Extracellular Endopeptidase Research Articles

Endopeptidase O promotes Streptococcus suis immune evasion by cleaving the host- defence peptide cathelicidins

ABSTRACT Streptococcus suis is a zoonotic Gram-positive bacterium that causes invasive infections such as sepsis and meningitis, threatening public health worldwide. For successful establishment of infection, the bacterium should subvert the innate effectors of immune defence, including the cathelicidin family of host-defence peptides that combat pathogenic bacteria by directly disrupting cell membranes and coordinating immune responses. Here, our study shows that an extracellular endopeptidase O (PepO) of S. suis contributes to assisting the bacterium to resist cathelicidin-mediated killing, as the deletion of the pepO gene makes S. suis more sensitive to the human cathelicidin LL-37, as well as its mouse equivalent, mCRAMP. This protease targets and cleaves both LL-37 and mCRAMP, degrading them into shorter peptides with only a few amino acids, thereby abrogating their ability to kill S. suis. By cleaving LL-37 and mCRAMP, PepO impairs their chemotactic properties for neutrophil migration and undermines their anti-apoptosis activity, which is required for prolonging neutrophil lifespan. Also, PepO inhibits the ability of LL-37 and mCRAMP to promote lysosome development in macrophages. Moreover, the loss of PepO attenuates organ injury and decreases bacterial burdens in a murine model of S. suis bacteraemia. Taken together, these data provide novel insights into the role of the intrinsic proteolytic characteristics of PepO in S. suis-host interaction. Our findings demonstrate that S. suis utilizes the PepO protease to cleave cathelicidins, which is an immunosuppressive strategy adopted by this bacterium to facilitate pathogenesis.

Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in EGF‐driven non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer cell protrusive activity.

Effect of subtilisin, a protease from Bacillus sp., on soil biochemical parameters and microbial biodiversity

Proteinaceous material constitute the main source of nitrogen in soil organic matter. Use of this nitrogen is limited by the breakdown of proteins into peptides and free amino acids. Proteases, therefore, are important in the use of protein N. The aim of this study is to assess the effect of subtilisin, which is a soil extracellular endopeptidase from Bacillus sp., both on soil microbial biostimulation and biodiversity. In addition to the protease, keratins, an insoluble-protein and low-bioavailable substrate, was added to enhance the effect of protease in soil. Protease, both on its own and in combination with keratins, has a biostimulant effect on soil microbial activity that was reflected in the protease, dehydrogenase and phosphatase activities.DNA metabarcoding analysis further revealed specific changes in soil biodiversity, mainly defined by the enrichment of plant-growth promoting microorganisms, specifically those producing hydrolase enzymes such as phosphatase and ammonifiers. Surprisingly, the addition of keratins specifically showed a rapid stimulation of soil proteolytic bacteria.Thus, the present work supports the use of subtilisin as biofertiliser and values the recycling of keratinous industrial waste in the field of biofertilisation.

Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates in response to Epidermal growth factor stimulation. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer protrusive activity.Support or Funding InformationWartburg Undergraduate Research Grants

Epigenetic landscapes suggest that genetic risk for intracranial aneurysm operates on the endothelium

BackgroundGenetics play an important role in intracranial aneurysm (IA) pathophysiology. Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) that are linked to IA but how they affect disease pathobiology remains poorly understood. We used Encyclopedia of DNA Elements (ENCODE) data to investigate the epigenetic landscapes surrounding genetic risk loci to determine if IA-associated SNPs affect functional elements that regulate gene expression and if those SNPs are most likely to impact a specific type of cells.MethodsWe mapped 16 highly significant IA-associated SNPs to linkage disequilibrium (LD) blocks within the human genome. Within these regions, we examined the presence of H3K4me1 and H3K27ac histone marks and CCCTC-binding factor (CTCF) and transcription-factor binding sites using chromatin immunoprecipitation-sequencing (ChIP-Seq) data. This analysis was conducted in several cell types relevant to endothelial (human umbilical vein endothelial cells [HUVECs]) and inflammatory (monocytes, neutrophils, and peripheral blood mononuclear cells [PBMCs]) biology. Gene ontology analysis was performed on genes within extended IA-risk regions to understand which biological processes could be affected by IA-risk SNPs. We also evaluated recently published data that showed differential methylation and differential ribonucleic acid (RNA) expression in IA to investigate the correlation between differentially regulated elements and the IA-risk LD blocks.ResultsThe IA-associated LD blocks were statistically significantly enriched for H3K4me1 and/or H3K27ac marks (markers of enhancer function) in endothelial cells but not in immune cells. The IA-associated LD blocks also contained more binding sites for CTCF in endothelial cells than monocytes, although not statistically significant. Differentially methylated regions of DNA identified in IA tissue were also present in several IA-risk LD blocks, suggesting SNPs could affect this epigenetic machinery. Gene ontology analysis supports that genes affected by IA-risk SNPs are associated with extracellular matrix reorganization and endopeptidase activity.ConclusionThese findings suggest that known genetic alterations linked to IA risk act on endothelial cell function. These alterations do not correlate with IA-associated gene expression signatures of circulating blood cells, which suggests that such signatures are a secondary response reflecting the presence of IA rather than indicating risk for IA.

