revealed important features of the binding mechanism of our inhibitors: (i) the ligand carboxylate group coordinating to the catalytic zinc ion and hydrogen bonding to the Glu219 side chain, (ii) one of the sulfonyl oxygens forming hydrogen bonds with the main chain NHs (Leu181 and Ala182), (iii) the sulfonyl substituent making extensive hydrophobic contact with the S1' subsite. The gauche conformation exclusively adopted by the sulfonamide C-N-S-C torsion plays an important role in achieving the third binding feature by properly directing the substituent into the S1' subsite. Improvement of the inhibitory activity according to straight elongation of the sulfonyl substituent was attributed to an increase of the hydrophobic contact between the substituent and the S1' subsite. Structural modifications which alter the straight shape of the substituent lead to deterioration of the activity. On the other hand, the two candidate models selected for MMP-2 differ in the bottom shape of the S1' subsite: one with a channel-like subsite and the other with a pocket-like subsite resembling that of the MMP-9 model. The bottom shape was experimentally probed by chemical synthesis of inhibitors having elongated sulfonyl substituents whose terminal alkyl groups were shown by MD simulations to protrude from the S1' subsite bottom into the solvent. Gelatinase assays of these inhibitors showed that elongation of the substituent significantly reduces activity against MMP-9 while retaining activity against MMP-2, consequently increasing the selectivity between MMP-2 and -9. The results confirm that MMP-9 has a pocket-like S1' subsite with a floorboard and MMP-2 has a channel-like S1' subsite.

Background: Basement membrane degradation is a critical component of tumor invasion and metastasis that is facilitated by the family of enzymes known as matrix metalloproteinases (MMPs). MMP-2 and MMP-9 are two subtypes that have specifically been identified in tumors of gastrointestinal origin. We have previously shown that broad inhibition of these enzymes with the MMP inhibitor BB-94 improves survival in a murine model of pancreatic cancer. The purpose of this study was to determine MMP-2 and MMP-9 activity in orthotopic tumors from mice treated with and without BB-94., Methods: Ten million cells of a moderately differentiated pancreatic cancer cell line (HPAC) were implanted orthotopically into Balb/c nu/nu mice. The mice were treated with BB-94 or vehicle control for 70 days or until death. At necropsy, tumors were harvested, total protein was extracted, and MMPs were purified from 400 microgram of crude protein extract by gelatin-Sepharose affinity chromatography. Relative enzyme levels and activity of MMP-2 and MMP-9 were determined by Western blot and gelatin zymography., Results: Tumors from treated animals were significantly smaller than those from nontreated animals. MMP-2 was present in greater amounts in both treated and nontreated animals than MMP-9. Active MMP-2 was present in both groups but significantly decreased in animals treated with BB-94. Active MMP-9 was absent in both groups, whereas levels of latent MMP-9 appeared lower than those of MMP-2 in all samples., Conclusions: Activated MMP-2 and not MMP-9 in HPAC cells grown in nude mice suggests that this MMP subtype is more critical in the phenotypic behavior of such tumors. Furthermore, attenuated levels of active MMP-2 in animals treated with the enzyme inhibitor BB-94 suggest that previously observed improvements in survival correlate with the level of MMP-2 activity., (Copyright 1999 Academic Press.)

A series of succinyl hydroxamate MMP inhibitors were prepared incorporating an aryl amino ketone moiety in place of the more typical C-terminal amino acid amides. Compounds of the C-terminal ketone series displayed potent inhibition of MMPs. Several compounds of the series were shown to be orally bioavailable.

Collagen gels in vitro can be contracted by fibroblasts. The role of matrix metalloproteinases (MMPs) in the contraction of collagen lattices by human neonatal foreskin fibroblasts (HuFFs) was investigated in tissue culture media supplemented by various doses of known gelatinase inhibitors. Fluorescent assays with model gelatinase substrates and media conditioned by fibroblasts apparently confirmed the ability of chemically modified tetracyclines (CMTs) to act as inhibitors of MMP2, and zymography demonstrated that this was the major cell-derived MMP activity. There were no observable effects on the rate of contraction of attached FPCLs containing 6 x 10(4) HuFFs (passages 18-25) with either CMT-5 or CMT-2 at all concentrations tested (0-100 micrograms/mL). However, at greater than 20 micrograms/mL doxycycline and greater than 5 micrograms/mL CMT-3, FPCL contraction was completely abolished. Quantitative assessment of cell viability by means of the MTT assay in monolayer and qualitatively within the FPCLs with CalceinAM suggested that differences were not due to cytotoxic effects. Seeding FPCLs with lower-passage fibroblasts produced identical trends. These results may implicate the involvement of MMPs in the process of gel contraction, although tetracyclines have effects additional to their ability to inhibit MMPs directly.

