Nuova ricerca


Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze,sede Istituti Anatomici (area Policlinico)

Home | Curriculum(pdf) | Didattica |


2023 - Amniotic Fluid and Placental Membranes as Sources of Stem Cells: Progress and Challenges 2.0 [Articolo su rivista]
Maraldi, Tullia; Russo, Valentina

: The aim of the second edition of this Special Issue was to collect both review and original research articles that investigate and elucidate the possible therapeutic role of perinatal stem cells in pathological conditions, such as cardiovascular and metabolic diseases, as well as inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...].

2023 - Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Gatti, Martina; Persico, Giuseppe; Bertucci, Emma; Giorgio, Marco; Maraldi, Tullia

Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit . Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGF, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL- 6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

2023 - Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity [Articolo su rivista]
Gatti, Martina; Dittlau, Katarina Stoklund; Beretti, Francesca; Yedigaryan, Laura; Zavatti, Manuela; Cortelli, Pietro; Palumbo, Carla; Bertucci, Emma; Van Den Bosch, Ludo; Sampaolesi, Maurilio; Maraldi, Tullia

: Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

2023 - Identification of Anti-Neuroinflammatory Bioactive Compounds in Essential Oils and Aqueous Distillation Residues Obtained from Commercial Varieties of Cannabis sativa L [Articolo su rivista]
Barbalace, Maria Cristina; Freschi, Michela; Rinaldi, Irene; Mazzara, Eugenia; Maraldi, Tullia; Malaguti, Marco; Prata, Cecilia; Maggi, Filippo; Petrelli, Riccardo; Hrelia, Silvana; Angeloni, Cristina

Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from 'Gorilla Glue' was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-kappa B pathway. Moreover, the sesquiterpenes (E)-caryophyllene, alpha-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils' anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of alpha-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation.

2022 - Amniotic Fluid and Placental Membranes as Sources of Stem Cells: Progress and Challenges [Articolo su rivista]
Maraldi, T.; Russo, V.

: The intention of this special edition is to collect review and original research articles that illustrate and stimulate growing efforts to understand the implication of perinatal stem cells in pathological conditions such as cardiovascular and metabolic diseases and inflammatory, autoimmune, musculoskeletal, and degenerative diseases [...].

2022 - Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aβ [Articolo su rivista]
Zavatti, M.; Gatti, M.; Beretti, F.; Palumbo, C.; Maraldi, T.

Background: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer’s disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aβ) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). Methods: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aβ were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. Results: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aβ. Conclusion: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.

2022 - Shared and Divergent Epigenetic Mechanisms in Cachexia and Sarcopenia [Articolo su rivista]
Yedigaryan, Laura; Gatti, Martina; Marini, Vittoria; Maraldi, Tullia; Sampaolesi, Maurilio

Significant loss of muscle mass may occur in cachexia and sarcopenia, which are major causes of mortality and disability. Cachexia represents a complex multi-organ syndrome associated with cancer and chronic diseases. It is often characterized by body weight loss, inflammation, and muscle and adipose wasting. Progressive muscle loss is also a hallmark of healthy aging, which is emerging worldwide as a main demographic trend. A great challenge for the health care systems is the age-related decline in functionality which threatens the independence and quality of life of elderly people. This biological decline can also be associated with functional muscle loss, known as sarcopenia. Previous studies have shown that microRNAs (miRNAs) play pivotal roles in the development and progression of muscle wasting in both cachexia and sarcopenia. These small non-coding RNAs, often carried in extracellular vesicles, inhibit translation by targeting messenger RNAs, therefore representing potent epigenetic modulators. The molecular mechanisms behind cachexia and sarcopenia, including the expression of specific miRNAs, share common and distinctive trends. The aim of the present review is to compile recent evidence about shared and divergent epigenetic mechanisms, particularly focusing on miRNAs, between cachexia and sarcopenia to understand a facet in the underlying muscle wasting associated with these morbidities and disclose potential therapeutic interventions.

2022 - Strategies to Counteract Oxidative Stress and Inflammation in Chronic-Degenerative Diseases [Articolo su rivista]
Prata, Cecilia; Maraldi, Tullia; Angeloni, Cristina

: The great increase in life expectancy is linked to the necessity of counteracting chronic-degenerative diseases, e [...].

2021 - Amniotic fluid stem cell-derived extracellular vesicles counteract steroid-induced osteoporosis in vitro [Articolo su rivista]
Gatti, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Luz, S. R.; Palumbo, C.; Maraldi, T.

Background—Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods—human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological as-says. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results—AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexa-methasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion— these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

2021 - Nadph oxidases: Redox regulators of stem cell fate and function [Articolo su rivista]
Maraldi, T.; Angeloni, C.; Prata, C.; Hrelia, S.

One of the major sources of reactive oxygen species (ROS) generated within stem cells is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs), which are critical determinants of the redox state beside antioxidant defense mechanisms. This balance is involved in another one that regulates stem cell fate: indeed, self-renewal, proliferation, and differentiation are decisive steps for stem cells during embryo development, adult tissue renovation, and cell therapy application. Ex vivo culture-expanded stem cells are being investigated for tissue repair and immune modulation, but events such as aging, senescence, and oxidative stress reduce their ex vivo proliferation, which is crucial for their clinical applications. Here, we review the role of NOX-derived ROS in stem cell biology and functions, focusing on positive and negative effects triggered by the activity of different NOX isoforms. We report recent findings on downstream molecular targets of NOX-ROS signaling that can modulate stem cell homeostasis and lineage commitment and discuss the implications in ex vivo expansion and in vivo engraftment, function, and longevity. This review highlights the role of NOX as a pivotal regulator of several stem cell populations, and we conclude that these aspects have important implications in the clinical utility of stem cells, but further studies on the effects of pharmacological modulation of NOX in human stem cells are imperative.

2021 - The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma [Articolo su rivista]
Beretti, Francesca; Farnetani, Francesca; Reggiani Bonetti, Luca; Fabbiani, Luca; Zavatti, Manuela; Maiorana, Antonino; Pellacani, Giovanni; Maraldi, Tullia

Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox).

2021 - Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations [Articolo su rivista]
Casciaro, Francesca; Zia, Silvia; Forcato, Mattia; Zavatti, Manuela; Beretti, Francesca; Bertucci, Emma; Zattoni, Andrea; Reschiglian, Pierluigi; Alviano, Francesco; Bonsi, Laura; Follo, Matilde Yung; Demaria, Marco; Roda, Barbara; Maraldi, Tullia

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

2020 - Comparison of the therapeutic effect of amniotic fluid stem cells and their exosomes on monoiodoacetate-induced animal model of osteoarthritis [Articolo su rivista]
Zavatti, M.; Beretti, F.; Casciaro, F.; Bertucci, E.; Maraldi, T.

