Gaetana A. Tonti

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.

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.

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.

Concentration of aluminium in breast cyst fluids collected from women affected by gross cystic breast disease

Gross cystic breast disease (GCBD) is the most common benign breast disorder, but the molecular basis of cyst formation remains to be identified. If the use of aluminium‐based antiperspirant salts is involved in the etiology of gross breast cyst formation, it might be expected that aluminium would be at elevated levels in human breast cyst fluid (BCF). Aluminium was measured by ICP‐MS in 48 samples of BCF, 30 samples of human blood serum and 45 samples of human breast milk at different stages of lactation (colostrum, intermediate, mature). The median level of aluminium in apocrine type I BCF (n = 27, 150 µg l−1) was significantly higher than in transudative type II BCF (n = 21, 32 µg l−1; P <0.0001). By comparison, aluminium measurements gave a median concentration of 6 µg l−1 in human serum and 25 µg l−1 in human breast milk, with no difference between colostrum, intermediate and mature milk. Levels of aluminium were significantly higher in both types of BCF than in human serum (P <0.0001). However when compared with human breast milk, aluminium levels were only significantly higher in apocrine type I BCF (P <0.0001) and not in transudative type II BCF (P = 0.152). It remains to be identified why such high levels of aluminium were found in the apocrine type I BCF and from where the aluminium originated. However, if aluminium‐based antiperspirants are found to be the source and to play any causal role in development of breast cysts, then it might become possible to prevent this common breast disorder. Copyright

Human gross cyst breast disease and cystic fluid: bio-molecular, morphological, and clinical studies.

SummaryFor more than one and a half century the cystic disease of the breast has been recognized as the most frequent female benign breast lesion. Although some conundrums and controversies exist about the relation between gross cysts and breast cancer, recent evidence suggests that the multidisciplinary study of gross cystic breast disease (GCBD) may be a powerful tool for predicting the natural history of the multifaceted gross cyst pathology. A lot of papers have been published on breast cyst fluids (BCF) concerning biochemical, hormonal and morphological aspects, demonstrating that the intracystic fluid contains a wide variety of components (such as ions, lipids, proteins, enzymes, growth factors and antigens) and suggesting that their profile provides additional knowledge on both physiopathology and etiologic pathways of human gross cystic breast disease. The aim of this overview is the critical evaluation of all data accumulated in the last thirty years, in order to highlight the utility of biochemical and epidemiological studies to identify gross cysts, if any, at higher breast cancer risk.

Gelatinase concentrations and zymographic profiles in human breast cancer: matrix metalloproteinases circulating in plasma are better markers for the subclassification and early prediction of cancer: the coagulation/fibrinolysis pathways alter the release, activation and recovery of different gelatinases in serum.

