Virginia Medda

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.

Biomarkers of oxidation, inflammation and cartilage degradation in osteoarthritis patients undergoing sulfur-based spa therapies

OBJECTIVES To investigate the effects of sulfur-based spa therapies on oxidation, inflammation and cartilage degradation biomarkers in osteoarthritis (OA) patients. DESIGN AND METHODS Analyses were performed before therapy (T0), after therapy (T1) and 1 month after its suspension (T2), in OA subjects undergoing mud bath treatments in combination (group A) or not (group B) with hydropinotherapy, and compared with those of patients not subjected to spa therapies (group C). RESULTS No modifications in plasma/serum biomarker concentrations were observed throughout the study in non-treated patients, while a significant reduction in oxidation, inflammation and cartilage degradation parameters was evidenced in patients of group A. Group B presented a favorable biochemical profile at T1 but not at T2. CONCLUSIONS To ensure the long term preservation of the chondroprotective effects of sulfur-based therapies, standard mud bath treatments should be associated with hydropinotherapy in order to maintain reduced oxidative, inflammatory and degradative stimuli longer.

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

Protein profile ana lysis of the breast microenvironment to differentiate healthy women from breast cancer patients

Protein and proteomic high-throughput technologies provide the polypeptide signatures of nipple aspirate fluid (NAF), a breast secretion collected noninvasively from healthy individuals and cancer patients. As breast cancer develops from ductal–lobular epithelium, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a useful tool for the analysis of metabolic pathways within the mammary gland, deepening our knowledge of the biomolecular mechanisms of breast cancer initiation and progression. The different protein expression of major NAF proteins, separated using 1D polyacrylamide gels, has proven valuable for the early detection of women with increased risk of cancer. The failure to recognize a single marker with sufficient clinical sensitivity and/or specificity has driven the identification of breast cancer multiple proteins by 2D electrophoresis. Mass spectrometry-based proteomic approaches (SELDI- and MALDI-TOF technologies) have allowed the characterization of differential NAF proteomic fingerprints between healthy individuals and breast cancer patients. The intraductal approach of protein and proteomic analyses may provide a panel of biomarkers to strengthen the armory against breast cancer.

Glycosaminoglycan Sulodexide Inhibition of MMP-9 Gelatinase Secretion and Activity: Possible Pharmacological Role Against Collagen Degradation in Vascular Chronic Diseases

We evaluated the effects of the glycosaminoglycan sulodexide (SDX; antithrombotic/profibrinolytic drug) on the activity and release of matrix metalloproteinases (MMPs) in human blood. This was a prospective non-randomized study, analyzing by zymography and ELISA the in vitro effects of SDX on pro-enzyme, complexed, and active MMP forms in plasma and serum from 60 healthy donors, and in U-937 leukemia cell line. The levels and zymographic profile of MMP-2 did not show significant changes among samples and during SDX treatments. However, pro- and complexed forms of MMP-9 were strongly affected by SDX treatment (P<0.001), with significant decrease of MMP-9 secretion from white blood cells in a dose-dependent fashion (P<0.0001), without any displacement of MMP prodomains. The mechanism of reduced release of MMP-9 forms from leukocytes and inhibition of proteolytic activity due to SDX treatment may support the hypothesis that drugs based upon inhibitors of MMP-9 activity may provide a therapeutic tool for the underlying pathological destruction of extracellular matrix, and offering novel pharmacologic applications for chronic inflammatory vascular diseases, including varicose vein and chronic venous diseases associated with enhanced MMP activation in blood and limbs.

Protein oxidation in breast microenvironment: Nipple aspirate fluid collected from breast cancer women contains increased protein carbonyl concentration

Background: Protein carbonyl levels are the most frequently used biomarker of protein oxidation in several human diseases, including cancer. Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium from which nipple aspirate fluid can be collected and analysed to assess tissue metabolic activity. Our aims were to perform an exploratory investigation on the protein carbonyl accumulation in breast secretions from healthy and cancer patients and its correlation with lipid peroxidation markers. Methods: Protein carbonyls were determined by ELISA in 288 Nipple Aspirate Fluids (NAF) from Control, Pre-malignant and Cancer patients. Results: Significantly higher protein carbonyl concentration was found in NAF from breast cancer (BC) patients compared to Control subjects. Cancer patients accumulated in NAF significantly higher levels of carbonyls in post-menopausal condition. A significant inverse relationship between carbonyls and 8-F2α-isoprostanes in NAF was found in Cancer patients. NAF levels of protein carbonyls are significantly higher in women with pre-malignant conditions than in healthy subjects. Conclusions: Our results support the hypothesis that oxidative stress in breast microenvironment plays a role in breast cancer; measurement of protein and lipid oxidative products in NAF may improve the identification of women at increased breast cancer risk.

