Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

Secretion of gelatinases and activation of gelatinase A (MMP-2) by human rheumatoid synovial fibroblasts.

In monolayer cultures human rheumatoid synovial fibroblasts (HRSF) secrete gelatinase A (MMP-2) and, unlike other human fibroblasts, to a minor extent also gelatinase B (MMP-9) as inactive proenzymes. In this regard HRSF resemble the fibrosarcoma cell line HT-1080. Unlike HT-1080, however, HRSF do not increase the secretion of MMP-9 in response to phorbol-12-myristate-13-acetate. This indicates that in HRSF the protein kinase C pathway for an enhanced MMP-9 secretion is inactive. None of the substances used in our study increased MMP-9 secretion, but some of them inhibited MMP-9 secretion. The secretion of MMP-2 could not be enhanced either, not even by dbcAMP, which has been reported to be effective in Sertoli and peritubular cells. Activation of MMP-2 in HRSF could be induced by treatment with concanavalin A (ConA) or cytochalasin D, as was shown for other cell types. This activation was not accompanied by a significant change in the amount of secreted TIMP-1 and TIMP-2. In contrast to reports on human skin fibroblasts, however, the activation of MMP-2 could not be induced in HRSF by treatment of the cells with monensin or sodium orthovanadate. Moreover, monensin was shown to act as an inhibitor of ConA- or cytochalasin D-mediated activation. Additionally, and in contrast to a report on a rat fibroblast cell line, MMP-2 activation is not mediated via the MAP kinase pathway in HRSF: PD 98059, a specific inhibitor of MAP kinase kinase, did not inhibit the activation of MMP-2. Similarly ineffective were PD 169316, an inhibitor for p38 MAP kinase, other inhibitors for protein kinases as lavendustin A, G? 6983, wortmannin, rapamycin, as well as the protein tyrosine kinase inhibitors herbimycin A and genistein. Only staurosporin, a broad spectrum inhibitor of protein kinases, and the ionophores monensin and A 23187 effectively inhibited MMP-2 activation in HRSF. Our results demonstrate that MMP-2 can be activated by quite different pathways, and that different cells, even when belonging to the fibroblast family, do not necessarily use the same activating pathways.     

Modulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by interferon-γ in a human salivary gland cell line

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-γ and TNF-α, have been demonstrated in the salivary gland microenvironment of patients with Sjo¨︁gren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-γ with or without (±) TNF-α could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-γ and (+) TNF-α in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-γ + TNF-α in the pathogenesis of SS. The HSG cell line, in the presence of IFN ± TNF-α, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-γ receptor, and this was associated with a complete inhibition of the previously described IFN-γ ± TNF-α antiproliferative effect. However, incubation of IFN-γ treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS. J. Cell. Physiol. 171:117–124, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.     

Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography

Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions.     

Matrix metalloproteinases (MMP-2 and MMP-9) activity in corneal ulcer and ocular surface disorders determined by gelatin zymography

The purpose of this paper is to determine the active form of matrix metalloproteinases (MMP-2 and MMP-9) in corneal ulcer and ocular surface disorder patients. A total of 35 patients of corneal ulcer, 20 patients of ocular surface disorders and 10 control subjects were included in this study and estimation of active form of MMP-2 and MMP-9 was done by gelatin zymography. Tear samples were collected by capillary tube method. Both pro- and active forms of MMP-9 were detected in 24 out of 35 patients with corneal ulcer and 15 out of 20 patients with ocular surface disorders. None of the patients were showing MMP-2 activity. Neither MMP-2 nor MMP-9 was detected in the control group. Active forms of MMP-9 are present in tears of severe ulcerative and ocular surface disorder patients. Thus, proteinase inhibitors have been recommended for the treatment of corneal ulcer and ocular surface disorders to reduced the progression of stromal ulcer and to minimize corneal scarring.     

New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2)

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).     

Localisation of gelatinase activity in epidermal hoof lamellae by in situ zymography

 In situ gelatin zymography is a technique, which utilises a gelatin-based emulsion overlay to detect and, more importantly, localise the gelatinase activity in underlying tissue. Gelatinase A [matrix metalloproteinase-2 (MMP-2)] and gelatinase B [matrix metalloproteinase-9 (MMP-9)] are present in equine hoof homogenates and supernatants from cultured hoof explants by SDS-PAGE gelatin zymography, and it has been assumed that the enzymes are derived solely from matrix and epithelia and not from other sources such as leucocytes. Using in situ zymography, gelatinases are shown to be localised within the equine epidermal hoof lamellae and, more specifically, are apparently produced by epidermal basal and/or parabasal cells. The pattern of expression correlates with that expected based on the progression of pathological changes observed during the onset of laminitis, thus providing further evidence that laminitis pathology probably arises as a result of inadequate local MMP regulation. Accepted: 15 June 1998     

