Matrix Metalloproteinases Activity Assay Research Articles

Novel biomimetic peptide-loaded chitosan nanoparticles improve dentin bonding via promoting dentin remineralization and inhibiting endogenous matrix metalloproteinases

ObjectiveThis study aims to synthesize novel chitosan nanoparticles loaded with an amelogenin-derived peptide QP5 (TMC-QP5/NPs), investigate their remineralization capability and inhibitory effects on endogenous matrix metalloproteinases (MMPs), and evaluate the dentin bonding properties of remineralized dentin regulated by TMC-QP5/NPs. MethodsTMC-QP5/NPs were prepared by ionic crosslinking method and characterized by dynamic light scattering method, scanning electron microscopy, transmission electron microscope, atomic force microscope, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The encapsulation and loading efficiency of TMC-QP5/NPs and the release of QP5 were examined. To evaluate the remineralization capability of TMC-QP5/NPs, the mechanical properties, and the changes in structure and composition of differently conditioned dentin were characterized. The MMPs inhibitory effects of TMC-QP5/NPs were explored by MMP Activity Assay and in-situ zymography. The dentin bonding performance was detected by interfacial microleakage and microshear bond strength (μSBS). ResultsTMC-QP5/NPs were successfully synthesized, with uniform size, good stability and biosafety. The encapsulation and loading efficiency of TMC-QP5/NPs was respectively 69.63 ± 2.22% and 13.21 ± 0.73%, with a sustained release of QP5. TMC-QP5/NPs could induce mineral deposits on demineralized collagen fibers and partial occlusion of dentin tubules, and recover the surface microhardness of dentin, showing better remineralization effects than QP5. Besides, TMC-QP5/NPs significantly inhibited the endogenous MMPs activity. The remineralized dentin induced by TMC-QP5/NPs exhibited less interfacial microleakage and higher μSBS, greatly improved dentin bonding. SignificanceThis novel peptide-loaded chitosan nanoparticles improved resin-dentin bonding by promoting dentin remineralization and inactivating MMPs, suggesting a promising strategy for optimizing dentin adhesive restorations.

Potential of Fluoride-Containing Zinc Oxide and Copper Oxide Nanocomposites on Dentin Bonding Ability.

Despite recent advances in bonding restorations, which are the basis of restorative dentistry, secondary caries are still able to form. Previously, a novel fluoride-containing zinc and copper (ZCF) nanocomposite was introduced to prevent the formation of caries due to its antibacterial activity. In this study, we studied the impact of ZCF nanoparticles on the adhesive strength of bonding restorations through micro-tensile bond strength (µTBS) testing. The impact of antibacterial and matrix metalloproteinase (MMP) inhibitors on the nanoparticles was also examined. The nanocomposites were prepared using a simple one-step homogeneous co-precipitation method at a low temperature. A self-etch adhesive was applied to 10 extracted caries-free human molars with (test group) and without (control group) the ZCF nanoparticles. This was followed by composite resin build-up and µTBS testing, MMP activity assays, and evaluation of the antibacterial effects. The results showed no significant differences in the µTBS between the ZCF and the control groups. However, the ZCF exhibited a significant inhibitory effect against MMP-2, MMP-8, and MMP-9, in addition to an antibacterial effect on Streptococcus mutans. Therefore, the present study demonstrated that the addition of ZCF nanoparticles to adhesive systems can result in MMP inhibition and antibacterial action while maintaining the mechanical properties of the bonding restorations.

Effect of chlorhexidine-loaded poly(amido amine) dendrimer on matrix metalloproteinase activities and remineralization in etched human dentin in vitro

