The matrix metalloproteinase-3 (MMP-3) 5A/6A promoter polymorphism is not associated with ischaemic heart disease: Analysis employing a family based approach

Matrix metalloproteinase-3 (MMP-3) has been proposed as an important mediator of the atherosclerotic process. The possible role of the functional -1612(.)5A/6A polymorphism of the MMP-3 gene in the susceptibility to ischaemic heart disease (IHD) was investigated in a well-defined Irish population using two recently described family based tests of association. One thousand and twelve individuals from 386 families with at least one member prematurely affected with IHD were genotyped. Using the combined transmission disequilibrium test (TDT)/sib-TDT and the pedigree disequilibrium test (PDT), no association between the MMP-3 -1612 5A/6A polymorphism and IHD was found. Our data demonstrate that, in an Irish population, the MMP-3 -1612 5A/6A polymorphism is not associated with IHD.

MMP-8 Activity Profiling In GCF Samples

Background: In healthy tissues a family of enzymes known as matrix metalloproteinases (MMPs) play an important role in regulating turnover and metabolism of connective tissue collagen. MMPs have been implicated in a wide variety of pathological conditions including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using enzyme-linked immunosorbent assay (ELISA). Although ELISA quantifies the presence of the MMP-8 protein, it is not possible to determine enzyme activity using this method. Furthermore, since members of the MMP family have poor substrate sequence specificity, a peptide substrate alone cannot differentiate the activity of MMP-8 from other MMPs that may be present in biological samples. Objectives: In the present study, a method to specifically measure MMP-8 activity in gingival crevicular fluid (GCF) samples was developed. Methods: GCF was collected from healthy patients and those with periodontal disease using Perio paper strips. Samples were stored frozen until required for analysis. A specific MMP-8 antibody was used to coat 96 well microtitre plates to selectively remove MMP-8 from the GCF samples. Following a washing step, the activity of bound MMP-8 was measured over 70 minutes using a fluorogenic (FRET) substrate. Results: GCF from healthy subjects exhibited basal MMP-8 activity but in diseased samples MMP-8 activity was significantly higher. Minimal binding of other recombinant MMPs to the specific MMP-8 antibody was observed in cross-reactivity studies. Conclusion: We show for the first time that MMP-8 activity was significantly increased in GCF from periodontitis sites compared with activity levels in healthy sites. Further studies of MMP-8 activity in GCF samples should improve our understanding of its destructive role in periodontal 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.

Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells ( an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.

Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Biomarkers of extracellular matrix metabolism (MMP-9 and TIMP-1) and risk of stroke, myocardial infarction and cause-specific mortality : cohort study

Objective: Turnover of the extracellular matrix in all solid organs is governed mainly by a balance between the degrading matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). An altered extracellular matrix metabolism has been implicated in a variety of diseases. We investigated relations of serum levels of MMP-9 and TIMP-1 to mortality risk from an etiological perspective. Design: The prospective Uppsala Longitudinal Study of Adult Men (ULSAM) cohort, followed from 1991–1995 for up to 18.1 years. A random population-based sample of 1,082 71-year-old men, no loss to follow-up. Endpoints were all-cause (n = 628), cardiovascular (n = 230), non-cardiovascular (n = 398) and cancer mortality (n = 178), and fatal or non-fatal myocardial infarction (n = 138) or stroke (n = 163). Results: Serum MMP-9 and TIMP-1 levels were associated with risk of all-cause mortality (Cox proportional hazard ratio [HR] per standard deviation 1.10, 95% confidence interval [CI] 1.03–1.19; and 1.11, 1.02–1.20; respectively). TIMP-1 levels were mainly related to risks of cardiovascular mortality and stroke (HR per standard deviation 1.22, 95% CI 1.09–1.37; and 1.18, 1.04–1.35; respectively). All relations except those of TIMP-1 to stroke risk were attenuated by adjustment for cardiovascular disease risk factors. Relations in a subsample without cardiovascular disease or cancer were similar to those in the total sample. Conclusion: In this community-based cohort of elderly men, serum MMP-9 and TIMP-1 levels were related to mortality risk. An altered extracellular matrix metabolism may be involved in several detrimental pathways, and circulating MMP-9 or TIMP-1 levels may be relevant markers thereof.

