MMP-2 and MMP-9 activities and TIMP-1 and TIMP-2 expression in the prostatic tissue of two ethanol-preferring rat models

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Differential MMP-2 and MMP-9 Activity and Collagen Distribution in Skeletal Muscle from pacu (Piaractus mesopotamicus) During Juvenile and Adult Growth Phases

Here, we evaluated collagen distribution and matrix metalloproteinases (MMPs) MMP-2 and MMP-9 activities in skeletal muscle of pacu (Piaractus mesopotamicus) during juvenile and adult growth phases. Muscle samples from juvenile and adult fishes were processed by histochemistry for collagen system fibers and for gelatin-zymography for MMP-2 and MMP-9 activities analysis. Picrosirius staining revealed a myosept, endomysium, and perimysium-like structures in both growth phases and muscle types, with increased areas of collagen fibers in adults, mainly in red muscle. Reticulin staining showed that reticular fibers in the endomysium-like structure were thinner and discontinuous in the red muscle fibers. The zymography revealed clear bands of the pro-MMP-9, active-MMP-9, intermediate-MMP-2, and active-MMP-2 forms in red and white muscle in both growth phases. MMP-2 activity was more intense in juvenile than adult muscle fibers. Comparing the red and white muscle types, MMP-2 activity was significantly higher in red muscle in adult phase only. The activity of MMP-9 forms was similar in juvenile red and white muscles and in the adult red muscle, without any activity in adult white muscle. In conclusion, our results show that, in pacu, the higher activities of MMP-2 and -9 are associated with the rapid muscle growth in juvenile age and in adult fish, these activities are related with a different red and white muscle physiology. This study may contribute to the understanding muscle growth mechanisms and may also contribute to analyse red and the white muscle parameters of firmness and softness, respectively, of the commercial product. Anat Rec, 292:387-395, 2009. (C) 2009 Wiley-Liss, Inc.

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. on the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium-stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Imbalanced matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 activities in patients with thromboangiitis obliterans

The pathogenic mechanisms of thromboangiitis obliterans (TAO) are not entirely known and the imbalance of matrix metalloproteinases (MMPs) plays a role in vascular diseases. We evaluated the MMP-2 and MMP-9 circulating levels and their endogenous tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in TAO patients with clinical manifestations. The study included 20 TAO patients (n = 10 female, n = 10 male) aged 38-59 years under clinical follow-up. The patients were classified into two groups: (1) TAO former smokers (n = 11) and (2) TAO active smokers (n = 9); the control group included normal volunteer non-smokers (n = 10) and active smokers without peripheral artery disease (n = 10). Patient plasma samples were used to analyze MMP-2 and MMP-9 levels using zymography, and TIMP-1 and TIMP-2 concentrations were determined by enzyme-linked immunosorbent assays. The analysis of MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios (which were used as indices of net MMP-2 and MMP-9 activity, respectively) showed significantly higher MMP-9/TIMP-1 ratios in TAO patients (p < 0.05). We found no significant differences in MMP-2/TIMP-2 ratios (p > 0.05). We found higher MMP-9 levels and decreased levels of TIMP-1 in the TAO groups (active smokers and former smokers), especially in active smokers compared with the other groups (all p < 0.05). MMP-2 and TIMP-2 were not significantly different in patients with TAO as compared to the control group (p > 0.05). In conclusion, our results showed increased MMP-9 and reduced TIMP-1 activity in TAO patients, especially in active smokers compared with non-TAO patients. These data suggest that smoke compounds could activate MMP-9 production or inhibit TIMP-1 activity.

Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities

This study evaluated the influence of fluoride on cell viability and activity of matrix metalloproteinases (MMP) -2 and -9 secreted by preosteoblasts. Preosteoblasts (MC3T3-E1 murine cell line) were cultured in MEM medium supplement with 10% Fetal Bovine Serum (FBS) and nucleosides/ribonucleosides without ascorbic acid. Adherent cells were treated with different concentrations of F (as sodium fluoride-NaF) in medium (5 x 10-6 M, 10-5 M, 10-4 M and 10-3 M) for 24, 48, 72 and 96 h at 37ºC, 5% CO2. Control cells were cultivated in MEM only. After each period, preosteoblast viability was assessed by MTT assay. MMP-2 and -9 activities were performed by gel zymography. Also, alkaline phosphatase (ALP) activity was quantified by colorimetry in all experimental groups. It was shown that cultured cells with the highest dose of F (10-3 M) for 96 h decreased preosteoblast viability while lower doses of F did not alter it, when compared to untreated cells. No differences were observed in ALP activity among groups. Moreover, compared to control, the treatment of cells with F at low dose slightly increased MMP-2 and -9 activities after 24 h. It was concluded that F modulates preosteoblast viability in a dose-dependent manner and also may regulate extracellular matrix remodeling.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