The cartilage tissue engineering associated with stem cell-related therapies is becoming very interesting since adult articular cartilage has limited intrinsic capacity for regeneration upon injury. Amniotic fluid stem cells (AFSC) have been shown to produce exosomes with growth factors and immunomodulating molecules that could stop tissue degradation and induce cartilage repair. Based on this state of the art, the main aim of this study was to explore the efficacy of the secreted exosomes, compared to their AFSC source, in MIA-induced animal model of osteoarthritis mimicking a chronic and degenerative process, where inflammation is also involved and lead to irreversible joint damage. Exosomes, obtained by the use of a commercial kit, prior to the injection in animal knee joints, were characterized for the presence of typical markers and HGF, TGFβ, and IDO. Then, analyses were performed by histology, immunohistochemistry, and behavioral scoring up to 3 weeks after the treatment. Exosome-treated defects showed enhanced pain tolerance level and improved histological scores than the AFSC-treated defects. Indeed by 3 weeks, TGFβ-rich exosome samples induced an almost complete restoration of cartilage with good surface regularity and with the characteristic of hyaline cartilage. Moreover, cells positive for resolving macrophage marker were more easily detectable into exosome-treated joints. Therefore, a modulating role for exosomes on macrophage polarization is conceivable, as demonstrated also by experiments performed on THP1 macrophages. In conclusion, this study demonstrates for the first time the efficacy of human AFSC exosomes in counteract cartilage damage, showing a positive correlation with their TGFβ content.

2020 - Natural compounds as a strategy to optimize “in vitro” expansion of stem cells [Articolo su rivista]
Maraldi, Tullia; Prata, Cecilia; Marrazzo, Pasquale; Hrelia, Silvana; Angeloni, Cristina

The efficient use of stem cells for transplantation is often limited by the relatively low numbers of stem cells collected. The ex vivo expansion of human stem cells for clinical use is a potentially valuable approach to increase stem cell number. Currently, most of the procedures used to expand stem cells are carried out using a 21% oxygen concentration, that is about 4- to 10-fold greater than the concentration characteristic of their natural niches. Hyperoxia might cause oxidative stress with a deleterious effect on the physiology of cultured stem cells. In this review, we investigate and critically examine the available information on the ability of natural compounds to counteract hyperoxia-induced damage in different type of stem cells ex vivo. In particular, we focused on proliferation and stemness maintenance in an attempt to draw up useful indications to define new culture media with a promoting activity on cell expansion in vitro.

2020 - Oxidative stress in Alzheimer's disease: In vitro therapeutic effect of amniotic fluid stem cells extracellular vesicles [Articolo su rivista]
Gatti, M.; Zavatti, M.; Beretti, F.; Giuliani, D.; Vandini, E.; Ottani, A.; Bertucci, E.; Maraldi, T.

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular β-amyloid proteins (Aβ), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aβ and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

2020 - Prolonged hypoxia delays aging and preserves functionality of human amniotic fluid stem cells [Articolo su rivista]
Casciaro, F.; Borghesan, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Follo, M. Y.; Maraldi, T.; Demaria, M.

Human amniotic fluid stem cells (hAFSCs) are an emerging tool in regenerative medicine because they have the ability to differentiate into various lineages and efficiently improve tissue regeneration with no risk of tumorigenesis. Although hAFSCs are easily isolated from the amniotic fluid, their expansion ex vivo is limited by a quick exhaustion which impairs replicative potential and differentiation capacity. In this study, we evaluate various aging features of hAFSCs cultured at different oxygen concentrations. We show that low oxygen (1% O2) extends stemness and proliferative features, and delays induction of senescence-associated markers. Hypoxic hAFSCs activate a metabolic shift and increase resistance to pro-apoptotic stimuli. Moreover, we observe that cells at low oxygen remain capable of osteogenesis for prolonged periods of time, suggesting a more youthful phenotype. Together, these data demonstrate that low oxygen concentrations might improve the generation of functional hAFSCs for therapeutic use by delaying the onset of cellular aging.

2020 - Role of mesenchymal stem cells in counteracting oxidative stress—related neurodegeneration [Articolo su rivista]
Angeloni, C.; Gatti, M.; Prata, C.; Hrelia, S.; Maraldi, T.

Neurodegenerative diseases include a variety of pathologies such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and so forth, which share many common characteristics such as oxidative stress, glycation, abnormal protein deposition, inflammation, and progressive neuronal loss. The last century has witnessed significant research to identify mechanisms and risk factors contributing to the complex etiopathogenesis of neurodegenerative diseases, such as genetic, vascular/metabolic, and lifestyle-related factors, which often co-occur and interact with each other. Apart from several environmental or genetic factors, in recent years, much evidence hints that impairment in redox homeostasis is a common mechanism in different neurological diseases. However, from a pharmacological perspective, oxidative stress is a difficult target, and antioxidants, the only strategy used so far, have been ineffective or even provoked side effects. In this review, we report an analysis of the recent literature on the role of oxidative stress in Alzheimer’s and Parkinson’s diseases as well as in amyotrophic lateral sclerosis, retinal ganglion cells, and ataxia. Moreover, the contribution of stem cells has been widely explored, looking at their potential in neuronal differentiation and reporting findings on their application in fighting oxidative stress in different neurodegenerative diseases. In particular, the exposure to mesenchymal stem cells or their secretome can be considered as a promising therapeutic strategy to enhance antioxidant capacity and neurotrophin expression while inhibiting pro-inflammatory cytokine secretion, which are common aspects of neurodegenerative pathologies. Further studies are needed to identify a tailored approach for each neurodegenerative disease in order to design more effective stem cell therapeutic strategies to prevent a broad range of neurodegenerative disorders.

