Ferdinando Mannello

Zymographic Analysis and Characterization of MMP-2 and -9 Forms in Human Sound Dentin

The role and function of dentin matrix metalloproteinases (MMPs) are not well-understood, but they may play a key role in dentinal caries and the degradation of resin-bonded dentin matrices. To test the null hypothesis that MMP-9 is not found in dentin matrix, we used gelatin zymography to extract and isolate all molecular forms of gelatinolytic MMPs in demineralized mature sound dentin powder obtained from extracted human molars, characterizing and identifying the enzymes by Western blotting. Gelatinolytic MMPs were detected in extracts of demineralized dentin matrix and identified as MMP-2 and MMP-9. Acidic extracts (pH 2.3) yielded 3–8 times more MMP activity than did EDTA (pH 7.4). Their activation may contribute to dentin matrix degradation, which occurs during caries progression and following resin bonding. Inhibition of MMP-2 and -9 proteolytic activity may slow caries progression and increase the durability of resin-dentin bonds.

Role and Function of Matrix Metalloproteinases in the Differentiation and Biological Characterization of Mesenchymal Stem Cells

Matrix metalloproteinases (MMPs), known as matrixins, are Ca‐ and Zn‐dependent endoproteinases involved in a wide variety of developmental and disease‐associated processes, proving to be crucial protagonists in many physiological and pathological mechanisms. The ability of MMPs to alter, by limited proteolysis and through the fine control of tissue inhibitors of metalloproteinases, the activity or function of numerous proteins, enzymes, and receptors suggests that they are also involved in various important cellular functions during development. In this review, we focus on the differentiation of mesenchymal stem cells (including those of the myoblastic, osteoblastic, chondroblastic, neural, and apidoblastic lineages) and the possible, if unexpected, biological significance of MMPs in its regulation. The MMP system has been implicated in several differentiation events that suggests that it mediates the proliferative and prodifferentiating effect of the matrixin proteolytic cascade. We summarize these regulatory effects of MMPs on the differentiation of mesenchymal stem cells and hypothesize on the function of MMPs in the stem cell differentiation processes.

From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera?

Bone marrow-derived mesenchymal stem cells are a multipotent adult cellular population endowed with broad differentiation potential. Their regeneration capability, ease to undergo gene modifications, and immuno-suppressive capacity makes them optimal tools for tissue engineering, gene- and immuno-therapy. Due to the ever-increasing number of studies on the clinical applications of mesenchymal stem cells in regenerative medicine, these cells have become attractive targets in clinical transplantation. However, the identification and definition of mesenchymal stem cell culture media for their clinical application in cell therapy is currently a matter of strong discussion. Up to now, clinical studies have been conducted with mesenchymal stem cells cultured in foetal calf serum, and the chance of contamination or immunological reaction towards xenogeneic compounds must be taken into consideration. On the other hand, a serum-free medium without the addition of growth factors is not able to expand these cells in vitro; so the evaluation of which is best, among foetal calf serum, human serum (whether autologous or allogeneic) and platelet-rich plasma, is a hot topic urgently needing further research efforts. The need for the establishment of standardized protocols for mesenchymal stem cell preparations, in order not to interfere with their self-renewal and differentiation processes, assuring durable engraftment and long-term therapeutic effects, is evidently crucial. Therefore, the search for optimal culture conditions for the effective clinical-scale production of vast numbers of mesenchymal stem cells for cellular therapy is of paramount importance and the need for a robust passage from basic to translational research is fundamental.

Nuclear localization of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) were originally identified as matrixin proteases that act in the extracellular matrix. Recent works have uncovered nontraditional roles for MMPs in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized matrixins participate in many physiological and pathological cellular processes, in which they can act as both degradative and regulatory proteases. In this review, we discuss the transcriptional and translational control of matrixin expression, their regulation of intracellular sorting, and the structural basis of activation and inhibition. In particular, we highlight the emerging roles of various matrixin forms in diseases. The activity of matrix metalloproteinases is regulated at several levels, including enzyme activation, inhibition, complex formation and compartmentalization. Most MMPs are secreted and have their function in the extracellular environment. MMPs are also found inside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is still poorly understood, although recent studies have unraveled some of their functions. The localization, activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycan core proteins and / or their glycosaminoglycan chains, as well as other molecules. Complexes formed between MMPs and various molecules may also include interactions with noncatalytic sites. Such exosites are regions involved in substrate processing, localized outside the active site, and are potential binding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential for understanding various physiological processes and pathogenesis of diseases, as well as for the development of new MMP targeting drugs.

Melatonin prevents apoptosis induced by UV-B treatment in U937 cell line.

