Neuroprotection from Tissue Inhibitor of Metalloproteinase-1 and its nanoparticles

Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28 kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30 min before treatment with KA and 6 h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30 min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition.     

Latent MMP-9 is bound to TIMP-1 before secretion

Expression patterns of matrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), are closely correlated with physiological and pathological processes characterized by the degradation and accumulation of the extracellular matrix (ECM). Both, activated MMP-9 and pro-MMP-9 can bind to TIMP-1, and most cell types secrete MMP-9 in complex with TIMP-1. Utilizing immunofluorescence, we observed intracellular co-localization of MMP-9 and TIMP-1 in stimulated human fibrosarcoma cells. In the present study we searched for the origin of the complex formation between the latent enzyme and its specific inhibitor on a subcellular level. Fluorescence resonance energy transfer (FRET) between the fluorescently labeled enzyme and its inhibitor in co-transfected cells were measured. MMP-9 and TIMP-1 were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein and transiently expressed in human hepatoma cells. The intracellular distribution of fluorescently labeled TIMP-1 and MMP-9 was analyzed by confocal laser scanning microscopy. Intracellular complex formation in the Golgi apparatus was verified, demonstrating FRET between MMP-9-CFP and TIMP-1-YFP. Our data provide evidence that the proMMP-9-TIMP-1 complex is already present in the Golgi apparatus. This may be of significance for a number of intracellular and extracellular biochemical processes involving proMMP-9. However, the magnitude and functional relevance of this finding remain unknown.     

Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.     

On-line chromatographic screening of matrix metalloproteinase inhibitors using immobilized MMP-9 enzyme reactor

Matrix metalloproteinase 9 (MMP-9) plays an important role in cancer invasion and metastasis and has been an attractive target for therapeutic intervention of cancer metastasis. However, considering the high cost and intricacy associated with the expression, isolation and purification of the recombinant enzyme for the screening of drug candidates, alternative methods that explore the recycling of enzymes become desirable. In this study, a new immobilized enzyme reactor (IMER) containing human recombinant MMP-9 enzyme was developed and characterized for the on-line screening of MMP-9 inhibitors. The MMP-9 IMER containing active unit of the enzyme (U = 0.08 μmol/min) on the disk was inserted into a HPLC system connected to a UV–vis detector for on-line chromatographic screening. The resulting conjugated enzyme was shown to be an active and stable catalyst for the hydrolysis of MMP-9 chromogenic thiopeptide substrate Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC₂H₅. The kinetics profile of the enzyme in IMER and free solution were determined and compared. Selected reversible MMP inhibitors, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH), doxycycline and minocycline were further characterized using the MMP-9 IMER and free enzyme solution assays. Our system demonstrated applicability as a rapid and cost-effective screening tool for inhibitors of the MMP-9 enzyme.     

ZAK induces MMP-2 activity via JNK/p38 signals and reduces MMP-9 activity by increasing TIMP-1/2 expression in H9c2 cardiomyoblast cells

Leucine-zipper and sterile-alpha motif kinase (ZAK) is the key intra-cellular mediator protein in cardiomyocyte hypertrophy induction by transforming growth factor beta 1 (TGF-β1) which has also been identified as a profibrotic cytokine involved in cardiac fibrosis progression. We hypothesized whether ZAK over-expression causes cardiac scar formation due to the extra-cellular matrix (ECM) degraded enzyme regulation in this paper. Using immuno-histochemical analysis of the human cardiovascular tissue array, we found a positively significant association between ZAK over-expression and myocardial scars. ZAK over-expression in H9c2 cardiomyoblast cells increases the metalloproteinase tissue inhibitor 1/2 (TIMP-1/2) protein level, which reduces matria metalloproteinase-9 (MMP-9) activity and also activates c-JNK N-terminal kinase 1/2 (JNK1/2) and p38 signaling, which induces MMP-2, possibly resulting in cardiac fibrosis. Taken together, ZAK activity inhibition may be a good strategy to prevent the cardiac fibrosis progression.     

Matrix Metalloproteinase-3 (MMP-3) Is an Endogenous Activator of the MMP-9 Secreted by Placental Leukocytes: Implication in Human Labor

Background

The activity of matrix degrading enzymes plays a leading role in the rupture of the fetal membranes under normal and pathological human labor, and matrix metalloproteinase-9 (MMP-9) it is considered a biomarker of this event. To gain further insight into local MMP-9 origin and activation, in this study we analyzed the contribution of human placental leukocytes to MMP-9 secretion and explored the local mechanisms of the pro-enzyme activation.