Chemically modified non-antimicrobial tetracycline derivatives and other low-molecular-weight synthetic matrix metalloproteinase (MMP) inhibitors inhibited keratinocyte migration. Since 72-kDa gelatinase-A (MMP-2) was the major gelatinase in our culture conditions, the results suggest that this MMP may be important in the regulation of keratinocyte mobility. On the other hand, we measured only gelatinase activities (MMP-2 and -9) present in culture medium, and therefore the results do not reveal how the inhibitors affect other MMPs as well as MMP levels close to the cell membranes. Overall, CMTs were found to be efficient in the inhibition of keratinocyte migration.

Collagenase and gelatinase are matrix metalloproteinases (MMPs) which play an important role in tissue destruction in arthritic joints. Studies have demonstrated that tetracyclines can inhibit MMPs and prevent tissue destruction independent of their antimicrobial activity. The purpose of this pilot study is to assess the potential therapeutic role of Doxycycline in patients with advanced osteoarthritis of the temporomandibular joint (TMJ). This ongoing investigation includes patients with a diagnosis of osteoarthritis of the TMJ based on clinical and diagnostic imaging findings, symptoms (localized TMJ pain, limited mobility, dysfunction) for a minimum of 36 months, and failure of previous non-surgical and surgical modalities to alleviate the symptoms. A synovial fluid sample is collected by a saline injection and aspiration technique, followed by diagnostic arthroscopy. Patients are placed on Doxycycline 50 mg BID for three months and then undergo repeat diagnostic arthroscopy and synovial fluid collection. The samples are stored at -80 degrees C. Collagenase activity is determined by a combination of SDS-polyacrylamide gel electrophoresis and fluorography and calculated based on the percentage of collagen alpha chains that are degraded into alphaA breakdown products. Three patients have completed the three-month course of Doxycycline thus far, and 5 joints with osteoarthritis have been analyzed. All patients were female (mean age = 35, mean duration of symptoms = 132 months) and had undergone previous bilateral arthroscopies. One patient had undergone unilateral arthroplasty. The mean collagenase activity showed 55% collagen lysis prior to Doxycycline treatment and 19% after three months of therapy. The mean gelatinase activity was 28% prior to Doxycycline treatment and 7% after three months of therapy. The mean interincisal opening was 33 mm initially and 41 mm after three months of Doxycycline. Subjectively, two of the three patients reported significant improvement in their overall symptoms, which they had not experienced over the previous three years. One patient did not experience any change in symptoms, in spite of a marked reduction in collagenase activity from 86.4% to 9.6%. Because of the very small numbers of patients enrolled in this pilot study so far, no statistically significant differences could be appreciated. However, the dramatic reduction in collagenase activity in these patients, with a long history of TMJ symptoms from osteoarthritis, suggests the potential promising role of Doxycycline in the management of osteoarthritis, and further investigation is warranted.

A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub-lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between the epidermal basal cells and the basement membrane therefore mimicking the lesion of laminitis. Electrophoresis of culture medium from control hoof explants into gradient polyacrylamide gels co-polymerised with gelatin revealed that the explants had produced 2 gelatinases of molecular weight 92 and 72 kDa corresponding to EqMMP-9 and EqMMP-2 respectively. Minor bands of lower molecular weight were the active forms of these enzymes. The zymograms of culture medium from APMA treated explants revealed an increase in the amount of active MMPs. Equine polymorphs cultured for 2 days produced only EqMMP-9. Lamellar explant medium from horses with acute laminitis contained increased amounts of zymogen and active EqMMP-2 and EqMMP-9 particularly in explants from the fore hooves. Zymography of homogenates of normal lamellar hoof tissue revealed only EqMMP-2 and a minor active band. However, homogenates of lamellar tissue from horses with laminitis showed that EqMMP-9 was present as well as increased EqMMP-2 in both zymogen and active forms. Addition of the MMP inhibitor batimastat (BB-94) to the culture medium of APMA treated explants prevented lamellar separation. BB-94 incubated with polyacrylamide strips containing the MMPs from laminitis affected lamellar explants inhibited enzymatic activity at a concentration of 1 mmol/l. It is concluded that activation of MMPs may be responsible for the lamellar separation seen in laminitis and that MMP inhibitors may be useful clinically for preventing this process.