Dear Sir, Somiari et al. recently published the measurements of serum levels and activities of matrix metalloproteinases (MMP) in patients classified as low and high risk (according to Gail score), benign disease or breast cancer. We read their article with concern, which described that total, active and activity of MMP were significantly increased in serum of cancer patients respect to benign diseases and control groups, concluding that serum MMP may classify patients with breast diseases. Surprisingly, the authors have just published an almost overlapping study using plasma samples following the same research protocol, reporting that also plasma MMP concentration and activity may permit subclassification of patients with breast disorders. Although the role of proteinases in cancer initiation and progression has been on a roller-coaster ride for the past years, there is no doubt that among proteinases, MMP family members are involved in human breast cancer (BC). Among Matrixins, Ca/Zn-dependent proteinases ubiquitously involved in metabolism regulation, MMPs are characterized by the ability to extensively degrade proteins, in particular almost all extracellular matrix proteins. To prevent the vast potential of MMPs, crucial in physiological cellular mechanisms (e.g., cell development, organogenesis, tissue remodelling and repair, apoptosis) from becoming destructive during pathological processes (e.g., inflammation, tissue degeneration, tumor growth, invasion and metastasis), a fine control is needed. The detrimental role of MMPs in cancer (not sufficiently counterbalanced by the endogenous specific inhibitors TIMP) has not been successfully blocked by the development of synthetic/natural MMP inhibitors that failed to limit the disease progression during several clinical trials. Although, the role of MMPs in early stages of cancer remains to be clearly established, the increased expression of MMPs in cancerrelated processes is also enhanced by the cross-talk signalling between stromal and inflammatory cells within the tumor microenvironment, leaving the way open for the multifaceted functions of proteinases in cancer development. No question is of greater interest to the clinicians dealing with breast diseases than the differentiation of BC subtypes through the determination of biomolecular markers useful for BC diagnosis and prognosis. Although the identification of the best source between plasma and serum for surrogate biomarkers is a matter of strenuous efforts and debate, the scientific interest in the measurement of MMP in plasma/serum continues to mount, enhancing their promising development as reliable biomarkers. The conclusions of Somiari et al. about the usefulness of MMP in serum samples strongly contrasts with the growing evidence that plasma MMP-9 is considered as a better marker for early diagnosis, progression and prognosis of BC. The faithful determination of MMP-2 and -9 circulating in blood depends on the procedure of blood sampling/handling, a methodological issue discussed extensively in this Journal and analytical journals. Although these articles unequivocally demonstrated the ‘‘artificially’’ higher levels of serum MMPs with respect to plasma, manuscripts ignore these statements and continue to publish based on the measurements of serum MMPs. A serious preanalytical flaw in this otherwise well-conceived study, undermines confidence in the authors’ conclusion, highlighting several caveats that are implicit, looking more correlative rather than mechanistic in nature. To correctly interpret data and to avoid pitfalls or wrong expectations in future studies in BC using circulating MMP-2 and -9 as surrogate biomarkers, we would like to draw the attention of interested clinicians to the crucial role of blood sampling/ handling when studying the functional involvement of MMPs in BC. We provide an explanation of why serum does not reflect the correct MMP expression, which should be the mirror of tumor microenvironment and not be due to the release by other cell sources, such as circulating white blood cells. The stroma surrounding the mammary terminal ductal/lobular unit is extensively infiltrated by migrating blood cells, mostly macrophages and eosinophils, capable of producing factors (e.g., angiogenic factors such as VEGF and angiopoietin, proteinases such as MMP-9 and uPA, growth factors such as EGF and CSF-1, cytokines and chemokines) that can promote both normal development (from postnatal morphogenesis through lactation) and escape of tumor cells from the local microenvironment, enhancing their metastatic potential. Although the origin/function of white blood cells in BC are not yet fully elucidated, an intensive interplay exists between breast tumor cells and leukocytes. Accordingly, also the count of peripheral leukocytes strongly influences BC prognosis. Platelets, eosinophils, neutrophils and monocytes contain high amounts of MMP-9 forms that may be extracellularly released upon activation or during aggregation. It is worth noting that BC infiltrating lymphocytes showed an increased

The 8‐epimer of prostaglandin F2α, a marker of lipid peroxidation and oxidative stress, is decreased in the nipple aspirate fluid of women with breast cancer

Breast cancer (BC), 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. Whether lipid peroxidation in the mammary gland promotes or prevents tumorigenesis is unclear. Malondialdehyde (MDA) and the 8‐epimer of Prostaglandin F2α (8‐iso‐PGF2α), two lipid peroxidation markers, were studied in milk (n = 10), NAF (n = 140) and plasma (n = 35) samples. MDA was detected in all plasma, in 80% of milk samples and in 95% of NAF samples. MDA levels in NAF and plasma were significantly higher than in milk (p = 0.016 and p = 0.029, respectively). We found no significant difference between levels of MDA in NAF samples from BC patients compared to healthy controls. 8‐iso‐PGF2α was detectable in all samples. 8‐iso‐PGF2α median levels in NAF were significantly higher than in both milk and plasma (p < 0.0001). The highest 8‐iso‐PGF2α levels were found in NAF from healthy women, significantly higher than in women with BC (p < 0.0001). No significant differences were found in both markers after the age‐adjustment. High levels of lipid peroxidation products in NAF suggest their in situ production in the nonlactating breast. Active lipid peroxidation may have a physiologic role in the normal mammary gland. Lower levels of 8‐iso‐PGF2α in NAF from BC patients suggest altered production of arachidonic acid metabolites during breast carcinogenesis.