2019 - A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering [Articolo su rivista]
Marassi, Valentina; Beretti, Francesca; Roda, Barbara; Alessandrini, Andrea; Facci, Paolo; Maraldi, Tullia; Zattoni, Andrea; Reschiglian, Pierluigi; Portolani, Marinella

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

2019 - Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche [Articolo su rivista]
Sola, Antonella; Bertacchini, Jessika; D'Avella, Daniele; Anselmi, Laura; Maraldi, Tullia; Marmiroli, Sandra; Messori, Massimo

The need for new approaches to investigate ex vivo the causes and effects of tumor and to achieve improved cancer treatments and medical therapies is particularly urgent for malignant pathologies such as lymphomas and leukemias, whose tissue initiator cells interact with the stroma creating a three-dimensional (3D) protective environment that conventional mono- and bi-dimensional (2D) models are not able to simulate realistically. The solvent-casting particulate leaching (SCPL) technique, that is already a standard method to produce polymer-based scaffolds for bone tissue repair, is proposed here to fabricate innovative 3D porous structures to mimic the bone marrow niche in vitro. Two different polymers, namely a rigid polymethyl methacrylate (PMMA) and a flexible polyurethane (PU), were evaluated to the purpose, whereas NaCl, in the form of common salt table, resulted to be an efficient porogen. The adoption of an appropriate polymer-to-salt ratio, experimentally defined as 1:4 for both PMMA and PU, gave place to a rich and interconnected porosity, ranging between 82.1 vol% and 91.3 vol%, and the choice of admixing fine-grained or coarse-grained salt powders allowed to control the final pore size. The mechanical properties under compression load were affected both by the polymer matrix and by the scaffold's architecture, with values of the elastic modulus indicatively varying between 29 kPa and 1283 kPa. Preliminary tests performed with human stromal HS-5 cells co-cultured with leukemic cells allowed us to conclude that stromal cells grown associated to the supports keep their well-known protective and pro-survival effect on cancer cells, indicating that these devices can be very useful to mimic the bone marrow microenvironment and therefore to assess the efficacy of novel therapies in pre-clinical studies.

2019 - Influence of selenium on the emergence of neuro tubule defects in a neuron-like cell line and its implications for amyotrophic lateral sclerosis [Articolo su rivista]
Maraldi, T.; Beretti, F.; Anselmi, L.; Franchin, C.; Arrigoni, G.; Braglia, L.; Mandrioli, J.; Vinceti, M.; Marmiroli, S.

Impairment of the axonal transport system mediated by intracellular microtubules (MTs) is known to be a major drawback in neurodegenerative processes. Due to a growing interest on the neurotoxic effects of selenium in environmental health, our study aimed to assess the relationship between selenium and MTs perturbation, that may favour disease onset over a genetic predisposition to amyotrophic lateral sclerosis. We treated a neuron-like cell line with sodium selenite, sodium selenate and seleno-methionine and observed that the whole cytoskeleton was affected. We then investigated the protein interactome of cells overexpressing αTubulin-4A (TUBA4A) and found that selenium increases the interaction of TUBA4A with DNA- and RNA-binding proteins. TUBA4A ubiquitination and glutathionylation were also observed, possibly due to a selenium-dependent increase of ROS, leading to perturbation and degradation of MTs. Remarkably, the TUBA4A mutants R320C and A383 T, previously described in ALS patients, showed the same post-translational modifications to a similar extent. In conclusion this study gives insights into a specific mechanism characterizing selenium neurotoxicity.

2018 - Amniotic fluid stem cell exosomes: Therapeutic perspective [Articolo su rivista]
Beretti, Francesca; Zavatti, Manuela; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; La Sala, Giovanni B.; Maraldi, Tullia

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158–167, 2018.

2018 - Combination of Epigallocatechin Gallate and Sulforaphane Counteracts In Vitro Oxidative Stress and Delays Stemness Loss of Amniotic Fluid Stem Cells [Articolo su rivista]
Marrazzo, Pasquale; Angeloni, Cristina; Freschi, Michela; Lorenzini, Antonello; Prata, Cecilia; Maraldi, Tullia; Hrelia, Silvana

Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.

2018 - Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Zavatti, Manuela; McCubrey, James A.; Ratti, Stefano; Marmiroli, Sandra; Follo, Matilde Y.; Maraldi, Tullia

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence.

2018 - Sulforaphane Modulates AQP8-Linked Redox Signalling in Leukemia Cells [Articolo su rivista]
Prata, Cecilia; Facchini, Carlotta; Leoncini, Emanuela; Lenzi, Monia; Maraldi, Tullia; Angeloni, Cristina; Zambonin, Laura; Hrelia, Silvana; Fiorentini, Diana

Sulforaphane, a biologically active isothiocyanate compound extracted from cruciferous vegetables, has been shown to exert cytotoxic effects on many human cancer cells, including leukemia. However, the exact molecular mechanisms behind the action of sulforaphane in hematological malignancies are still unclear. Like other cancer cells, leukemia cells produce high level of reactive oxygen species; in particular, hydrogen peroxide derived from Nox family is involved in various redox signal transduction pathways, promoting cell proliferation and survival. Recent evidence show that many tumour cell types express elevated level of aquaporin isoforms, and we previously demonstrated that aquaporin-8 acts as H2O2 transport facilitator across the plasma membrane of B1647 cells, a model of acute myeloid human leukemia. Thus, the control of AQP8-mediated H2O2 transport could be a novel strategy to regulate cell signalling and survival. To this purpose, we evaluated whether sulforaphane could somehow affect aquaporin-8-mediated H2O2 transport and/or Nox-mediated H2O2 production in B1647 cell line. Results indicated that sulforaphane inhibited both aquaporin-8 and Nox2 expression, thus decreasing B1647 cells viability. Moreover, the data obtained by coimmunoprecipitation technique demonstrated that these two proteins are linked to each other; thus, sulforaphane has an important role in modulating the downstream events triggered by the axis Nox2-aquaporin-8. Cell treatment with sulforaphane also reduced the expression of peroxiredoxin-1, which is increased in almost all acute myeloid leukemia subtypes. Interestingly, sulforaphane concentrations able to trigger these effects are achievable by dietary intake of cruciferous vegetables, confirming the importance of the beneficial effect of a diet rich in bioactive compounds.

2017 - Apoptosis and inflammatory response in human astrocytes are induced by a transmissible cytotoxic agent of neurological origin [Articolo su rivista]
Beretti, Francesca; Ardizzoni, Andrea; Cermelli, Claudio; Guida, Marianna; Maraldi, Tullia; Pietrosemoli, P; Paulone, Simona; De Pol, Anto; Blasi, Elisabetta; Portolani, Marinella

We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating:a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis;an early and late p38 MAPK activation;an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.

Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; DE POL, Anto; LA SALA, Giovanni Battista; Maraldi, Tullia

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank.