To investigate the effect of chlorhexidine (CHX)-loaded carboxyl-terminated poly (amido amine) dendrimer (CHX-PAMAM-COOH) on matrix metalloproteinase (MMP) activities and remineralization in human dentin, CHX-PAMAM-COOH was prepared and characterized by Fourier-transform infrared spectroscopy. The inhibitory effects of CHX, PAMAM-COOH, and CHX-PAMAM-COOH on soluble recombinant human matrix metalloproteinase (rhMMP-2) and dentin-bound endogenous MMP activity were measured using an MMP Activity Assay Kit. In situ zymography was performed to evaluate the gelatinase activity in dentin pretreated with CHX, PAMAM-COOH, and CHX-PAMAM-COOH. The remineralization of etched dentin pretreated with CHX, PAMAM-COOH, and CHX-PAMAM-COOH was evaluated by field emission-scanning electron microscopy (SEM) and energy disperse spectroscopy (EDS) after incubation in artificial saliva for 14 days. The results of the rhMMP-2 activity assay showed that the MMP-2 activity in the CHX-PAMAM-COOH group and the CHX group decreased significantly to 5.58 ± 0.85% (P < 0.05) and 4.86 ± 1.12% (P < 0.05), respectively, but that in the PAMAM-COOH group increased significantly to 213.38 ± 0.11% (P < 0.05). The results of total MMP activity and in situ zymography showed a significant reduction in endogenous gelatinase activity in dentin in the CHX-PAMAM-COOH group and the CHX group. The SEM and EDS results showed that rod-like crystals were formed on the etched dentin surface in the PAMAM-COOH group and the CHX-PAMAM-COOH group, and their Ca/P ratios were 1.73 and 1.71, respectively. In conclusion, CHX-PAMAM-COOH can inhibit dentin-bound endogenous MMPs and induce remineralization in etched dentin simultaneously. However, it is important to note that the catalytic role of PAMAM dendrimers may have an undesired excitatory effect on MMP activity, which cannot be ignored if PAMAM dendrimers were used alone in the oral environment.

Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models.

BackgroundWe developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia.MethodsA morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-β2 (TGF-β2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice.FindingsIn the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes.InterpretationStepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials.FundingThis research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.

The effect of phytic acid on enzymatic degradation of dentin.

We evaluated the effect of phytic acid on matrix metalloproteinase (MMP)- or cysteine cathepsin (CC)-mediated dentin degradation. Demineralized dentin beams were divided into five groups (n=12) and treated with 1%, 2%, or 3% phytic acid or with 37% phosphoric acid. Untreated demineralized beams served as controls. After incubation for 1 or 3wk, dry mass loss was determined and aliquots of incubation media were analysed for cross-linked telopeptide of type I collagen (ICTP) fragments for MMP-mediated and c-terminal telopeptide of type I collagen (CTX) for cathepsin-k-mediated degradation. The direct effect of phytic acid was evaluated using MMP activity assay. Data were analysed using repeated-measures anova. ICTP releases with 1% and 2% phytic acid treatment were statistically significantly lower than those following phosphoric acid treatment at 3wk. The CTX release for phytic acid-treated beams at 3wk was not significantly different from that of untreated control beams, but it was significantly lower than that of phosphoric acid-treated beams. Their MMP activities at 3wk were not significantly different from those of the controls but they were significantly lower than those seen for phosphoric acid-treated beams. Compared to phosphoric acid, phytic acid treatment resulted in a reduced dentinal host-derived endogenous enzymatic activity and collagen degradation.

Effect of myricetin on odontoblast-like cells and its potential to preserve resin–dentin Bonds

Stabilization of the resin–dentin interface to increase the durability of adhesive dental restorations is a challenging task. The use of naturally occurring collagen crosslinking agents has been proposed to prevent degradation of the hybrid layer. Myricetin (MYR) is a flavonoid with a wide variety of beneficial effects and it has been used for the treatment of different systemic pathologies. The chemical structure of MYR makes it a powerful antioxidant, an inhibitor of matrix metalloproteinase (MMP) activity, and a collagen cross-linker. This study presents MYR as a novel treatment in operative dentistry to stabilize the resin–dentin interface by inhibiting MMPs and crosslinking the collagen. Viability tests carried out using a resazurin assay showed that MYR had no cytotoxic effects on human odontoblast-like cells and the phenotype was preserved. Fluorometric MMP activity assay and fluorescence microscopy revealed that the MMPs in the demineralized dentin were effectively inhibited by the application of MYR (600 μM for 120 s). A microtensile bond strength test was performed immediately and after six months of storage. The bond strength to dentin was not affected by MYR and was preserved over time. Demineralized dentin beams were evaluated to determine the dentin biomodification using microtensile strength and elastic modulus assays. MYR improved the biomechanical behavior of the demineralized dentin similarly to glutaraldehyde, a recognized crosslinking agent. These findings indicated that MYR acts as an MMP inhibitor, collagen cross-linker, and preserver of the bond strength. Furthermore, MYR is an ethanol-soluble molecule with a lower molecular weight than the other polyphenols; hence, it can be applied for a short time and diffuses deeply through the dentin without any associated cytotoxicity. This molecule has beneficial effects on the biological and mechanical behavior of the resin–dentin interface and may be used to effectively stabilize the hybrid layer in a clinical setting.