Methods

Placental blood leukocytes were obtained from women at term gestation without labor and maintained in culture up to 72 h. MMP-9 activity in the culture supernatants was determined by zymography and using a specific substrate. The presence of a potential pro-MMP-9 activator in the culture supernatants was monitored using a recombinant biotin-labeled human pro-MMP-9. To characterize the endogenous pro-MMP-9 activator, MMP-1, -3, -7 and -9 were measured by multiplex assay in the supernatants, and an inhibition assay of MMP-9 activation was performed using an anti-human MMP-3 and a specific MMP-3 inhibitor. Finally, production of MMP-9 and MMP-3 in placental leukocytes obtained from term pregnancies with and without labor was assessed by immunofluorescence.

Results

Placental leukocytes spontaneously secreted pro-MMP-9 after 24 h of culture, increasing significantly at 48 h (P≤0.05), when the active form of MMP-9 was detected. Culture supernatants activated the recombinant pro-MMP-9 showing that placental leukocytes secrete the activator. A significant increase in MMP-3 secretion by placental leukocytes was observed since 48 h in culture (P≤0.05) and up to 72 h (P≤0.001), when concentration reached its maximum value. Specific activity of MMP-9 decreased significantly (P≤0.005) when an anti-MMP-3 antibody or a specific MMP-3 inhibitor were added to the culture media. Placental leukocytes from term labor produced more MMP-9 and MMP-3 compared to term non-labor cells.

Conclusions

In this work we confirm that placental leukocytes from human term pregnancies are able to secrete large amounts of MMP-9, and that the production of the enzyme it is enhanced by labor. We also demonstrate for the first time that endogenous MMP-3 plays a major role in MMP-9 activation process. These findings support the contribution of placental leukocytes to create the collagenolytic microenvironment that induces the rupture of the fetal membranes during human labor.     

Genetic polymorphism c.1562C>T of the MMP-9 is associated with macroangiopathy in type 2 diabetes mellitus

Objective

To determine whether the matrix metalloproteinase-9 (MMP-9) c.1562C>T polymorphism has an effect on the plasma MMP-9 levels and the macroangiopathic complications in type 2 diabetes mellitus (T2DM).

Methods

The genotypes and allelic frequencies of the MMP-9 c.1562C>T were examined with polymerase chain reaction and restriction fragment length polymorphism in 320 patients with T2DM and 160 unrelated healthy subjects. The plasma concentrations of MMP-9 were determined in all subjects.

Results

The mean plasma concentrations of MMP-9 of patients with T2DM were significantly higher than that of controls and the plasma levels of MMP-9 were higher in diabetic patients with macroangiopathy than in patients without macroangiopathy (P < 0.05). The genotype (CC, CT, and TT) distribution of c.1562C>T polymorphism of the MMP-9 gene was 60.0%, 31.3%, and 8.8% in diabetic patients with macroangiopathy, 76.3%, 21.3%, and 2.5% in patients without macroangiopathy, and 77.5%, 21.3%, 1.3% in controls, respectively, a significant difference was found between diabetic patients with and without macroangiopathy (P < 0.05). The frequency of the allele T was higher in patients with macroangiopathy than in patients without macroangiopathy (24.4% vs 13.1%; P < 0.05). Moreover, the plasma MMP-9 levels were markedly higher in patients with TT genotype than those with CC or CT genotype in patients with macroangiopathy (P < 0.05).

Conclusion

The MMP-9 c.1562C>T gene polymorphism associated with a predisposition to increased plasma MMP-9 levels could constitute a useful predictive marker for diabetic macroangiopathy.     

Matrix metalloproteinase-9 activity detected in body fluids is the result of two different enzyme forms

In vitro activation of matrix metalloproteinase-9 (MMP-9) (Gelatinase B) with MMP-3 shows the presence of two different forms: an 82 kDa, N-terminal truncated form, and a 65 kDa, N- and C-terminal truncated form. So far the presence of the 65 kDa form has not been reported in vivo. Affinity chromatography was performed to separate MMP-9 from MMP-2 and immunoprecipitation to isolate ～65 kDa MMP-9 from 82 kDa MMP-9 in sera of healthy donors. The presence of ～65 kDa active MMP-9 was demonstrated both with gelatin zymography and western blot analysis. The ～65 kDa MMP-9 lacks the haemopexin domain required for the high-affinity binding of the tissue inhibitor TIMP-1, and can be evaluated by activity assay in the presence of TIMP-1. This opens the possibility to investigate the role of this form of MMP-9 that escapes physiological regulation.     