The levels of matrix metalloproteinase-9 and neutrophil gelatinase-associated lipocalin in different stages of endometriosis

This study was designed to explore matrix metalloproteinase-9 (MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) levels and MMP-9/NGAL ratio in women with and without endometriosis diagnosed surgically and/or histopathologically. The correlation between biomarkers and the severity of the disease is analysed. The revised American Fertility Society classification system was used to determine the severity of endometriosis. Serum MMP-9 and Ca125, urine NGAL levels were measured in all participants. Serum MMP-9 levels were significantly higher in the study group (n = 60) compared to controls (n = 31) (15.0 pg/mL (6.0–143.0) vs. 12.0 (4.0–18.0), respectively; p=.002). MMP-9 levels were significantly higher in severe endometriosis compared to mild endometriosis subgroups (p<.001). No significant difference was found between NGAL levels in study and control groups (p>.05). The diagnostic value of MMP-9 and NGAL is not superior than CA-125 for endometriosis. Nevertheless, MMP-9 might be a potential predictive marker for advanced stage of the disease.Impact StatementWhat is already known on this subject? The gold standard diagnostic test for diagnosis of endometriosis is laparoscopy combined with histopathological confirmation of eutopic endometrial glands and/or stroma. Both invasiveness and possible accompanying complications limit the preference regarding the surgical approach. Among non-invasive markers none has been accepted as gold standard neither for diagnosis nor for determining the severity of the disease. MMPs are extracellular endopeptidases, which have a significant role in degradation and remodelling of extracellular matrix for cellular migration and invasion. Among these, MMP-9 has been shown to be higher in eutopic/ectopic endometrial tissue in women with endometriosis and has been suggested to have a role in pathogenesis of endometriosis by promoting invasion of the endometriotic lesions. NGAL is an acute phase protein, which is involved in a variety of physiological and pathophysiological processes. The molecule has also been revealed to correlate with endometriosis pathophysiology through the epithelial–mesenchymal transition process which is the basis for the onset of endometriosis. But also, NGAL which composes a complex with MMP-9 (MMP-9 and NGAL complex), has been shown to protect MMP-9 from autodegradation in vitro which might be a contributing factor for endometriosis pathophysiology.What the results of this study add? MMP-9 cut-off level for prediction of severe endometriosis is a novel finding obtained from this study with acceptable sensitivity and specificity. On the other hand, NGAL seems to have no significant value either for diagnosis of for determining severity of the disease. After all, MMP-9 might be an easy use acceptable biomarker for endometriosis but further studies on larger populations are needed.What the implications are of these findings for clinical practice and/or further research? MMP might be a potential non-invasive predictive marker for advanced stage disease.

Different inflammatory cytokines release after open and endovascular reconstructions influences wound healing.