2017 - Glycosides from stevia rebaudiana Bertoni possess insulin-mimetic and antioxidant activities in rat cardiac fibroblasts [Articolo su rivista]
Prata, Cecilia; Zambonin, Laura; Rizzo, Benedetta; Maraldi, Tullia; Angeloni, Cristina; Dalla Sega, Francesco Vieceli; Fiorentini, Diana; Hrelia, Silvana

Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

2017 - Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Beretti, Francesca; Resca, Elisa; Carpino, Guido; Cardinale, Vincenzo; Gentile, Raffaele; Ardizzoni, Andrea; Murgia, Alba; Alvaro, Domenico; Gaudio, Eugenio; DE POL, Anto

Aim: Human biliary tree stem/progenitor cells (hBTSC) are multipotent epithelial stem cells with the potential for allogenic transplant in liver, biliary tree, and pancreatic diseases. Human mesenchymal stem cells, but also epithelial stem cells, are able to modulate immune responses with different types of secretion molecules. Methods: The initial aim of the present study was to develop for the first time a culture protocol in order to expand hBTSC invitro through passages, allowing to maintain a similar stem cell and secretome profile. Furthermore, we investigated the secretome profile of the hBTSC to assess the production of molecules capable of affecting immune feedback. Results: We found that hepatocyte growth factor produced by hBTSC exerts its cytoprotective role inducing apoptosis in human immune cells, such as lymphocytes. Conclusions: The present study, therefore, supports the hypothesis that hBTSC can be useful for the purpose of regenerative medicine, as they can be banked and expanded, and they can secrete immunoregulatory factors.

2016 - Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells [Articolo su rivista]
Zavatti, Manuela; Guida, M; Maraldi, Tullia; Beretti, Francesca; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto

Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.

2016 - NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging [Articolo su rivista]
Maraldi, Tullia; Resca, Elisa; Nicoli, Alessia; Beretti, Francesca; Zavatti, Manuela; Capodanno, Francesco; Morini, Daria; Palomba, Stefano; LA SALA, Giovanni Battista; DE POL, Anto

Aims Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. Materials and methods MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age ≥ 40 years (cases) and with age ≤ 37 years (controls). Key findings The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. Significance The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging.

2015 - Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats [Articolo su rivista]
Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; De Pol, Anto

Aims: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. Main methods: In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 pm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. Key findings: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistly performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ER alpha. and GPR30) in all-groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. Significance: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen-AFSCs constructs.

2015 - Dietary polyphenols and their effects on cell biochemistry and pathophysiology 2014 [Articolo su rivista]
Angeloni, Cristina; Maraldi, Tullia; Milenkovic, Dragan; Vauzour, David

Epidemiological studies suggest that high dietary intake of phytochemicals and in particular of polyphenols is associated with decreased risk of a multitude of diseases states including cardiovascular disease, cancer, and neurodegenerative diseases. As oxidative stress is involved in all these pathological conditions, the antioxidant properties of polyphenols and other natural compounds have attracted the interest of many authors.

2015 - Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Maraldi, Tullia; Bertoni, Laura; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; Guillot, P. V; David, A. L; Sebire, N. J; De Pol, Anto; De Coppi, P.

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

2015 - Nuclear Nox4 role in stemness power of human amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; La Sala, Giovanni Battista; De Pol, Anto

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

2015 - Reactive oxygen species in stem cells [Articolo su rivista]
Maraldi, Tullia; Angeloni, Cristina; Giannoni, Elisa; Sell, Christian

tem cells are defined by their unique ability to self-renew and their multipotent differentiation capacity, thus maintaining tissue homeostasis throughout the life of a multicellular organism. Stem cells reside in niches characterized by hypoxia and low reactive oxygen species (ROS), both of which are critical for maintaining the potential for self-renewal and stemness. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, that is, regulation of the cell cycle. In fact, ROS at low levels function as signaling molecules to mediate cell proliferation, migration, and differentiation and gene expression. ROS levels in stem and progenitor cells have a clear correlation with cellular functions and are regulated by a fine-tuning of the balance between ROS-generating and antioxidant defense systems. This special issue tries to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, which is critical to address the important role of redox homeostasis in the regulation of both self-renewal and differentiation of stem cells.

2015 - Role of hepatocyte growth factor in the immunomodulation potential of amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Beretti, Francesca; Guida, Marianna; Zavatti, Manuela; De Pol, Anto

Human amniotic fluid stem cells (hAFSCs) may be useful for regenerative medicine because of their potential to differentiate into all three germ layers and to modulate immune response with different types of secretion molecules. This last issue has not been completely elucidated. The aim of this study was to investigate the secretome profile of the hAFSC, focusing on the role of hepatocyte growth factor (HGF) in immunoregulation through short and long cocultures with human peripheral blood mononuclear cells. We found that HGF produced by hAFSCs exerts a cytoprotective role, inducing an increase in caspase-dependent apoptosis in human immune cells. This study provides evidence supporting the hypothesis that amniotic fluid is an ideal source of stem cells for expansion and banking properties for therapeutic use. hAFSCs not only are less immunogenic but also can secrete immunoregulatory factors that may be useful in autoimmune diseases or allogenic implants. SIGNIFICANCE: New information about the secretome pattern is reported in this paper. Human amniotic fluid stem cells (hAFSCs) possess immunomodulatory properties involving hepatocyte growth factor production. hAFSCs could be used in immunotherapies and might be able to avoid allogenic rejection

2014 - Dietary Polyphenols and Their Effects on Cell Biochemistry and Pathophysiology 2013 [Articolo su rivista]
Maraldi, Tullia; Vauzour, D.; Angeloni, C.

Dietary polyphenols, along with other natural compounds occurring in fruits and vegetables, have been reported to exert beneficial effects in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. Many of the biological actions of polyphenols have been attributed to their antioxidant properties; however, during the last years, a new realization of how nutritional antioxidants may function has been envisaged, and recent findings have suggested that they affect several cellular pathways exerting a pleiotropic effect. This special issue analyzes and expands our knowledge on the new mechanisms of actions of polyphenols and other natural compounds with the aim to better understand what could be defined as “the network” of their different biological effects.

2014 - Human amniotic fluid stem cells: neural differentiation in vitro and in vivo [Articolo su rivista]
Maraldi, Tullia; Bertoni, Laura; Riccio, Massimo; Zavatti, Manuela; Carnevale, Gianluca; Resca, Elisa; Guida, Marianna; Beretti, Francesca; LA SALA, Giovanni Battista; DE POL, Anto

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, β-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

2014 - Nuclear Nox4-derived reactive oxygen species in myelodysplastic syndromes [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Beretti, Francesca; Follo, Matilde Y; Manzoli, Lucia; DE POL, Anto

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML.

2014 - Redox signaling in degenerative diseases: from molecular mechanisms to health implications [Articolo su rivista]
Angeloni, Cristina; Maraldi, Tullia; Vauzour, David

Maintenance of normal intracellular redox status plays an important role in regulating many physiological processes. The cellular oxidation and reduction environment is influenced by the production and removal of reactive oxygen species (ROS). Unbalanced levels of ROS are a common characteristic of many acute and chronic degenerative diseases such as cancer, cardiovascular diseases, type II diabetes, acute liver and renal failure, and neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases and strokes. On the other hand, in the last years it has been shown that not only are ROS detrimental to cells but at physiological level they regulate a myriad of cellular processes including transcription regulation and cell signaling. Several reports support the hypothesis that cellular ROS levels could function as “second messengers.” The second messenger properties of ROS are believed to activate signaling pathways by regulating kinases, phosphatases, transcription factors, or ion channels to coordinate the final response of the cell. Understanding the crosstalk between signaling, ROS, and cell homeostasis is fundamental for understanding redox biology and disease pathogenesis.