MMP-9 and the epidermal growth factor signal pathway in non-small cell lung cancer

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Expression and prognostic influence of MMP-2, MMP-9 and TIMP-2 in non-small cell lung cancer

Background Matrix metalloproteinases (MMPs) are a family of endopeptidases that digest the extracellular matrix (ECM). Overexpression of different MMPs has been shown to promote tumour cell invasion in vitro. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs that also possess growth-promoting properties. Aims To analyse the expression profile of MMP-2, MMP-9 and TIMP-2 in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Patient follow up was a minimum of 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-2, MMP-9 and TIMP-2. Results The results of the immunohistochemistry are set out below. marker tumour expression log-rank survival stromal expression log-rank survival MMP-2 9/72 (13%) p=0.10 34/72 (47%) p=0.34 MMP-9 79/152 (52%) p=0.04* 69/152 (45%) p=0.84 TIMP-2 28/90 (31%) p=0.04* 66/90 (73%) p=0.90 Two or more 16/59 (27%) p=0.007* There were no associations between expression and clinicopathological findings for any tumour marker. There was co-expression of MMP-2 and MMP-9 in tumour cells (p=0.01). Conclusions MMP-2, MMP-9 and TIMP-2 are expressed in NSCLC. MMP-9 and TIMP-2 tumour expression correlate with a poor outcome (both p=0.04) and are potential prognostic markers for NSCLC. Cumulative expression of two or more MMPs/TIMPs may also have increased prognostic significance. Proteases and their inhibitors are novel targets for therapeutic intervention and should be evaluated in NSCLC.

Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK.

MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits

We have investigated the gelatinase profiles and invasiveness of clonal tumour sublines derived from a spontaneously arising mammary tumour in a Balb/cfC3H mouse. The 67NR, 66c14 and 4T1.2 sublines have low, intermediate and high metastatic potential respectively. In Boyden chamber studies, Matrigel invasion was seen to be progressively higher in the more metastatic lines 4T1.2>66c14>67NR, consistent with MMP-2 activation potential, MMP-9 secretion, and migration over either type I or IV collagen, which were low in both 67NR and 66c14 cells compared to 4T1.2 cells. These attributes are consistent with those seen in human breast cancer cell lines which appear to have undergone an epithelial-mesenchymal transition (EMT) as indicated by vimentin expression. We were, however, surprised to find vimentin expression, MT1-MMP expression and stellate Matrigel outgrowth in the non-invasive, non-metastatic 67NR cells, indicating that they had undergone an EMT despite not being invasive. We conclude that the EMT is manifested to differing degrees in these three clonal cell lines, and that the 67NR cells have either undergone a partial EMT or have since lost certain important attributes of the EMT-derived phenotype. This model should prove useful in further characterizing the regulation of MT1-MMP mediated MMP-2 activation and delineating the EMT in breast cancer progression.

PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling

Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor-kappa B (NF-kappa B) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of uppressor of Hairless (CSL) and NF-kappa B to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

High MMP-9 activity characterizes pleural tuberculosis correlating with granuloma formation

Tuberculosis (TB) pleural disease is complicated by extensive tissue destruction. Matrix metalloproteinase (MMP)-1 and -9 are implicated in immunopathology of pulmonary and central nervous system TB. There are few data on MMP activity in TB pleurisy. The present study investigated MMP-1, -2 and -9 and their specific inhibitors (tissue inhibitor of metalloproteinase (TIMP)-1 and -2) in tuberculous effusions, and correlated these with clinical and histopathological features. Clinical data, routine blood tests, and pleural fluid/biopsy material were obtained from 89 patients presenting with pleural effusions in a TB-endemic area. MMP-1, -2 and -9 were measured by zymography or western blot, and TIMP-1 and -2 by ELISA. Pleural biopsies were examined microscopically, cultured for acid–alcohol fast bacilli and immunostained for MMP-9. Tuberculous pleural effusions contained the highest concentrations of MMP-9 compared with malignant effusions or heart failure transudates. MMP-9 concentrations were highest in effusions from patients with granulomatous biopsies: median (interquartile range) 108 (61–218) pg·mL-1 versus 43 (12–83) pg·mL-1 in those with nongranulomatous pleural biopsies. MMP-1 and -2 were not upregulated in tuberculous pleural fluid. The ratio of MMP-9:TIMP-1 was significantly higher in TB effusions. Tuberculous pleurisy is characterised by a specific pattern of matrix metalloproteinase-9 upregulation, correlating with the presence of granulomas and suggesting a specific role for matrix metalloproteinase-9 in inflammatory responses in tuberculous pleural disease.

Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity

Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. AcetylL-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underling its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METHtriggered MMPs’ activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use.

MMP-9/CD44 : un nouveau complexe ligand/récepteur impliqué dans la régulation de la fonction des cellules musculaires lisses bronchiques humaines

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.