Imbalance of MMP-2 and MMP-9 expression versus TIMP-1 and TIMP-2 reflects increased invasiveness of human testicular germ cell tumours.

The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.

The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins.

Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.

Sclerostin Deficiency Promotes Reparative Dentinogenesis.

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.

The role of matrix metalloproteinases (MMPs) in human caries.

The objective of this review is to summarize our understanding of the role of host matrix metalloproteinases (MMPs) in the caries process and to discuss new therapeutic avenues. MMPs hydrolyze components of the extracellular matrix and play a central role in many biological and pathological processes. MMPs have been suggested to play an important role in the destruction of dentin organic matrix following demineralization by bacterial acids and, therefore, in the control or progression of carious decay. Host-derived MMPs can originate both from saliva and from dentin. They may be activated by an acidic pH brought about by lactate release from cariogenic bacteria. Once activated, they are able to digest demineralized dentin matrix after pH neutralization by salivary buffers. Furthermore, the degradation of SIBLINGs (Small Integrin-binding Ligand N-linked Glycoproteins) by the caries process may potentially enhance the release of MMPs and their activation. This review also explores the different available MMP inhibitors, natural or synthetic, and suggests that MMP inhibition by several inhibitors, particularly by natural substances, could provide a potential therapeutic pathway to limit caries progression in dentin.

Enthalphy changes associated with the denaturation of collagens of different imino acid content.

The enthalpy changes associated with the denaturation of acid-soluble and insoluble collagens prepared from sheep, cod, halibut and pike skin were determined by differential scanning calorimetry. The enthalpy change associated with the soluble collagens decreased with decreasing imino acid content (from 1420 cal/mol for sheep to 736 cal/mol for cod) while the value for insoluble collagens was approximately constant at 1360 cal/mol. A possible explanation for these values in terms of the nautre of the bonds present in collagen is discussed.

Deletion of extracellular matrix metalloproteinase inducer/CD147 induces altered cardiac extracellular matrix remodeling in aging mice.

Extracellular matrix metalloproteinase inducer (EMMPRIN), known for its ability to induce matrix metalloproteinase (MMP) expression, was proposed to play a role in the adverse cardiac extracellular matrix remodeling. After observing an age-associated increase in cardiac EMMPRIN expression in both mice and rats, the role and mechanism of action of EMMPRIN was investigated in the myocardial age-associated changes using 3, 12 and 24 month old EMMPRIN knock-out (KO) vs. wild-type (WT) mice, by cardiac echocardiography, Western blots, immunohistochemistry, ELISA and histology. Adilated cardiomyopathy characterized by a decreased ejection fraction and an enlargement of left ventricular chamber (LV) associated with LV hypertrophy, occurred in KO mice as soon as 12 month old. The increase in interstitial collagen deposition during aging in WT mice could not be detected in KO mice. This may be related to the reduced activation (48% reduction; P < 0.05) and signaling (smad2/3 nuclear translocation) of TGF-ß in the 12 month old KO mice which paralleled with a greater reduction in the TGF-ß known activating enzymes such as MT1-MMP and MMP-1 (33% and 37% reduction respectively, between 3 and 12 month old in KO mice; P < 0.05) as well as uPA. These findings demonstrate that EMMPRIN gene silencing is associated with an aberrant extracellular matrix remodeling, characterized by the absence of a detected age-associated fibrosis and consequently to dilated cardiopathy, indicating that a fine regulation of EMMPRIN is essential for the coordinated ECM remodeling during aging.

AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice.

TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.

Upregulation of MMP-9 expression in MDA-MB231 tumor cells by platelet granular membrane.

The interaction between tumor cells and platelets facilitates the formation of metastasis in a way depending on the platelet aggregating ability of the tumor cell, but the mechanism remains to be elucidated. We have shown, by zymography and Western blot, that platelets greatly increased the secretion to the culture medium of MMP-9 by human mammary tumor cells MDA-MB231. This increase, which was dependent on protein synthesis, was caused by the platelet aggregates interacting with the tumor cells and not by the soluble factors released during platelet activation. Platelet subcellular fractionation allowed the localization of the inducing factor to the membrane fraction of the platelet granules, thus requiring platelet aggregation in order to become accessible on the platelet surface.

The extracellular matrix produced by bovine corneal endothelial cells contains progelatinase A.

Progelatinase A is a matrix metalloproteinase involved in the turnover of extracellular matrix (ECM). We report that the ECM produced by bovine corneal endothelial (BCE) cells contains progelatinase A free of tissue inhibitor of metalloproteinase (TIMP2). The matrix-bound progelatinase A can be activated by APMA to generate a 62 kDa and a 45 kDa species with enzymatic activity and is inhibited by TIMP2. The bound progelatinase can be released after treatment of the ECM with gelatinase B. These studies suggest that the ECM can function as a reservoir for progelatinase A which may be readily available for cells in processes such as metastasis, angiogenesis, inflammation and wound healing.