2013 - Enrichment in c-Kit(+) enhances mesodermal and neural differentiation of human chorionic placental cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; DE BIASI, Sara; Pisciotta, Alessandra; A., Nicoli; LA SALA, Giovanni Battista; P. V., Guillot; A. L., David; N. J., Sebire; P. D., Coppi; DE POL, Anto

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

2013 - Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells. [Articolo su rivista]
Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; Maraldi, Tullia; Guida, Marianna; Carnevale, Gianluca; Ferrari, Adriano; DE POL, Anto

The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs).AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17β-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed.MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations.The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

2013 - Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Pisciotta, Alessandra; Zavatti, Manuela; Carnevale, Gianluca; Beretti, Francesca; LA SALA, Giovanni Battista; A., Motta; DE POL, Anto

INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells (DPSC) and amniotic fluid stem cells (AFSC), combined with collagen scaffold, to reconstruct critical size cranial bone defects in animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all the samples but the most relevant difference in defect correction were shown by stem cell-collagen samples at 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSIONS: All these data confirmed the strong potential of bioengineered constructs of stem cell-collagen scaffold for correcting large cranial defects in animal model and highlighting the role of stem cells in neo vascularization during skeletal defect reconstruction.

2013 - In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp. [Articolo su rivista]
Carnevale, Gianluca; Riccio, Massimo; Pisciotta, Alessandra; Beretti, Francesca; Maraldi, Tullia; Zavatti, Manuela; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Ferrari, Adriano; DE POL, Anto

AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

2013 - Inhibition of nuclear nox4 activity by plumbagin: effect on proliferative capacity in human amniotic stem cells. [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Zavatti, Manuela; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto

Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage.

2013 - Natural compounds as modulators of NADPH oxidases. [Articolo su rivista]
Maraldi, Tullia

Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols.

2013 - Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis. [Articolo su rivista]
Angeloni, C; Turroni, S; Bianchi, L; Fabbri, D; Motori, E; Malaguti, M; Leoncini, E; Maraldi, Tullia; Bini, L; Brigidi, P; Hrelia, S.

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases

2012 - Dietary Polyphenols and Their Effects on Cell Biochemistry and Pathophysiology [Articolo su rivista]
C., Angeloni; L., Pirola; D., Vauzour; Maraldi, Tullia

Polyphenols, occurring in fruit and vegetables, wine, tea, extra virgin olive oil, chocolate, and other cocoa products, have been demonstrated to exert beneficial effects in a large array of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. Many of the biological effects of polyphenols have been attributed to their antioxidant properties, either through their reducing capacities per se or through their possible influences on intracellular redox status. As such, polyphenols may protect cell constituents against oxidative damage and have been reported to limit the risk of various degenerative diseases associated with oxidative stress, including cardiovascular diseases, type 2 diabetes, and cancer. However, accumulating evidence suggests that the classical hydrogen-donating antioxidant activity is unlikely to be the sole explanation for their cellular effects in vivo. Indeed, it has recently become clear that, in complex biological systems, polyphenols are able to exhibit several additional properties which are yet poorly understood. It is evident that polyphenols are potent bioactive molecules and a clear understanding of their precise mechanisms of action as either antioxidants or modulators of cell signaling is crucial to the evaluation of their potential as chemopreventive or anticancer agents and inhibitors of neurodegeneration. This special issue comprises 14 original research articles that further expand our understanding of the biological functions of polyphenols from different sources and 9 review articles that summarize the current knowledge on the beneficial effects of polyphenols on health.

2012 - Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells. [Articolo su rivista]
Riccio, Massimo; Maraldi, Tullia; Pisciotta, Alessandra; LA SALA, Giovanni Battista; Ferrari, Adriano; G., Bruzzesi; A., Motta; C., Migliaresi; DE POL, Anto

The main aim of this study was the comparative evaluation of fibroin scaffolds combined with human stem cells, such as dental pulp stem cells (hDPSCs) and amniotic fluid stem cells (hAFSCs), used to repair critical-size cranial bone defects in immunocompromised rats. Two symmetric full-thickness cranial defects on each parietal region of rats have been replenished with silk fibroin scaffolds with or without preseeded stem cells addressed toward osteogenic lineage in vitro. Animals were euthanized after 4 weeks postoperatively and cranial tissue samples were taken for histological analysis. The presence of human cells in the new-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Fibroin scaffolds induced mature bone formation and defect correction, with higher bone amount produced by hAFSC-seeded scaffolds. Our findings demonstrated the strong potential of stem cells/fibroin bioengineered constructs for correcting large cranial defects in animal model and is likely a promising approach for the reconstruction of human large skeletal defects in craniofacial surgery.

2012 - Human serum promotes osteogenic differentiation of Human Dental Pulp Stem Cells in vitro and in vivo. [Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

2011 - Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Resca, Elisa; Pisciotta, Alessandra; LA SALA, Giovanni Battista; Ferrari, Adriano; Bruzzesi, G; Motta, A; Migliaresi, C; Marzona, Laura; DE POL, Anto

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.

2011 - Low levels of selenium compounds are selectively toxic for a human neuron cell line through ROS/RNS increase and apoptotic process activation [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; L., Zambonin; Vinceti, Marco; DE POL, Anto; G., Hakimb

Organic and inorganic selenium compounds were used to examinewhether low selenium concentrationis able to trigger apoptotic degeneration in a human neuron cell line in vitro and to explore changes inreactive oxygen and nitrogen species and antioxidant protein content during the apoptotic processes.The results indicated that: (1) SKNBE neuroblastoma cells treated with sodium selenite, sodium selenateand seleno-methionine (0.1, 0.5 and 0.5 mM, respectively) for 24 h exhibited a viability decrease, unlikekidney or prostatic cells; (2) the PARP (poly-ADP-ribose-polymerase) degradation and caspase activationdetected by Western blot and flow cytometry fluorimetric examination showed induction of apoptosis;(3) during selenium treatment, a ROS/RNS increase occurred despite the GSH increment, as revealed byfluorimetric analysis; (4) the RNS production could be blocked by a peroxynitrite scavenger; (5) afterexposure to selenium compounds, the concentration of nitric oxide synthase, manganese superoxidedismutase (SOD2), P-NF-kB (phospho nuclear factor kB), glutathione reductase and glutathioneperoxidase increased, whereas that of P-ERK (phospho extracellular signal-regulated kinase) decreased;(6) selenium presence induced copper/zinc superoxide dismutase (SOD1) translocation into mitochondria,in a way similar to what is observed in amyotrophic lateral sclerosis (ALS). This study supportsepidemiologic studies showing the possibility that excess environmental exposure to Se represents a riskfactor for a devastating human neurodegenerative disease.