Matrix metalloproteinases are a family of highly regulated peptidases that are collectively responsible for the degradation of extracellular matrix during tissue remodeling. Dysregulated activity has long been implicated in the pathologies of cancer and arthritis, and the number of diseases more recently associated with these enzymes has been increasing. In the past year, new transgenic models of matrix metalloproteinase knockouts have been described, allowing the direct assessment of specific enzyme activity in particular disease models. In addition, more selective inhibitors with improved pharmacokinetic profiles have entered clinical trials, allowing the assessment of the safety and efficacy of this strategy.

The close correlation observed between matrix metalloproteinase 2 (MMP-2) activation and metastatic progression in various tumors suggests that MMP-2 is a 'master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1-MMP) was identified as a potential physiological activator of MMP-2. Like all other MMPs, MT1-MMP possesses a pro-domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin-like convertases at the end of its pro-domain suggests a potential role for these proteinases in MT1-MMP processing. In order to evaluate the implication of furin in pro-MT1-MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro-MMP-2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro-MT1-MMP processing as well as pro-MMP-2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.

Various N-sulfonylamino acid derivatives were synthesized and evaluated for their in vitro and in vivo activities to inhibit type IV collagenase (MMP-9 and MMP-2). When the amino acid residue and the sulfonamide moiety were modified, their inhibitory activities were greatly affected by the structure of the sulfonamide moiety. A series of aryl sulfonamide derivatives containing biaryl, tetrazole, amide, and triple bond were found to be potent and highly selective inhibitors of MMP-9 and MMP-2. In addition, these compounds were orally active in animal models of tumor growth and metastasis. These results revealed the potential of the N-sulfonylamino acid derivatives as a new type of candidate drug for the treatment of cancer.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs. The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta. Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster. In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies. Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids. Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities. The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E. coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2. The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.

The synthesis and biological evaluation of orally active inhibitors of matrix metalloproteinase are reported. Modifications of the P2' position and the alpha-substituent of hydroxamic acid derivatives were carried out, and revealed that the P2' substituent influenced the MMP inhibitory activities in vitro and in plasma after oral administration. The hydroxamates with phenylglycine at the P2' position were absorbed well orally. Compound 15e, which exhibited the longest duration of inhibitory activity in plasma after oral administration among the phenylglycine derivatives (5a-5d, 15a, 15c, 15e), was evaluated in a rat adjuvant arthritis model. A reduction in hind foot pad swelling and improvements of some inflammatory parameters were demonstrated when the compound was administered orally. These results indicate the potential of MMP inhibitors for rheumatoid arthritis.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Pancreatic islets contain trace amounts of zinc to form insulin dimer, and matrix metalloproteinases (MMPs) are single-chain zinc-containing metallo-enzymes. By immunocytochemically staining pancreatic tissue, which contained exocrine duct adenocarcinoma, normal islets were incidentally found positive for gelatinase-A (MMP-2) and gelatinase-B (MMP-9), and for tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2). Normal islets from five pancreata were exclusively stained for two each of gelatinases and TIMPs. Twenty-two islet cell tumors were also stained for pancreatic hormones, gelatinases, and TIMPs, which included insulinomas, gastrinomas, glucagonomas, pancreatic polypeptide-omas (PPomas), and nonfunctioning tumor. In general, islet cell tumors were weakly stained for gelatinases and TIMPs, compared with normal islets in the adjacent pancreatic tissue. No clear difference in staining intensity among five kinds of islet cell tumors was observed. The selective immunolocalization of gelatinases and TIMPs in islet cells and islet cell tumors may suggest possible a structure-function relationship among zinc, gelatinases-TIMPs, and pancreatic hormones.

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases are implicated in various steps of development of metastasis, through their ability to degrade the extracellular matrix. Increased matrix metalloproteinase activity of tumor cells has been associated with a higher metastatic potential. Inhibitors of metalloproteinases have been shown to effectively reduce or prevent the formation of metastases. The family of tetracyclines is able to inhibit matrix metalloproteinase activity through chelation of the zinc ion at the active site of the enzyme. Using tumor cell lines relevant to bone metastases, i.e. PC-3, MDA-MB-231, Hs696, B16/F1, we showed that tetracycline and derivatives of tetracycline, namely doxycycline and minocycline, also induced cytotoxicity. The effective concentrations are relatively high for plasma, but are clinically achievable in the bone, since tetracyclines are osteotropic. All four bone-metastasizing tumor cells produced and secreted various matrix metalloproteinases. Doxycycline was able to inhibit the activity of 72- and 92-kDa type IV collagenase secreted by bone-metastasizing cells by 79-87%. These characteristics could make tetracycline a unique candidate as a therapeutic agent to prevent bone metastases in cancer patients with a high likelihood for development of bone metastasis. Studies using animal models of experimental bone metastasis will be necessary to confirm this.