Native TIMP-free 70 kDa progelatinase (MMP-2) secreted at elevated levels by RSV transformed fibroblasts

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed. © 1994 Wiley-Liss, Inc.     

Plasma storage at -80 degrees C does not protect matrix metalloproteinase-9 from degradation

Recently, matrix metalloproteinase-9 (MMP-9) has been identified as a cardiovascular risk marker and is increasingly being determined in clinical studies. Among other matrix metalloproteinases, MMP-9 is known to be self-activable, as the cleavage of the propeptide leads to the formation of an active enzyme. In such a case the issue of storage of biological samples such as plasmas is of outstanding importance, as an enzymatic activity, although minimal, may remain at common storage temperature, i.e., -80 degrees C. Since 2000 our institute has created a plasma library from patients presenting with acute myocardial infarction. Recently, the evaluation of the MMP-9 led to the surprise of finding a dramatically low level of detectable enzyme in the oldest plasma samples. By using zymography, enzyme-linked immunosorbent assay and Western blots, we evaluated new and old samples and found that MMP-9 degrades over time. After 2 years, the detectable total MMP-9 dropped by 65%, and the asymptotic profile of the curve reached a residual 1% level after 43 months. These results were confirmed by zymography and western blotting. TIMP-1, the natural inhibitor of MMP-9 and MMP-2, remained rather stable over time. The results suggest that human plasma MMP-9 levels should be determined as soon as possible after sampling.     

Elevated serum MMP-9/TIMP-1 ratio in patients with homozygous familial hypercholesterolemia: Effects of LDL-apheresis

ObjectiveMatrix degradation within an atherosclerotic plaque is an important pathogenic factor in atherosclerosis, and is largely modulated by the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors (i.e., tissue inhibitor of MMPs [TIMPs]). Familial hypercholesterolemia (FH) is a rare inherited disorder associated with premature coronary heart disease. The aim of the present study was to examine MMP-9 and TIMP-1 on plasma and cellular mRNA levels in homozygous FH patients (n = 7) compared with age- and sex-matched heterozygous FH patients (n = 6), and with healthy subjects (n = 7), and to test whether once-weekly LDL-apheresis (three consecutive sessions) of homozygous FH patients show short-term effects on these variables.ResultsThe main findings were that (i) Compared to healthy control subjects, homozygous FH patients have significantly higher serum levels of MMP-9 and lower levels of TIMP-1, and consequently significantly higher MMP-9/TIMP-1 ratio, potentially reflecting higher MMP-9 activity. (ii) Peripheral blood mononuclear cells (PBMC) isolated from FH homozygotes have significantly higher mRNA levels of MMP-9 compared to cells from heterozygotes. (iii) TNFα-stimulated PBMC from FH homozygotes released borderline-significantly more MMP-9 than cells from heterozygotes and healthy controls. (iv) LDL-apheresis (one day before treatment versus fifteen days later, on the day after the weekly treatment) had no significant short-term effect on any of the MMP-9 and TIMP-1 variables measured in serum and cells.ConclusionsThe data may suggest that homozygous FH patients have an enhanced matrix degrading potential as compared with heterozygous FH patients and healthy controls, potentially contributing to the increased cardiovascular risk observed in these patients.     

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.     

BMP-2 treatment of C3H10T1/2 mesenchymal cells blocks MMP-9 activity during chondrocyte commitment

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. We explored the underlying mechanism of BMP-2-induced chondrocyte commitment of C3H10T1/2 cells. Treating cells with exogenous BMP-2 was tied to chondrocyte commitment by inhibiting matrix metalloproteinase-9 activity (MMP-9: 92 kDa type IV collagenase/gelatinase B). Glycogen synthase kinase (GSK)-3β inhibition by its specific inhibitor blocked BMP-2-induced chondrocyte commitment by stimulating MMP-9 activity. These findings indicate that the downregulation of MMP-9 by BMP-2 is associated with chondrocyte commitment, and that the GSK-3β signaling pathway is involved in this process.     