2011 - Reverse-phase protein microarrays (RPPA) as a diagnosticand therapeutic guide in multidrug resistant leukemia [Articolo su rivista]
Maraldi, Tullia; Bertacchini, Jessika; Benincasa, Marta; M., Guida; DE POL, Anto; L., Liotta LA; E., Petricoin; L., Cocco; Marmiroli, Sandra

Abstract. Reverse-phase microarray assays using phosphospecificantibodies (RPPA) can directly measure levels ofphosphorylated protein isoforms. In the current study, lysatesfrom parental and multidrug resistant (MDR) CEM leukemiacells were spotted onto reverse-phase protein microarraysand probed with a panel of phospho-antibodies to ERK, PCKand Akt pathways. In particular, the Akt pathway is consideredto play significant roles in leukemia and Akt inhibitor therapyhas been proposed as a potential tool in the treatment of thisdisease. The RPPA data prompted us to investigate deeperthis pathway. Here, we found that whereas total Akt1 proteinlevel is higher in parental CEM cells, the activated isoformcontent, p-Akt1, increases in doxorubicin-selected CEM cells(MDR-CEM). This was backed up by Western blot analysis,confirming that Akt1 activity/phosphorylation may be upregulatedin MDR-CEM cells. Further exploration of inhibitorytherapy in this system was evaluated. The TNF-relatedapoptosis-inducing ligand, TRAIL, has been shown toselectively kill tumor cells. Herein, we describe that in MDRCEMcells TRAIL responsiveness correlates with a reducedexpression of endogenous Akt1, suggesting that the MDRphenotype associated to P-gp sensitizes cells to TRAIL therapy.

2010 - Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures [Articolo su rivista]
Riccio, Massimo; Resca, Elisa; Maraldi, Tullia; Pisciotta, Alessandra; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, MatrigelTM and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in MatrigelTM DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

2010 - VEGF-induced ROS generation from NAD(P)H oxidases protects human leukemic cells from apoptosis [Articolo su rivista]
Maraldi, Tullia; Prata, C; Caliceti, C; Vieceli Dalla Sega, F; Zambonin, L; Fiorentini, D; Hakim, G.

Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular pathophysiology and in hematological malignancies. VEGF is supposed to utilize ROS as messenger intermediates downstream of the VEGF receptor-2. NAD(P)H oxidase (Nox) family is a major source of cellular ROS and is implicated in increased ROS production in tumor cells. We previously demonstrated that B1647 cells, a human leukemic cell line, express Nox2 and Nox4, both at mRNA and protein level. We suggest here that the VEGF-induced increase in ROS can be related to Nox2 and Nox4 activities. Nox-derived ROS are involved in early signaling events such as the autophosphorylation of VEGF receptor-2, and in the modulation of glucose uptake, a cellular activity strictly bound to VEGF-induced leukemic cell proliferation, as shown by experiments with antioxidants and Nox inhibitors and siRNA. Nox-generated ROS are required to sustain B1647 cell viability and proliferation; in fact, antioxidants such as EUK-134 or Nox inhibitors and siRNA direct cells to apoptotic cell death, suggesting that manipulation of cellular Nox2 and Nox4 could affect survival of leukemic cells.

2009 - Induction of apoptosis in a human leukemic cell line via reactive oxygen species modulation by antioxidants [Articolo su rivista]
Maraldi, Tullia; Prata, C; Fiorentini, D; Zambonin, L; Landi, L; Hakim, G.

In the human acute myeloid leukemia cell line M07e, the growth factor interleukin-3 (IL-3) induces ROS formation, positively affecting Glut1-mediated glucose uptake and cell survival. The effect of IL-3 and exogenous hydrogen peroxide on cell viability seems to be mediated through inhibition of the cell death commitment, as shown by apoptotic markers such as caspase activities, apoptotic nuclei, and changes in the amount of proteins belonging to the Bcl-2 family. The pivotal role of ROS is confirmed using various antioxidants, such as EUK-134, ebselen, TEMPO, and hydroxylamine probe. In fact, these antioxidants, acting through different mechanisms, decrease glucose transport activity and cell proliferation activated by IL-3 or by low concentrations of hydrogen peroxide. Moreover, antioxidants foster programmed cell death commitment, as shown by the cited apoptotic parameters. EUK-134, a combined superoxide dismutase/catalase mimetic, opposes the effects of IL-3 and H(2)O(2), decreasing phosphorylation levels of signaling enzymes such as Akt, Src tyrosine kinase, and ERK. Results show that ROS production induced by IL-3 can protect leukemic cells from apoptosis, the effect being counteracted by antioxidants. This mechanism may play an important role in supporting acute myeloid leukemia treatment, thus representing a novel therapeutic strategy.

2009 - MATER protein as substrate of PKCepsilon in human cumulus cells. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Sena, Paola; Marzona, Laura; A., Nicoli; Marca, A. L.; Marmiroli, Sandra; Bertacchini, Jessika; LA SALA, Giovanni Battista; DE POL, Anto

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

2009 - Multipotent stem cells in vitro differentiation on 3-D scaffolds. [Abstract in Rivista]
Palumbo, Carla; Riccio, M.; Resca, E.; Maraldi, Tullia; Bertoni, Laura; Sena, Paola; DE POL, Anto

Goal of the study is to obtain cell/scaffold complexes to use in regenerative medicine.

2009 - NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells [Articolo su rivista]
Maraldi, Tullia; Prata, C; Vieceli Dalla Sega, F; Caliceti, C; Zambonin, L; Fiorentini, D; Hakim, G.

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.

Resca, Elisa; Riccio, Massimo; Bertoni, Laura; Maraldi, Tullia; Palumbo, Carla; DE POL, Anto

The investigation concerns the selection of mesenchymal stem cells, derived from adult human dental pulp (DPSC), to commit towards osteogenic differentiation in order to perform new strategies for the regenerative medicine. The final aim of this study is to use DPSC in regenerative medicine approaches for recovering wide gaps of bone tissue due to post-traumatic locomotor apparatus damages.