Prodromal signs of a non-healing wound after revascularisation, which might be strictly linked with impending failure of vascular reconstructions, are associated with an inflammatory response mediated by several circulating adhesion molecules, extracellular endopeptidases, and cytokines. The aim of our study was to investigate the role of selected plasma biomarkers in the prediction of both wound healing and failure of infrapopliteal vein graft or percutaneous trans-luminal angioplasty (PTA) with selective stent positioning of the superficial femoral artery (SFA) in a population affected with critical limb ischaemia. A total of 68 patients who underwent either surgical or endovascular revascularisation of the inferior limb with autologous saphenous vein infrapopliteal bypass or PTA and selective stenting of the SFA were enrolled in our study. Patients were divided into two groups according to treatment: 41 patients were included in Group 1 (open surgery) and 27 in Group 2 (endovascular procedure). Plasma and blood samples were collected on the morning of surgery and every 6 months thereafter for up to 2 years of follow-up or until an occlusion occurred of either the vein bypass graft or the vessel treated endovascularly. Fifteen age-matched healthy male volunteers were considered a reference for biological parameters. Vascular cell adhesion molecule 1 [VCAM-1]/CD106, inter-cellular adhesion molecule-1 [ICAM-1]/CD54), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), and metalloproteinases (MMP)-2 and -9 plasma levels were measured with enzyme-linked immunosorbent assay (ELISA) kits. The mean observed time to heal of 54 wounds was 13 ± 4 months, with no statistically significant differences among the groups. The healing failure of the remaining wounds was strictly related to an unsuccessful open (n = 12) or endovascular (n = 8) treatment. The 2-year primary patency rate was 65% (SE = .09) in Group 1 and 52% (SE = .1) in Group 2. When compared with mean concentration values of Group 1, VCAM-1 and ICAM-1 were always significantly higher during follow-up in patients of Group 2 (P < .05). Furthermore, in the same group, IL-6 and tumour necrosis factor alpha (TNF-α) were found to be significantly higher at 6- and 12-month (P < .05) when compared with surgically treated patients. Cox regression analysis showed that elevated plasma levels of VCAM-1, ICAM-1, IL-6, and TNF-α during follow up were strongly related to impaired wound healing and/or revascularisation failure (P < .05). Elevated plasma levels of inflammatory markers VCAM-1, ICAM-1, IL-6, and TNF-α may be related to the failure of wound healing and revascularisation procedures. Interestingly, we have observed that endovascular treatments cause a higher level of these inflammation biomarkers when compared with a vein graft, although wound-healing and patency and limb salvage rates are not influenced.

Two Heat Resistant Endopeptidases from Pseudomonas Species with Destabilizing Potential during Milk Storage

In the current study, the extracellular endopeptidases from Pseudomonas lundensis and Pseudomonas proteolytica were investigated. The amino acid sequence identity between both endopeptidases is 68%. Both endopeptidases were purified to homogeneity and partially characterized. They were classified as metallopeptidases with a maximum activity at pH 10.0 ( P. lundensis) or 8.5 ( P. proteolytica) at 35 °C. Both remained active in skim milk with 39.7 ± 2.4% and 24.5 ± 3.3%, respectively, of the initial enzyme activity after UHT processing (138 °C for 20 s), indicating the relevance for milk destabilization. The transition points in buffer were determined at 50 °C ( P. lundensis) and 43 °C ( P. proteolytica) using circular dichroism spectroscopy. The loss of the secondary structure at different temperatures was correlated with residual peptidase activities after heat treatment. The ability to destabilize UHT milk was proven by supplementation of skim milk with endopeptidase and storage for 4 weeks.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion.

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24–32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.

Stromelysin-2 (MMP-10) facilitates clearance and moderates inflammation and cell death following lung exposure to long multiwalled carbon nanotubes.

Multiwalled carbon nanotubes (MWCNTs) are nanomaterials composed of multiple layers of graphene cylinders with unique properties that make them valuable for a number of industries. However, rising global production has led to concerns regarding potential occupational exposures to them as raw materials during handling. This is especially true for long MWCNT fibers, whose aspect ratio has been posited to initiate pathology similar to that of asbestos. Matrix metalloproteinases (MMPs) are a class of extracellular endopeptidases that control various processes related to tissue repair, inflammation, and more. Stromelysin-2 (MMP-10) has roles in modulating macrophage activation and function, and hence, we used an MMP-10 null (Mmp10−/−) mouse model to assess its role in controlling lung responses to inhaled long MWCNTs. Oropharyngeal aspiration of long MWCNTs (80 µg/mouse) by wild-type mice induced expression of Mmp10 mRNA, which was accompanied by a robust inflammatory response characterized by elevated expression of Tnfa, Il6, and Il1b. In Mmp10−/− mice, we found that absence of MMP-10 led to impaired pulmonary clearance of MWCNTs and reduced macrophage cell survival. Exposure of wild-type bone marrow-derived macrophages (BMDMs) and alveolar macrophages to MWCNTs caused a rapid, dose-dependent upregulation of Mmp10 mRNA expression, which was accompanied by expression of pro-inflammatory products (Il6 and Il1b). These products were further enhanced in Mmp10−/− macrophages, resulting in increased caspase-3-dependent cell death compared with wild-type cells. These findings indicate that MMP-10 facilitates the clearance of MWCNTs and moderates the pro-inflammatory response of exposed alveolar and infiltrated macrophages.

Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies

The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

Expression of MMP-2 and MMP-9 in odontogenic myxoma in a child: report of a clinical case

Odontogenic myxoma (OM) is a benign, locally invasive, non-metastasizing neoplasm of the jaw bones. Despite the benign nature of these lesions, there is a high rate of recurrence and the current recommended therapy, depending on the size and behaviour of the lesion, can vary from curettage with peripheral ostectomy, segmental resection up to radical resections for more aggressive lesions. OM is a rare tumour which occurs predominantly in the third decade of life and it is rare in children. Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases responsible for the degradation and remodelling of extracellular matrix, they are known to be involved in the progression and invasiveness of many types of tumour. MMPs have been studied in OM because of their well-known role in extracellular matrix degradation, tumour invasion and recurrence. We report a case of OM in a 6-year-old boy. A conservative excision was accomplished. The mass was excised without affecting the mandibular bone and the inferior alveolar nerve. Curettage and removal of the first right inferior molar were performed. After 6-month follow-up, no evidence of recurrence was found. We investigated the expression of MMP-2 and MMP-9 in this case of OM in a child. RT-PCR showed the expression of both MMP-2 and MMP-9 mRNAs. Immunohistochemistry showed a weak MMP-2 protein expression while MMP-9 protein was not detected. In this case of OM in a child, we report lack of recurrence after excision associated with low MMP-2 protein expression and absence of MMP-9. We believe it is worthy to deeply investigate the relationship between MMPs expression and OM behaviour with the aim to use MMPs as prognostic and/or therapeutic markers in OM.

Inhibition of Matrix Metalloproteinases Prevents the Potentiation of Nondeprived-Eye Responses after Monocular Deprivation in Juvenile Rats

The ocular dominance (OD) shift induced by monocular deprivation (MD) during the critical period is mediated by an initial depression of deprived-eye responses followed by an increased responsiveness to the nondeprived eye. It is not fully clear to what extent these 2 events are correlated and which are their physiological and molecular mediators. The extracellular synaptic environment plays an important role in regulating visual cortical plasticity. Matrix metalloproteinases (MMPs) are a family of activity-dependent zinc-dependent extracellular endopeptidases mediating extracellular matrix remodeling. We investigated the effects of MMP inhibition on OD plasticity in juvenile monocularly deprived rats. By using electrophysiological recordings, we found that MMP inhibition selectively prevented the potentiation of neuronal responses to nondeprived-eye stimulation occurring after 7 days of MD and potentiation of deprived-eye responses occurring after eye reopening. Three days of MD only resulted in a depression of deprived-eye responses insensitive to MMP inhibition. MMP inhibition did not influence homeostatic plasticity tested in the monocular cortex but significantly prevented an increase in dendritic spine density present after 7 days MD in layer II-III pyramids.

Matrix metalloproteinases are elevated in the urine of patients with endometriosis

In a prospective, blinded, longitudinal study MMP-2, MMP-9, and MMP-9/neutrophil gelatinase-associated lipocalin were significantly more likely to be detected in the urine of patients with endometriosis than in controls.

Biochemical characterization of an alkaline metallopeptidase secreted by a Pseudomonas fluorescens isolated from soil

The extracellular endopeptidase synthesized by soil bacterium Pseudomonas fluorescens was purified to homogeneity in a four-step procedure. The enzyme was purified 45-fold, with a 20% recovery. The endopeptidase appeared to be a monomer with a molecular mass of approx. 50 kDa. The enzyme was active in the pH range of 7 to 10. The optimal activity was detected at pH 9.0 and at 42 degrees C. Enzyme activity was inhibited by EDTA, EGTA and 1,10 phenanthroline, typical metalloprotease inhibitors. Ca(2+) activated the enzyme while Zn(2+), Co(2+), Cd(2+)(in high concentration) strongly inhibited it. The presence of calcium ions strongly stabilized the enzyme with regard to thermal resistance. The amino acid sequence of fragments of the enzymatic protein determined by MS analysis revealed a high similarity to the sequences of other alkaline metalloendopeptidases of bacteria belonging to the genus Pseudomonas.