The Effect of PAMAM Dendrimer on the Dentin Remineralization and Matrix Metalloproteinases Activity

The purpose of this study was to investigate the effect of G4.5 carboxyl-terminated poly dendrimer (PAMAM-COOH) on the dentin remineralization and the matrix metalloproteinases (MMPs) activity. The dentine samples were averagely divided into four groups: 100 mg/mL PAMAM-COOH group (A group), 10 mg/mL PAMAM-COOH group (B group), 2% (wt) chlorhexidine (CHX) group (C group) and deionized water group (Control group). MMP Activity Assay Kit was used to detect the activity of dentin endogenous MMPs in the four groups. The loss of dry mass of dentin after 30 d were measured. In situ zymography analysis was performed to detect the effects of PAMAM dendrimer in each group (except A group) on gelatinase activity in dentin. After incubation in artificial saliva for 7 and 14 d incubated, the remineralization of each group (except A group) in dentin surfaces were examined using a field emission-scanning electron microscope (FESEM). Compared with the control group, the dentin endogenous MMPs activity in A, B and C groups were all decreased ( P<0.05). The activity of endogenous MMPs in C group was lower than that of A and B groups ( P<0.001), but the difference between A and B groups was not statistically significant. The loss of dry mass in A, B and C groups were lower than that in control group ( P<0.05), but there were no significant difference in A, B and C groups. The in situzymography analysis showed that 48 h later, the dentin gelatinase activity in B group was inhibited compared with the control group, but the inhibitory effect was weaker than that of CHX. After 7 d and 14 d, there were no obvious mineralization in the control group, while distinct mineralization were observed in B group. The mineralization effect in group B was better than group C. G4.5 PAMAM-COOH could introduce remineralizationin and demineralizeddentin by effectively inhibiting endogenous MMPs and gelatinase, thus contributes as novel material to enhancing durability of adhesion.

Lipoxin A4 interferes with embryo implantation via suppression of epithelial-mesenchymal transition.

To test whether lipoxin A4 (LXA4) interferes with embryo implantation via suppression of epithelial-mesenchymal transition (EMT). We developed a mouse model of LXA4 blocking embryo implantation and detected the indicators of EMT to confirm that LXA4 inhibits EMT might be a mechanism of interfering with the embryo implantation. We detected integrin-linked kinase (ILK), N-formylpeptide receptor 2 (FPR2), vascular endothelial growth factor, matrix metalloproteinases (MMPs), Akt, GSK3β, NF-ĸB, twist, vimentin, fibronectin, and β-catenin mRNA expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR; localized protein expression using immunohistochemistry and Western blotting assay; MMPs activity assay by gelatin zymography; and the status of implantation in pregnant animals assessed by pontamine blue reaction test. Preimplantation administration of LXA4 resulted in implantation failure. LXA4 has a time- and dose-dependent effect on embryo implantation. Day 0.5 after fertilization is the most effective time to use LXA4 to block embryo implantation. (a) LXA4 reduced endometrial stroma edema; (b) LXA4 inhibited the activity of MMP9 and significantly upregulated the expression of β-catenin, and downregulated the expression of vimentin, fibronectin, twist, NF-κB, Akt, and Gsk-3β in the endometrium and TEV-1 cells; (c) LXA4 upregulated the expression of FPR2, and downregulated the expression of ILK; FPR2-overexpressing had an inhibitory effect on ILK in TEV-1 cells. LXA4 inhibits EMT which attenuates ILK action by enhancing FPR2; therefore, this might be a mechanism of interfering with embryo implantation.