A fully automated peptide synthesizer was used to generate tetrapeptide sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their linear range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine-epsilon-amino caproic acid-beta-alanine-histidine (His-epsilon Ahx-beta Ala-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibition by selected tetrapeptides was also evaluated with two other techniques, a native type IV collagen degradation assay and a fluorogenic enzymatic assay, confirming the tetrapeptide potency. The His-epsilon Ahx-beta Ala-His tetrapeptide also inhibited purified human MMP-2 and MMP-9 and the corresponding enzymes present in conditioned media from human tumour cells. Finally, the length of the spacer between the two terminal histidines was found to be crucial to the inhibitory potential. This approach may thus be considered as a-successful strategy to yield specific peptide or pseudopeptide inhibitors, although their potency remains moderate, since it was measured before any chemical optimization was undertaken.

The anti-metastatic ruthenium complex Na[trans-RuCl4(DMSO)Im] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying s.c. implants of MCa mammary carcinoma. The aim of the study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-1 and TIMP-2 mRNAs. Na[trans-RuCl4(DMSO)Im] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCl4(DMSO)Im] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour-infiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in controls.

Membrane type 1-matrix metalloproteinase (MT1-MMP) initiates the activation of the zymogen progelatinase A/ 72-kDa type IV collagenase by cleavage of the Asn66-Leu peptide bond. We previously pointed out that MT1-MMP possesses a unique amino acid sequence Arg-Arg-Lys-Arg111 which is a potential recognition sequence for furin-like proteases (Nature, 370 (1994) 61-65). Here, using a recombinant MT1-MMP expressed in Escherichia coli we demonstrated that furin specifically cleaves MT1-MMP between Arg111-Tyr in vitro, which resulted in a stimulation of progelatinase A-activation function. Tissue inhibitor of metalloproteinases (TIMP)-2 inhibited activation of progelatinase A by forming a stable complex with activated MT1-MMP.

Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion by degrading extracellular matrix proteins. However, little is known about the in situ expression of MMP in human normal livers and primary liver tumors. In this study, we therefore examined the in situ expression of immunoreactive MMP and tissue inhibitors of MMP (TIMP) in 10 normal livers, 11 surgically resected intrahepatic cholangiocarcinomas (CCs), and 6 surgically resected hepatocellular carcinomas (HCCs). In normal livers, MMP and TIMP were infrequently and faintly expressed in bile ducts, but were not expressed in hepatocytes. In the 11 CCs, MMP-1, MMP-2, MMP3, MMP-9, TIMP-1, and TIMP-2 were expressed in tumor cells and/or tumor stroma in 11 (100%), 5 (45%), 8 (73%), 3 (27%), 9 (82%), and 9 (82%), respectively. The expression of MMP and TIMP in tumor cells was located in the cytoplasm with a diffuse or granular pattern; that in the tumor stroma was situated in fibroblasts, leukocytes, and extracellular matrix. Their expression was stronger in CC cases with severe invasion than in CC cases with mild invasion. In contrast, MMP and TIMP were not expressed in any cases of HCC. These results show that intrahepatic bile duct cells may neoexpress or overexpress MMP and TIMP after malignant transformation but that hepatocytes do not, and suggest that MMP and TIMP play an important role in CC cell invasion by degrading extracellular matrix proteins.

Matrix metalloproteinases (MMPs), which degrade the components of the extracellular matrix, are key enzymes involved in the tissue remodeling of multicellular organisms. Since MMPs are secreted as inactive zymogens (pro-MMPs), they have to be activated to function. We identified a membrane-type MMP (MT-MMP) that activated proMMP-2 (pro-gelatinase A = 72 kDa type IV pro-collagenase) and described its expression on the invasive tumor cell surface. In this study we further examined the expression and role of MT-MMP in the activation of proMMP-2 during mouse embryogenesis. Northern blotting demonstrated that MT-MMP expression was increased together with that of MMP-2 and its inhibitor gene, TIMP-2, in embryos depending upon the number of days after gestation, and decreased with maturation after birth. In situ hybridization and immunohistochemistry localized MT-MMP mRNA and protein in the cells of ossifying tissues where both MMP-2 and TIMP-2 were expressed. Activated MMP-2 was detected by gelatin zymography in the lysates prepared from the micro dissected tissues that expressed the three genes. The activation rate of proMMP-2 was proportional to the expression of MMP-2 and MT-MMP. These results indicated that proMMP-2 activation through its activator, MT-MMP, is a physiological system used by organisms to initiate tissue remodeling on the cell surface.