Abstract:  Melatonin influences circadian rhythms and acts as antioxidant and free radical scavenger. UV irradiation triggers multiple cellular events which lead to cell death, in particular to apoptosis; this process involves reactive oxygen species. Apoptotic machinery involves several pathways, in which mitochondria play crucial roles. In this work we have evaluated by means of cytometric, biochemical and ultrastructural approaches, if incubation of U937 promonocytic leukemia cells with melatonin may affect apoptotic behavior induced by UV‐B. The cell line was treated with 1 mm melatonin before and after UV‐B exposure. Melatonin pretreatment significantly reduced the number of apoptotic cells, as revealed by FITC Annexin‐V and propidium iodide assays (P < 0.005), as well as attenuated mitochondria alterations, as shown by ultrastructural morphology, Mito Tracker and JC‐1 staining, and cytochrome c (cyt c) release (P < 0.005). On the contrary, incubation with melatonin after UV‐B exposure significantly protect U937 cells from UV‐B induced alterations, showing a possible delay of the apoptotic machinery (as revealed by the presence of earlier stages of apoptosis and significant cyt c release). Our results suggest that, in our experimental model, melatonin may play a role as noncytotoxic anti‐apoptotic compound and, at least in part, may protect U937 cells from UV‐B induced mitochondria dysfunction/damage.

Effects of Blood Collection Methods on Gelatin Zymography of Matrix Metalloproteinases

Matrix metalloproteinases (MMPs), zinc-dependent proteinases belonging to the family of matrixins, are involved in the processes of normal development and growth as well as in several pathologic states (1). MMP-2 (gelatinase A; EC 3.4.24.24) and MMP-9 (gelatinase B; EC 3.4.24.35) play crucial roles in the proteolytic cascade leading to extracellular matrix degradation (2). To evaluate the usefulness of MMPs as tumor markers, several authors have measured MMP-2 and MMP-9 in the blood of cancer patients, using serum or plasma samples, and reached contradictory conclusions (3)(4). The preanalytical steps of blood sampling and processing influence the concentrations of MMPs and their inhibitors (5)(6), but no data are available concerning the effect of blood specimen collection methods on the appearance of the full spectrum of MMP isoforms on gelatin zymograms. We simultaneously collected blood samples from 20 healthy volunteers (median age, 36 years; range, 23–55 years) into plastic tubes with no additive, plastic tubes with a silica gel-coated surface as coagulation accelerator, lithium heparin-coated …

Aluminium and breast cancer: Sources of exposure, tissue measurements and mechanisms of toxicological actions on breast biology.

This review examines recent evidence linking exposure to aluminium with the aetiology of breast cancer. The human population is exposed to aluminium throughout daily life including through diet, application of antiperspirants, use of antacids and vaccination. Aluminium has now been measured in a range of human breast structures at higher levels than in blood serum and experimental evidence suggests that the tissue concentrations measured have the potential to adversely influence breast epithelial cells including generation of genomic instability, induction of anchorage-independent proliferation and interference in oestrogen action. The presence of aluminium in the human breast may also alter the breast microenvironment causing disruption to iron metabolism, oxidative damage to cellular components, inflammatory responses and alterations to the motility of cells. The main research need is now to investigate whether the concentrations of aluminium measured in the human breast can lead in vivo to any of the effects observed in cells in vitro and this would be aided by the identification of biomarkers specific for aluminium action.

Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice

ObjectiveTo characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis. Design and methodsTat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines. ResultsTat protein was detected in tumour cell lines in amounts from 600–7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice, induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect. ConclusionsTat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Neural stem cells at the crossroads: MMPs may tell the way

Matrix metalloproteinases (MMP) constitute a family of more than 25 enzymes which process a large number of pericellular substrates. Even though initially reported to have an ability to degrade almost all of the extracellular components, MMP are now known to play roles which are not limited to the breakdown of extracellular barriers. In fact, MMPs regulate many biological processes, being involved not only in physiological events, but also in pathological processes. Strikingly, MMPs have been found to be involved in the physiology of the Central Nervous System (CNS), taking part and playing important roles in several processes such as repair and ontogeny, as well as in pathological conditions of the CNS. Initially considered to be a static structure, lacking regenerative capability, the CNS has been considered for a long time to be a system without renewal capabilities. Recently, the discovery of constant neural replacement has changed our way of considering the adult brain, and the finding of the existence of neural stem cells has opened the way to exciting and fascinating perspectives of the CNS. So, could MMPs, originally found during metamorphosis in tadpoles, and now amazingly identified in the CNS, have something to do in neuronal function? In this review we take into consideration the possible roles of two metalloproteinases, MMP-2 and MMP-9, also called gelatinases, in controlling several aspects of CNS organization, including the modulation of neural stem cell properties and the differentiation of their progeny, both under normal and pathophysiological conditions.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer‐affected patients

Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron‐binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer‐affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP‐mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 μg l−1; n = 19) than in NoCancer NAF (131.3 ± 9.6 μg l−1; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 μg l−1) than in NoCancer NAF (55.5 ± 7.2 μg l−1), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium‐based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues. Copyright