2009 - Risk of chronic low-dose selenium overexposure in humans: insights from epidemiology and biochemistry. [Articolo su rivista]
Vinceti, Marco; Maraldi, Tullia; Bergomi, Margherita; Malagoli, Carlotta

The latest developments of epidemiologic and biochemical research suggest that current upper limits of intake for dietary selenium and for overall selenium exposure may be inadequate to protect human health. In particular, recent experimental and observational prospective studies indicate a diabetogenic effect of selenium at unexpectedly low levels of intake. Experimental evidence from laboratory studies and veterinary medicine appears to confirm previous epidemiologic observations that selenium overexposure is associated with an increased risk of amyotrophic lateral sclerosis, and a recent large trial indicated no beneficial effect in preventing prostate cancer. Moreover, the pro-oxidant properties of selenium species and the observation that the selenium-containing enzymes glutathione peroxidases are induced by oxidative stress imply that the increase in enzymatic activity induced by this metalloid may represent at least in part a compensatory response. Taken together, the data indicate that the upper safe limit of organic and inorganic selenium intake in humans may be lower than has been thought and that low-dose chronic overexposure to selenium may be considerably more widespread than supposed.

2008 - Effect of radical stress and ageing on the occurrence of trans fatty acids in rats fed a trans-free diet [Articolo su rivista]
Laura, Zambonin; Cecilia, Prata; Luciana, Cabrini; Maraldi, Tullia; Diana, Florentini; Francesco Vieceli Dalla, Sega; Gabriele, Hakim; Laura, Landi

In a previous paper, we demonstrated that tissue trans fatty acids can not only derive from the diet but also be endogenously formed. The central focus of this study was to prove that the in vivo isomerization occurs via a radical process. Two different models of radical insult were used: CCl4 and AAPH injection to rats fed a diet completely free of trans isomers. Following this acute radical stress, a significant increase in unnatural trans fatty acid content of erythrocyte, kidney, and heart, but not liver, was observed. These results can be mainly explained by the high content, particularly in the liver, of antioxidant vitamins A and E that exhibit also an ``anti-isomerizing{''} effect. Since during ageing cellular components are exposed to increasing radical insults, the observation of a significant trans fatty acid accumulation in 30-month-old rats could confirm that the in vivo formation of unnatural isomers is due to a radical process. Trans fatty acids can influence the physical characteristics of bilayer microdomains, affecting membrane properties and functions; thus, knowledge of biological radical species responsible for cis/trans isomerization and their possible sources can provide protective systems for preserving lipid geometry.

2008 - Nox-generated ROS modulate glucose uptake in a leukaemic cell line [Articolo su rivista]
Cecilia, Prata; Maraldi, Tullia; Diana, Fiorentini; Laura, Zambonin; Gabriele, Hakim; Laura, Landi

The discovery of superoxide-generating enzymes homologues of phagocytic NAD(P)H oxidase, the Nox family, has led to the concept that reactive oxygen species (ROS) are `intentionally' generated with biological functions in various cell types. In this study, by treating an acute leukaemic cell line with different antioxidants, ROS generation was shown to be crucially involved in the modulation of glucose transport (mediated by Glut 1), which is frequently up-regulated in cancer cells. Then, this study tried to elucidate ROS source(s) and mechanisms by which ROS are involved in Glut1 activity regulation. Results prove that Nox2 and Nox4 are the candidates and that phosphorylation processes are important in the regulation of glucose uptake on which cancer cells rely. On the whole, data suggest that both Glut1 and Nox homologues may be considered new potential targets in the treatment of leukaemia.

2008 - The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect [Articolo su rivista]
C., Jane Hanson; Martin D., Bootman; Clark W., Distelhorst; Maraldi, Tullia; H., Llewelyn Roderick

Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2. (C) 2007 Elsevier Ltd. All rights reserved.

2007 - Glucose-transport regulation in leukemic cells: how can H2O2 mimic stem cell factor effects? [Articolo su rivista]
Maraldi, Tullia; Diana, Fiorentini; Cecilia, Prata; Laura, Landi; Gabriele, Hakim

In leukemic cells, glucose transport is activated by SCF and H2O2 through a common signal cascade involving Akt, PLC gamma, Syk, and the Src family, in this order. An explanation can be provided by the phosphorylation of c-kit, the SCF receptor, elicited by either SCF or H2O2. Moreover, antioxidants prevent the SCF effect on glucose transport, confirming the involvement of H2O2 in the pathway leading to glucose-transport activation and suggesting a potential role for reactive oxygen species in leukemia proliferation.

2007 - Green tea modulates alpha(1)-Adrenergic stimulated glucose transport in cultured rat cardiomyocytes [Articolo su rivista]
Cristina, Angeloni; Maraldi, Tullia; Anna, Ghelli; Michela, Rugolo; Emanuela, Leoncini; Gabriele, Hakim; Silvana, Hrelia

alpha(1)-Adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha(1)-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of P and 6 isoforms, but not alpha- and epsilon, supporting the notion that GTE directly affects PKC activation and is a and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/ counteraction of these diseases.

2007 - Mild exercise training, cardioprotection and stress genes profile [Articolo su rivista]
Marina, Marini; Rosa, Lapalombella; Vittoria, Margonato; Raffaella, Ronchi; Michele, Samaja; Cristina, Scapin; Luisa, Gorza; Maraldi, Tullia; Paolo, Carinci; Carlo, Ventura; Arsenio, Veicsteinas

To improve current knowledge of the molecular mechanisms underlying exercise-induced cardioprotection in a rat model of mild exercise training, Sprague-Dawley rats were trained to run on a treadmill up to 55\% of their maximal oxygen uptake for 1 h/day, 3 days/week, 14 weeks, with age-matched sedentary controls (n = 20/group). Rats were sacrificed 48 h after the last training session. Despite lack of cardiac hypertrophy, training decreased blood hemoglobin (7.94 +/- 0.21 mM vs. 8.78 +/- 0.23 mM, mean +/- SE, P = 0.01) and increased both plasma malondialdehyde (0.139 +/- 0.005 mM vs. 0.085 +/- 0.009 mM, P = 0.05) and the activity of Mn-superoxide dismutase (11.6 +/- 0.6 vs. 16.5 +/- 1.6 mU/mu g, P = 0.01), whereas total superoxide dismutase activity was unaffected. When subjected to 30-min ischemia followed by 90-min reperfusion, hearts from trained rats (n = 5) displayed reduced infarct size as compared to controls (37.26 +/- 0.92\% vs. 49.09 +/- 2.11\% of risk area, P = 0.04). The biochemical analyses in the myocardium, which included gene expression profiles, real-time PCR, Western blot and determination of enzymatic activity, showed training-induced upregulation of the following mRNAs and/or proteins: growth-arrest and DNA-damage induced 153 (GADD153/CHOP), heme-oxygenase-1 (HO-1), cyclooxygenase-2 (Cox-2), heat-shock protein 70/72 (HSP70/72), whereas heat-shock protein 60 (HSP60) and glucose-regulated protein 75 (GRP75) were decreased. As a whole, these data indicate that mild exercise training activates a second window of myocardial protection against ischemia/reperfusion by upregulating a number of protective genes, thereby warranting further investigation in man.