Serum gelatinases (MMP-2 and MMP-9) and VCAM-1 as a guideline in a therapeutic approach in Graves' ophthalmopathy

INTRODUCTION: The aim of this study was to estimate the influence of corticosteroids on soluble MMP-2, MMP-9 and VCAM-1 in patients with Graves ophthalmopathy (GO) in order to assess their usefulness as a guideline in a therapeutic approach. MATERIAL AND METHODS: Serum gelatinases and VCAM-1 were detected in three groups of subjects: 20 patients with GO (CAS > or = 3, anamnesis of GO > or = 1 yr), 12 patients with no clinical symptoms of ophthalmopathy (Gd) and 10 healthy volunteers. Corticosteroid therapy consisted of intravenous infusions (2 series, 3 grams each time) of methylprednisolone (MP) and subsequent treatment with oral prednisone (60 mg per day) in a tapering schedule. The serum samples were collected 24 hours before MP, 24 hours after MP, after 14 days of treatment with prednisone and at the end of corticosteroid therapy. The levels of soluble MMP-2, MMP-9 and VCAM-1 were determined by the ELISA method. RESULTS: We have found no differences in serum MMP-2 between the groups studied and a significant reduction after MP only in corticosteroid-resistant GO patients. Soluble MMP-9 was highest in the GO group compared with both the Gd and control individuals. Moreover serum MMP-9 decreased in corticosteroid-responsive GO patients after MP and remained at the lower level at the end of the study. Positive correlations between MMP-2 and MMP-9 before and after MP administration were observed. Serum VCAM-1 was significantly elevated both in GO and Gd subjects and pre-treatment VCAM-1 levels were elevated in corticosteroid-responders compared with non-responders. CONCLUSIONS: Our results suggest that serum VCAM-1 may serve as a marker predicting the efficacy of corticosteroids and that soluble MMP-9 may be helpful in monitoring corticosteroid administration and in decision-making with regard to further GO treatment.     

Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo

We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.     

The gelatinases,MMP-2 and MMP-9, as fine tuners of neuroinflammatory processes

This review focuses on the complementary roles of MMP-2 and MMP-9 in leukocyte migration into the brain in neuroinflammation, studied mainly in a murine model of experimental autoimmune encephalomyelitis (EAE) that has similarity to the human disease multiple sclerosis. We discuss the cellular sources of MMP-2/MMP-9 in EAE, their sites of activity, and how cleavage of the to-date identified MMP-2/MMP-9 substrates at the blood-brain barrier facilitate leukocyte filtration of the central nervous system (CNS). Where necessary, comparisons are made to inflammatory processes in the periphery and to other MMPs relevant to neuroinflammation. While the general principles concerning MMP-2 and MMP-9 function discussed here are relevant to all inflammatory situations, the details regarding substrates and molecular mechanisms of action are likely to be specific for neuroinflammation.     

MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors

The goal of our study was to analyse the prognostic values for some matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in breast cancer. We evaluated the activity and the expression levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 in malignant versus benign fresh breast tumor extracts. For this purpose, gelatinzymography, immunoblotting and ELISA were used to analyse the activity and expression of MMPs and TIMPs. We found that MMP-9 expression level and activity are increased in malignant tumors. In addition, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratio values obtained by us were significantly different in malignant tumors compared to benign tumors. We suggest that the abnormal MMP-9/TIMP-1 balance plays a role in the configuration of breast invasive carcinoma of no special type and also in tumor growth, while altered MMP-2/TIMP-2 ratio value could be associated with lymph node invasion and used as a prognostic marker in correlation with Nottingham Prognostic Index. Finally, we showed that in malignant tumors high expression of estrogen receptors is associated with enhanced activity of MMP-2 and increased bcl- 2 levels, while high expression of progesterone receptors is correlated with low TIMP-1 protein levels.     

Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols.

Proteases are essential for protein catabolism, regulation of a wide range of biological processes, and in the pathogenesis of many diseases. Several techniques are available to localize activity of proteases in tissue sections or cell preparations. For localization of the activity of matrix metalloproteinases, in situ zymography was introduced some decades ago. The procedure is based on zymography using SDS polyacrylamide gels containing gelatin, casein, or fibrin as substrate. For in situ zymography, either a photographic emulsion containing gelatin or a fluorescence-labeled proteinaceous macromolecular substrate is brought into contact with a tissue section or cell preparation. After incubation, enzymatic activity is revealed as white spots in a dark background or as black spots in a fluorescent background. However, this approach does not allow precise localization of proteinase activity because of limited sensitivity. A major improvement in sensitivity was achieved with the introduction of dye-quenched (DQ-)gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched. After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that are visible against a weakly fluorescent background. The incubation with DQ-gelatin can be combined with simultaneous immunohistochemical detection of a protein on the same section. To draw valid conclusions from the findings with in situ zymography, specific inhibitors need to be used and the technique has to be combined with immunohistochemistry and zymography. In that case, in situ zymography provides data that extend our understanding of the role of specific proteinases in various physiological and pathological conditions.     

Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and two-dimensional electrophoresis.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) play a key role in the proteolytic cascade leading to ECM degradation during invasion and metastasis. The enzyme activity is regulated both at the intra- and extra-cellular level. Extracellular regulation is achieved mainly through the balance between proenzyme activation and inhibition, which appears to be altered in cancer patients. One of the mechanisms of MMP inhibition is the binding of the enzymes to appropriate tissue inhibitors (TIMP). In the recent literature, it has been suggested that MMP-2 and/or MMP-9 are indeed over-produced in many carcinomas, while the identity of the various enzymatic forms (latent, activated and enzyme/inhibitor complexes) remains to be elucidated. In this study we have analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of patients with colon carcinoma, as well as the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissue). Proteins were separated by means of mono-dimensional or bidimensional electrophoresis, and the enzymes detected by gelatin zymography and immunological assays. The results of densitometric analyses demonstrate that proMMP-9, but not proMMP-2, is significantly higher in the oncologic sera vs. the normal sera. In addition, several oligomeric circulating and tissue forms of MMP-9 are preferentially found in the oncologic samples, both in mono- and second-dimension zymograms. The activated forms of MMP-2 and MMP-9 are uniquely present in the primary tumor extracts, thus confirming the involvement of the tissue microenvironment in gelatinase activation and function.     

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.     

Simultaneous presence of unsaturation and long alkyl chain at P'1 of Ilomastat confers selectivity for gelatinase A (MMP-2) over gelatinase B (MMP-9) inhibition as shown by molecular modelling studies

Structural analogues of Ilomastat (Galardin), containing unsaturation(s) and chain extension carrying bulky phenyl group or alkyl moieties at P'1 were synthesized and purified by centrifugal partition chromatography. They were analyzed for their inhibitory capacity towards MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14, main endopeptidases involved in tumour progression. Presence of unsaturation(s) decreased the inhibitory potency of compounds but, in turn increased their selectivity for gelatinases. 2b and 2d derivatives with a phenyl group inhibited preferentially MMP-9 with IC50 equal to 45 and 38 nM, respectively, but also display activity against MMP-2 (IC50 equal to 280 and 120 nM, respectively). Molecular docking computations confirmed affinity of these substances for both gelatinases. With aims to obtain a specific gelatinase A (MMP-2) inhibitor, P'1 of Ilomastat was modified to carry one unsaturation coupled to an alkyl chain with pentylidene group. Docking studies indicated that MMP-2, but not MMP-9, could accommodate such substitution; indeed 2a proved to inhibit MMP-2 (IC50=123 nM), while displaying no inhibitory capacity towards MMP-9.     

Effect of activation of viral receptors on the gelatinases MMP-2 and MMP-9 in human mesothelial cells

BackgroundExtracellular matrix (ECM) not only provides molecular and spatial information that influence cell proliferation, differentiation and apoptosis but also has the potential to bind and present or release cytokines and cytotactic factors. Synthesis and degradation of extracellular matrix components are balanced by matrix metalloproteinases (MMP) and their inhibitors. In the pericardium as well as in the pleural and peritoneal cavities a multitude of clinically relevant disease states ranging from inflammation to fibrosis and tumor invasion result from altered regulation of MMP activity and are known to be associated with viral disease.MethodsTherefore, the functional linkage between viral receptors of the innate immune system, the toll-like receptors (TLR), and control of MMP activity was exemplarily analyzed by stimulating human mesothelial cells with poly (I:C) RNA.ResultsWe hereby show that human mesothelial cells (MC) express TLR3. After stimulation of MC with the cytokines TNF-α, IL-1β and IFN-γ alone or in combination to simulate a proinflammatory milieu as would occur during immune-mediated inflammatory disease, an upregulation of TLR3 is seen. Furthermore, a selectively TLR3 mediated, time- and dose-dependent upregulation of MMP-9 and TIMP-1 is found, whereas MMP-2 expression is not significantly affected by TLR3 stimulation.ConclusionsWith these results we provide evidence for a mechanism by which infectious agents can mediate processes of the final common path of inflammation as fibrosis via regulation of MMP and TIMP.