Myosin-X Silencing in the Trabecular Meshwork Suggests a Role for Tunneling Nanotubes in Outflow Regulation.

PurposeThe actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM.MethodsShort hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed.ResultsCK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake.ConclusionsInhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.

Abstract 678: microRNA 146a Reduces Activity of Matrix-Metalloproteinases in the Context of Arterial Stiffness

Background: Arterial stiffness is part of arterial aging and strongly associated with increased risk for cardiovascular events. Unfortunately, until today there is no treatment available effectively counteracting arterial stiffening. One key feature of arterial stiffening is elastin fragmentation. Breakdown of elastin is mediated by matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9 play a pivotal role in this process. Non-coding micro RNAs (miRs) were found to effectively regulate gene expression, consequentially representing potential therapeutic targets. We hypothesize that miR146a inhibits MMP-2 and MMP-9, thus leading to decreased elastin fragmentation in the vascular extracellular matrix. Methods and Results: RNA Sequencing found miR146a to be upregulated in a stiffness-dependent manner in vitro and in an age dependent manner in vivo . Since both MMP-2 and MMP-9 are predicted targets of miR146a, we transfected aortic smooth muscle cells with constructs either increasing (mimicking construct) or decreasing (inhibitory anti-sense construct) effective miR146a levels and evaluated the effect on MMP-2 and MMP-9 expression and activity in vitro . qRT-PCR analysis demonstrated significantly de-repressed MMP-2 and MMP-9 levels upon transfection with inhibitor constructs, while mimic constructs further repressed the expression of MMP-2 and MMP-9. Overall MMP activity was significantly altered accordingly after transfection with the respective constructs as determined by a fluorimetric MMP Activity Assay. MMP-2 activity could be specifically assessed using Zymography. After transfection with miR146a inhibitor, both active MMP-2 as well as pro-MMP-2 activity were found to be significantly increased. Conclusion: Our study provides first evidence that miR146a reduces the levels and activity of MMP-2 and MMP-9 in vascular smooth muscle cells. We conclude that miR146a might have protective function on the integrity of the elastin network in the arterial media. We therefore consider miR146a a potential drug target in the context of elastin fragmentation and arterial stiffness, thus potentially alleviating the development of aortic aneurysm.

Complement 5a Receptor deficiency does not influence adverse cardiac remodeling after pressure-overload in mice

Hypertension is one of the most common risk factors for the development heart failure in the general population. Inflammation plays a central role in this adverse remodeling and eventually to the development of heart failure. Circulating levels of Complement factor 5a (C5a) are increased in hypertensive patients and the C5a receptor is associated with the presence of cardiac fibrosis and inflammation in an experimental hypertension model. To test if C5aR is involved in adverse cardiac remodeling following pressure-overload, we induced transverse aortic constriction (TAC) in wildtype and C5a receptor deficient mice (C5aR−/−). Six weeks after TAC, C5aR-/- animals showed a similar degree of cardiac hypertrophy and decrease in cardiac function as wild type mice (End Systolic Volume; 50.30±5.32 µl vs. 55.81±8.16 µl). In addition, other features of adverse cardiac remodeling like cardiomyocyte cell size (WGA staining), fibrosis (picrosirius red staining) or collagen degradation (matrix metalloproteinase activity assay) did not differ either. In conclusion, full body C5aR deficiency does not affect adverse cardiac remodeling after pressure-overload. However, our finding are in contrast with C5a inhibition studies. Our observations do present the role of C5a-C5aR in adverse cardiac remodeling and heart failure as controversial at the least.

Use of crosslinkers to inactivate dentin MMPs

ObjectivesThis study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity. MethodsSix different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography. ResultsMMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples’ activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release. SignificanceThe result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.