Modulation of endothelial cells fibrinolytic activity by platelets.

Interaction between endothelial cells (EC) and platelets in culture was shown to regulate the fibrinolytic system of the aortic EC. Untreated porcine EC from aorta exhibited almost no net fibrinolytic activity and zymographic assay have shown a single fibrin lysis band of 105 kDa corresponding to a tPA-PAI complex. Incubation of aortic EC with intact platelets stimulated a cell-associated fibrinolytic activity of the urokinase type as evidenced by a plasminogen-dependent fibrin independent amidolytic activity, and the appearance of a new 48 kDa lysis band on zymography. However, in the culture medium of platelet-treated aortic EC, a new lysis band of 92 kDa appeared with no associated amidolytic activity suggesting that the 48 kDa plasminogen activator secreted by the aortic EC after treatment with platelets is complexed to the inhibitor PAI1. This modulation of fibrinolytic activity depends on the EC origin since it is not observed with pulmonary artery EC, and represents a new concept in fibrinolysis regulation by cell-cell interaction.

High levels of circulating thrombomodulin in human foetuses and children.

Thrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.

Elastase-like activity in cultured aortic endothelial cells.

Cultured porcine aortic endothelial cells were studied for cellular and secreted elastase activity. We describe an activity hydrolyzing the synthetic elastase substrate, succinyl(alanine)3 nitroanilide, but not elastin, which was shown to be membrane located and was not secreted to the culture medium. A different neutral proteinase activity degrading insoluble elastin was demonstrated in the culture medium following its fractionation by gel filtration high performance liquid chromatography (HPLC). Since no elastinolytic activity could be directly detected in the conditioned medium, it is likely that the chromatographic separation removed an endogenous inhibitor.

Fibrinolysis potentiating activity following endothelial cells-platelet interaction.

When porcine endothelial cells in culture are incubated in the presence of human platelets, a 90kDa neutral proteinase activity is generated on casein gel (PECAP-Platelet Endothelial Cell Activated Protease). This activity was undetected when platelet extract or serum free EC conditioned medium were analysed under similar conditions. The optimum pH, isoelectric point, molecular weight and inhibitory profile of this activity were similar to Glu-plasmin. However, the low plasminogen content (less than 50ng/ml) in the conditioned medium of endothelial cells incubated with platelet could not contribute alone to this activity and the presence of a plasmin potentiating factor was suggested. This factor was separated from plasminogen by lysine-Sepharose chromatography.

Comparative study of fibrinolytic activity on 937 line after stimulation by interferon gamma, 1,25 dihydroxyvitamin D3 and their combination.

Previous study showed that the secretion of urokinase (UK) by monoblastic cell line U 937 and the number of binding sites for urokinase and for plasminogen (Plg) on the cell surfaces were augmented by interferon gamma (INF tau). This induction led to an increase in fibrinolytic activity on cell surfaces. A similar increase was also observed when treating the U 937 cells with 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3. Here we report that the combination of these two agents induced a 2.7 fold increase in the plasminogen activator activity on U 937 cell surfaces in comparison with 1 fold increase induced by INF tau and 1.3 fold increase by 1,25(OH)2D3. As evaluated by a flow cytometer, the increased fibrinolytic activity induced by the combination of INF tau and 1,25(OH)2D3 could be attributed to the increase of the number of binding sites both for UK (3.7 x 10(4) vs 1.2 x 10(4) per cell) and for Plg (16.2 x 10(4) vs 3.6 x 10(4) per cell), accompanied by an increased expression of CD 14, which is an antigen of differentiation on cell surfaces. These results suggest that the expression of urokinase receptors and plasminogen receptors may be coupled together by unknown intracellular mechanisms during cell differentiation, and support the idea that the concomitant regulation of these two receptors for UK and Plg is an important aspect in cell associated-fibrinolytic activity.

Antisense inhibition of amphiregulin expression reduces EGFR phosphorylation in transformed human breast epithelial cells.

The activation of epidermal growth factor receptor (EGFR) by its ligands constitutes an important step in the metastatic process but the clinical response to its inhibition in breast cancer patients has so far been very low. In this work, we investigated the role of the EGFR ligand amphiregulin (AR) in modulating EGFR activation. For this, transformed epithelial mammary tumor cells NS2T2A1 were used in which AR or EGFR expression was down-regulated by antisense cDNA technique. This down-regulation was associated with a significant inhibition of matrix metalloproteinase-9 production as well as cell proliferation, but this inhibition was only minimally reversed by exogenously added AR or EGF. EGFR protein levels were not affected but EGFR-tyrosine phosphorylation in response to EGF was markedly reduced. Thus, the inhibition of AR expression, which impairs EGFR response to its exogenously available ligands, may represent an alternative anti-EGFR therapeutic strategy in breast cancer.