The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.     

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2)

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 μg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 μg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 μg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.     

Increased levels of leukocyte-derived MMP-9 in patients with stable angina pectoris

Objective

There is a growing interest for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in plasma as novel biomarkers in coronary artery disease (CAD). We aimed to identify the sources of MMP-8, MMP-9, TIMP-1 and TIMP-2 among peripheral blood cells and further explore whether gene expression or protein release was altered in patients with stable angina pectoris (SA).

Methods

In total, plasma MMP-9 was measured in 44 SA patients and 47 healthy controls. From 10 patients and 10 controls, peripheral blood mononuclear cells (PBMC) and neutrophils were isolated and stimulated ex vivo. MMPs, TIMPs and myeloperoxidase were measured in plasma and supernatants by ELISA. The corresponding gene expression was measured by real-time PCR.

Results

Neutrophils were the dominant source of MMP-8 and MMP-9. Upon moderate stimulation with IL-8, the neutrophil release of MMP-9 was higher in the SA patients compared with controls (p<0.05). In PBMC, the TIMP-1 and MMP-9 mRNA expression was higher in SA patients compared with controls, p<0.01 and 0.05, respectively. There were no differences in plasma levels between patients and controls except for TIMP-2, which was lower in patients, p<0.01.

Conclusion

Measurements of MMPs and TIMPs in plasma may be of limited use. Despite similar plasma levels in SA patients and controls, the leukocyte-derived MMP-9 and TIMP-1 are significantly altered in patients. The findings indicate that the leukocytes are more prone to release and produce MMP-9 in symptomatic and angiographically verified CAD—a phenomenon that may have clinical implications in the course of disease.     

Matrix metalloproteinase (MMP)-2 gene polymorphisms affect circulating MMP-2 levels in patients with migraine with aura

Matrix metalloproteinases (MMP) are involved in the disruption of blood–brain barrier (BBB) during migraine attacks. In the present study, we hypothesized that two functional polymorphisms (C^− 1306T and C^− 735T) in MMP-2 gene and MMP-2 haplotypes are associated with migraine and modify MMP-2 and tissue inhibitor of MMP (TIMP)-2 levels in migraine. Genotypes for MMP-2 polymorphisms were determined by real time-PCR using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by gelatin zymography and ELISA, respectively, in 148 healthy women without history of migraine and in 204 women with migraine (153 without aura; MWA, and 51 with aura; MA). Patients with MA had higher plasma MMP-2 concentrations and MMP-2/TIMP-2 ratios than patients with MWA and controls (P < 0.05). While MMP-2 genotype and haplotype distributions for the polymorphisms were similar among the groups (P > 0.05), we found that the CC genotype for C^− 735T polymorphism and the CC haplotype were associated with higher plasma MMP-2 concentrations in MA group (P < 0.05). Our findings may help to understand the role of MMP-2 and its genetic variants in the pathophysiology of migraine and to identify a particular group of migraine patients with increased MMP-2 levels that would benefit from the use of MMP inhibitors.     

Interferon-gamma and lipopolysaccharide stimulation increases matrix metalloproteinase-9 expression and enhances invasion activity in ras/myc-transformed serum-free mouse embryo cells

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma, 100 units/ml) and/or lipopolysaccharide (LPS, 0.5 microg/ml) for 24 h to investigate the effects of these ligands on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Aminoguanidine (AG, 1mM; a nitric oxide synthase [NOS] inhibitor) was also added along with IFN-gamma and LPS to analyze a possible association of NO with invasiveness. Treatment of cells with IFN-gamma alone did not alter MMP-9 mRNA expression or pro-MMP-9 production, but LPS alone and IFN-gamma+LPS co-treatment enhanced them significantly. TIMP-1 mRNA expression remained unchanged with or without treatment and the mRNA expression of MMP-9 exceeded that of TIMP-1 in LPS- or IFN-gamma+LPS-treated cells. Co-treatment of cells with IFN-gamma and LPS up-regulated invasiveness and indicated a possible involvement of NO in the enhancement of invasiveness. These results suggest that ras/myc SFME cells respond to inflammatory and infectious conditions and that they may possibly modulate their characteristics as cancer cells due to their increase in MMP-9 expression and invasion activity.