Characterization of TAMRA- and biotin-conjugated peptide arrays for on-chip matrix metalloproteinase activity assay

Peptide arrays have been widely used for high-throughput determination of protease activities in cells and tissues because specific peptides have high binding affinity for the active site of enzymes. Designing peptide substrate probes for enzyme activity assays have been considered to be important; however, the significance of its reporter tag for detecting enzymatic reactions is relatively underestimated. Thus, we investigated the effect of the reporter tag of peptide substrate probes on on-chip protease activity assays. We optimized and characterized proteolytic activity assay of matrix metalloproteinase-3 using direct and indirect substrate probes, tetramethyl-6-carboxyrhodamine (TAMRA)- and biotin-conjugated peptide arrays, respectively. Proteolytic activity assays using both substrate probes demonstrated similar sensitivity, ratio of maximal to minimal FI, IC50 of GM6001, and inter-array reproducibility. However, biotin-conjugated substrate arrays showed a wider dynamic range than TAMRA-conjugated substrate arrays. Thus, this comparative study provides a wealth of information for developing optimal probes necessary for effective analysis of enzyme activity and kinetics.

Rhinovirus‐induced basic fibroblast growth factor release mediates airway remodeling features

BackgroundHuman rhinoviruses, major precipitants of asthma exacerbations, induce lower airway inflammation and mediate angiogenesis. The purpose of this study was to assess the possibility that rhinoviruses may also contribute to the fibrotic component of airway remodeling.MethodsLevels of basic fibroblast growth factor (bFGF) mRNA and protein were measured following rhinovirus infection of bronchial epithelial cells. The profibrotic effect of epithelial products was assessed by DNA synthesis and matrix metalloproteinase activity assays. Moreover, epithelial cells were exposed to supernatants from cultured peripheral blood mononuclear cells, obtained from healthy donors or atopic asthmatic subjects and subsequently infected by rhinovirus and bFGF release was estimated. bFGF was also measured in respiratory secretions from atopic asthmatic patients before and during rhinovirus-induced asthma exacerbations.ResultsRhinovirus epithelial infection stimulated mRNA expression and release of bFGF, the latter being positively correlated with cell death under conditions promoting rhinovirus-induced cytotoxicity. Supernatants from infected cultures induced lung fibroblast proliferation, which was inhibited by anti-bFGF antibody, and demonstrated increased matrix metalloproteinase activity. Rhinovirus-mediated bFGF release was significantly higher in an in vitro simulation of atopic asthmatic environment and, importantly, during rhinovirus-associated asthma exacerbations.ConclusionsRhinovirus infection induces bFGF release by airway epithelium, and stimulates stroma cell proliferation contributing to airway remodeling in asthma. Repeated rhinovirus infections may promote asthma persistence, particularly in the context of atopy; prevention of such infections may influence the natural history of asthma.

Comparison of metalloproteinase protein and activity profiling

Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.

Matrix metalloproteinase activity assays: Importance of zymography

Introduction: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. Methods: For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Results and Discussion: Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

Coordinated Expression of MMPs and TIMPs in Rat Knee Intra-Articular Tissues After ACL Injury

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.

The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases

Matrix metalloproteinase (MMP) sequestering polymer microspheres were prepared by a post-polymerization hydroxamic acid derivatization of poly(methyl methacrylate- co-methacrylic acid). The microspheres were designed to selectively bind MMPs from solutions on contact through a direct interaction between the polymer-bound hydroxamic acid groups and the characteristic catalytic site zinc atom common to all MMPs. MMP activity assays showed that the hydroxamic acid microspheres reduce MMP activity on contact in a time and concentration-dependent fashion. This effect was observed for several MMP subclasses (MMP-2, -3, -8 and -13) suggesting that the microspheres possess a broad-spectrum MMP binding capacity. However, inactive pro-forms of MMPs showed little binding affinity for the microspheres indicating that the interaction was dependent on MMP activation. The preferential binding of active MMPs was confirmed by MMP-3 and MMP-8 activation studies, which demonstrated significant increases in microsphere binding on activation. The MMP sequestering effect of the microspheres was also demonstrated in a physiologically relevant solution (human chronic wound fluid extract) indicating that the binding interaction was effective in a multi-component, competitive adsorption environment. Thus, the hydroxamic acid-containing microspheres may find use as localized, broad-spectrum MMP inhibitors for the treatment of a number of disease conditions characterized by elevated MMP activity.

Coordinated Expression of MMPs and TIMPs in Rat Knee Intra-Articular Tissues After ACL Injury

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.