Expression of matrix metalloproteinases in full-term human umbilical cord and Human Umbilical Vein Endothelial Cells.

background Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in breakdown and remodelling of extracellular matrix in physiological and pathological processes. MMPs have also a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. For these reasons we believe it is interesting to investigate the expression of some remodelling enzymes in this tissue. Human Umbilical Vein Endothelial cells (HUVEC) have shown to be endowed with an high degree of plasticity; they have the potentiality to differentiate into different cell types and can be considered one of the territories of umbilical cord more dynamic and involved in remodelling/renewal phenomena, for this reason we decided to extend our research investigating the expression of MMPs also in these cells. Aim In this study we investigated by immunohistochemistry and RT-PCR the expression of the gelatinases MMP-2 and MMP-9, the collagenases MMP-8 and MMP-13 and the stromelysin MMP-3 and MMP-10 in the whole human umbilical cord and in HUVEC. Results MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton’s jelly mesenchymal cells. Immunohistochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured Human Umbilical cord Vein Endothelial Cells. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC cells only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected.

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.

Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury

BackgroundMatrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that degrade the extracellular matrix and other extracellular proteins. Studies in experimental animals demonstrate that MMPs play a number of roles in the detrimental as well as in the beneficial events after spinal cord injury (SCI). In the present correlative investigation, the expression pattern of several MMPs and their inhibitors has been investigated in the human spinal cord.MethodsAn immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.ResultsIn the unlesioned human spinal cord, MMP and TIMP immunoreactivity was scarce. After traumatic SCI, a lesion-induced bi-phasic pattern of raised MMP-1 levels could be found with an early up-regulation in macrophages within the lesion epicentre and a later induction in peri-lesional activated astrocytes. There was an early and brief induction of MMP-2 at the lesion core in macrophages. MMP-9 and -12 expression peaked at 24 days after injury and both molecules were mostly expressed in macrophages at the lesion epicentre. Whereas MMP-9 levels rose progressively from 1 week to 3 weeks, there was an isolated peak of MMP-12 expression at 24 days. The post-traumatic distribution of the MMP inhibitors TIMP-1, -2 and -3 was limited. Only occasional TIMP immuno-positive macrophages could be detected at short survival times. The only clear induction was detected for TIMP-3 at survival times of 8 months and 1 year in peri-lesional activated astrocytes.ConclusionThe involvement of MMP-1, -2, -9 and -12 has been demonstrated in the post-traumatic events after human SCI. With an expression pattern corresponding largely to prior experimental studies, they were mainly expressed during the first weeks after injury and were most likely involved in the destructive inflammatory events of protein breakdown and phagocytosis carried out by infiltrating neutrophils and macrophages, as well as being involved in enhanced permeability of the blood spinal cord barrier. Similar to animal investigations, the strong induction of MMPs was not accompanied by an expression of their inhibitors, allowing these proteins to exert their effects in the lesioned spinal cord.

Effects of matrix metalloproteinase inhibition on short‐ and long‐term plasticity of schaffer collateral/CA1 synapses

It is increasingly evident that matrix metalloproteinases (MMPs), a family of zinc containing extracellular endopeptidases, participate in processes supporting hippocampal synaptic plasticity. The purpose of this study was to further the understanding of MMPs involvement in hippocampal plasticity. Acute hippocampal slices, generated from 20- to 30-day-old male Sprague-Dawley rats, were subjected to various electrophysiologic stimulatory paradigms to produce either short-term or long-term modifications to synaptic efficacy. Slices exposed to broad-spectrum MMP inhibitor, FN-439, exhibited impairments in paired-pulse facilitation, theta-burst facilitation, and long-term depression. Additionally, we observed that MMP inhibition impaired both the induction and stability of long-term potentiation (LTP). Furthermore, evidence indicated that the effect of MMP inhibition on LTP maintenance is dependent upon integrin-directed adhesion, whereas the effects of MMP inhibition on LTP induction are independent of integrin-directed adhesion. Together, these data support a generalized role for MMPs in short-term and long-term hippocampal plasticity and indicate that MMPs are a necessary facet of integrin-mediated cell adhesion supporting LTP stabilization.