Modeling of enzyme–substrate complexes for the metalloproteases MMP-3, ADAM-9 and ADAM-10

The matrix metalloproteases (MMPs) and the ADAMs (A Disintegrin And Metalloprotease domain) are proteolytic enzyme families containing a catalytic zinc ion, that are implicated in a variety of normal and pathological processes involving tissue remodeling and cancer. Synthetic MMP inhibitors have been designed for applications in pathological situations. However, a greater understanding of substrate binding and the catalytic mechanism is required so that more effective and selective inhibitors may be developed for both experimental and clinical purposes. By modeling a natural substrate spanning P4-P4‘ in complex with the catalytic domains, we aim to compare substrate-specificities between Stromelysin-1 (MMP-3), ADAM-9 and ADAM–10, with the aid of molecular dynamics simulations. Our results show that the substrate retains a favourable antiparallel beta-sheet conformation on the P-side in addition to the well-known orientation of the P'-region of the scissile bond, and that the primary substrate selectivity is dominated by the sidechains in the S1' pocket and the S2/S3 region. ADAM-9 has a hydrophobic residue as the central determinant in the S1' pocket, while ADAM-10 has an amphiphilic residue, which suggests a different primary specificity. The S2/S3 pocket is largely hydrophobic in all three enzymes. Inspired by our molecular dynamics calculations and supported by a large body of literature, we propose a novel, hypothetical, catalytic mechanism where the Zn-ion polarizes the oxygens from the catalytic glutamate to form a nucleophile, leading to a tetrahedral oxyanion anhydride transition state.

Measurement of a MMP-2 degraded Titin fragment in serum reflects changes in muscle turnover induced by atrophy.

In this study we sought to determine whether a Titin peptide fragment can serve as a clinical biomarker for changes in muscle mass.

Expressão imuno-histoquímica das proteínas MMP-9, VEGF e FVW em lesões centrais e periféricas de células gigantes

Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) of the jaws have a distinct clinical behavior, although they share histopathologic features. It is still unclear whether these clinical differences are supported by a distinct pattern of immunoexpression of markers for multinucleated giant cells (GC) and mononuclear cells (MC). The purpose of this study was to compare the immunohistochemical expression of VEGF, MMP-9 in CG and MC and measure the vascularization by vWF to check whether there are differences in expression of these biomarkers between CGCL and PGCL. Paraffin wax blocks of 20 cases of LCCG and 20 LPCG were retrieved. MMP-9 immunoreactivity was greater in the CM of PGCL compared to VEGF (p<0.05). VEGF expression was greater in the CM of CGCL compared to PGCL (p<0.05) and it was greater in the overall expression of CGCL compared to PGCL (p<0.05). Vascularity was quantified by microvascular counting (MVC). MVC was greater in the PGCL compared CGCL (p<0.05). MMP-9 showed a greater tendency of expression in CGCL, though was not significant (p>0.05). We tested correlation between the proteins studied in each group and found a significant negative correlation between VEGF and vWF in CGCL (p<0.05). These results suggest that there are differences in the expression of VEGF in CM and overall expression between the lesions, although no statistically significant difference in the overall expression of the MMP-9. Then, there was a trend in increased expression of MMP-9 and VEGF in CGCL, possibly by the involvement of both proteins in osteoclastogenesis. Additionally, the results of this study indicate a higher degree of vascularization in PGCL compared to CGCL, fact that can be directly linked to the reactive nature of the PGCL, where the inflammatory process with its rich angiogenesis contributes significantly to these findings.