Increased shedding of soluble fragments of P-cadherin in nipple aspirate fluids from women with breast cancer

Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess breast metabolic activity. P‐cadherin is frequently over‐expressed in high‐grade invasive breast carcinomas and has been reported to be an enhancer of migration and invasion of breast cancer cells, being correlated with tumor aggressiveness. The present study analyzed the soluble fragment of P‐cadherin in milk, NAF and matched plasma samples of healthy subjects and in women with precancer conditions and breast cancer. Soluble P‐cadherin was detected in all plasma and milk samples, and in about 31.3% of NAF samples. The lowest levels of soluble P‐cadherin were found in plasma, with no significant difference among NoCancer, PreCancer and Cancer patients. The highest concentration of soluble P‐cadherin was detected in milk collected during the first trimester of lactation, significantly with respect to all NAF samples. There were significantly higher levels of soluble P‐cadherin in NAF from Cancer patients than those in women with NoCancer and PreCancer (P < 0.0001). Although no significant difference was found between in situ and invasive breast cancer, soluble P‐cadherin levels were found at high concentrations in c‐erbB‐2‐positive tumors, showing a positive correlation with disease stage grouping and tumor grade, and an inverse relationship with estrogen/progesterone receptor status. High levels of the soluble fragment of P‐cadherin in Cancer NAF suggest its possible release via proteolytic processing, favoring cancer cell detachment from breast duct, and suggesting that measuring soluble P‐cadherin in NAF may improve the identification of women with increased breast cancer risk. (Cancer Sci 2008; 99: 2160–2169)

Intracrinology of breast microenvironment: hormonal status in nipple aspirate fluid and its relationship to breast cancer

Breast cancer, a complex and multifactorial disease, is the most commonly diagnosed malignancy affecting women. Methods currently available for breast cancer detection have well-described limitations; in this respect, the intraductal approaches directly assess the microenvironment of the breast. Nipple aspirate fluid (NAF) can be noninvasively obtained from the breast in most women and represents a promising biological tool to assess metabolic, hormonal and molecular changes occurring in the cells lining the ducts, from which breast cancer arises. The aim of this review is to highlight the application of NAF studies in the field of biomarker discovery, which provide results useful for early detection and prevention of breast cancer risk; in fact, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a reliable method for assessment of metabolic/hormonal pathways within the mammary gland, identifying biomolecular mechanisms of breast cancer initiation and progression. The intracrinology of breast microenvironment (i.e., hormonal status in NAF) may provide independent diagnostic/prognostic factors, highlighting the importance of early altered hormonal metabolism (e.g., aromatase, estrogen sulfotransferase and steroid sulfatase pathway) in relation to breast cancer initiation. The possible application of targeted therapies through the inhibition of intratumoral enzymes involved in steroid metabolism is also discussed. The intraductal approach to hormone analyses may provide a further panel of biomarkers providing clinical benefits and strengthening the armory against breast cancer.

The matrix metalloproteinase inhibitor marimastat promotes neural progenitor cell differentiation into neurons by gelatinase-independent TIMP-2-dependent mechanisms.

Metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs), produced in the brain by cells of non-neural and neural origin, including neural progenitors (NPs), are emerging as regulators of nervous system development and adult brain functions. In the present study, we explored whether MMP-2, MMP-9, and TIMP-2, abundantly produced in the brain, modulate NP developmental properties. We found that treatment of NPs, isolated from the murine fetal cerebral cortex or adult subventricular zone, with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected the NP differentiation fate. Marimastat treatment allowed for an enrichment of our cultures in neuronal cells, inducing NPs to generate higher percentage of neurons and a lower percentage of astrocytes, possibly affecting NP commitment. Consistently with its proneurogenic effect, Marimastat early downregulated the expression of Notch target genes, such as Hes1 and Hes5. MMP-2 and MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not expressed under these conditions, whereas MMP-2 increased in the medium as pro-MMP-2 (72 kDa) during differentiation; its active form (62 kDa) was not detectable by gel zymography. MMP-2 silencing or administration of recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal differentiation, nor it is involved in the Marimastat proneurogenic effect. We also found that TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic potential; however, the proneurogenic action of Marimastat was mediated by TIMP-2, as demonstrated by silencing experiments. In conclusion, our data exclude a major involvement of MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of TIMP-2 to act as key effector of the proneurogenic response to an inducing stimulus such as Marimastat.

Differential expression of MMP-2 and MMP-9 activity in megakaryocytes and platelets

To the editor: In the May 31, 2011 online issue of Blood , Cecchetti et al presented a transcriptome analysis of matrix metalloproteinase (MMP) expression in megakaryocytes and platelets.[1][1] Their results demonstrate that megakaryocytes and platelets differentially express mRNAs and proteins for