2007 - Signal processes and ROS production in glucose transport regulation by thrombopoietin and granulocyte macrophage-colony stimulation factor in a human leukaemic cell line [Articolo su rivista]
Maraldi, Tullia; Cecilia, Prata; Diana, Fiorentini; Laura, Zambonin; Laura, Landi; Gabriele, Hakim

In M07e cells, a human megakaryocytic leukaemia line, reactive oxygen species (ROS) are generated in response to cytokines acting as intracellular messengers to modulate glucose transport. The aim of this work was to study the signal cascade involved in the acute glucose transport activation in cells exposed to growth factors, such as granulocyte macrophage-colony stimulation factor (GM-CSF) and thrombopoietin (TPO), to better understand some aspects of the aberrant proliferation in leukaemia. Results confirm ROS involvement in modulation of glucose transport in this cell line. Furthermore, GM-CSF and TPO produced changes in Glut1 phosphorylation and specific inhibitors employed to identify protein kinases involved in Glut activation by these cytokines proved that Akt, PLC gamma, Syk and the Src family take part in signal transduction leading to Glut1 activation.

2006 - Are 4-thiaflavans "double-faced" antioxidants in model membranes and cells? [Articolo su rivista]
L., Zambonin; R., Amorati; C., Prata; Maraldi, Tullia; D., Fiorentini; G., Hakim; L., Landi


Maraldi, Tullia; Rugolo, M.; Fiorentini, D.; Landi, L.; Hakim, G.

The aim of this work was to investigate the role of cytosolic calcium and calmodulin-dependent systems in the activation of glucose uptake in the human megakaryocytic cell line M07e. Glucose uptake was significantly raised by elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) with thapsigargin, this effect being additive to the activation induced by cytokines (SCF, GM-CSF and TPO) and hydrogen peroxide. Intracellular Ca(2+) chelation by BAPTA decreased basal and activated glucose uptake in a dose-dependent manner. BAPTA reduced the GLUT1 translocation induced by SCF and H(2)O(2), suggesting a major role for Ca(2+) in GLUT1 intracellular trafficking. In the absence of extracellular Ca(2+), 2-aminoethoxydiphenyl-borate (2-APB) abolished the activation of glucose uptake induced by cytokines and H(2)O(2) suggesting an involvement in GLUT1 regulation in responses related to InsP(3)-induced Ca(2+) release. Under our experimental conditions, all the stimuli inducing glucose uptake activation failed to increase [Ca(2+)](c) suggesting that cytosolic Ca(2+) plays a permissive role in the regulation of GLUT1. The calmodulin antagonist W-7 and the inhibitor of Ca(2+)-calmodulin dependent protein kinase II (CAMK II) KN-62 removed the glucose transport activation by all the tested stimuli. These results suggest that in M07e cells calmodulin and CAMKII are involved in GLUT1 stimulation by cytokines and ROS.

2006 - Is Nox the source of ROS involved in Glut1 activity in B1647 cells? [Relazione in Atti di Convegno]
Prata, C.; Maraldi, Tullia; Fiorentini, D.; Zambonin, L.; Hakim, G.; Landi, L.


2006 - Regulation of ROS-induced Ca2+ signalling by Bcl-2 [Articolo su rivista]
Maraldi, Tullia; D., Fiorentini; C., Prata; L., Zambonin; G., Hakim; L., Landi; M. D., Bootman


2005 - Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms [Articolo su rivista]
G., Agnetti; Maraldi, Tullia; D., Fiorentini; E., Giordano; C., Prata; G., Hakim; C., Muscari; C., Guarnieri; Cm, Caldarera

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-D-{[}2,6-H-3]-glucose uptake was increased (57\% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19\%, p < 0.001) followed also treatment with Go6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31\%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process. (c) 2005 Elsevier Inc. All rights reserved.

2005 - Induction of apoptosis in a human leukaemic cell line by a combined superoxide dismutase/catalase mimetic [Articolo su rivista]
Maraldi, Tullia; Prata, C.; Fiorentini, D.; Landi, L.; Hakim, G.


2005 - NAD(P)H oxidase as a potential source of ROS involved in the regulation of glucose uptake in a human leukaemic cell line (B1647) [Articolo su rivista]
Prata, C.; Fiorentini, D.; Maraldi, Tullia; Zambonin, L.; Hakim, G.; Landi, L.


2004 - Acute regulation of the facilitative glucose transporter GLUT1 in a hematopoietic cell line [Articolo su rivista]
Hakim, G; Fiorentini, D; Maraldi, Tullia; Landi, L.

This brief review is focused on the short-term regulation of the facilitative glucose transporter GLUT1 in megakaryocytic cells M07e. The effects of cytokines such as TPO, GM-CSF and SCF and of a low dose of H202 on the transport activity and its kinetic parameters are compared. The possible mechanisms and the signalling pathways involved in the glucose uptake activation are discussed. A role for the cellular redox status in glucose uptake control, possibly related to the status of redox-sensitive enzymes such as tyrosine phosphatases, is suggested.

2004 - Contribution of reactive oxygen species to the regulation of Glut1 in two hemopoietic cell lines differing in cytokine sensitivity [Articolo su rivista]
D., Fiorentini; C., Prata; Maraldi, Tullia; L., Zambonin; L., Bonsi; G., Hakim; L., Landi

Glucose transport activity and its possible regulation by reactive oxygen species in two Glut1-expressing megakaryocytic cell lines, MO7e and B1647, differing in cytokine sensitivity were compared. Results show that: (1) In MO7e, cells, glucose transport rate increased in response to thrombopoietin, granulocyte-macrophage colony-stimulating factor, or stem cell factor, due to a decreased K-m. (2) A higher V-max value was determined in B1647 cells, owing to the relative higher abundance of Glut1 on the plasmalemma; in these cells no change in glucose transport rate was observed on cytokine treatment. (3) The basal level of intracellular ROS was higher in B1647 than in M07e cells, where ROS production was enhanced on cytokine exposure. (4) Basal or stimulated ROS production and Glut1 activity were significantly reduced by pretreating both cell lines with EUK-134, a superoxide dismutase and catalase mimetic. (5) In M07e cells, EUK-134 brought back to control levels the K-m values obtained on cytokine treatment, whereas in B 1647 cells the antioxidant drastically reduced V-max by decreasing the Glut1 content of the plasma membrane. Our data suggest that differences in acute regulation of glucose transport activity in the two cell lines may be related to differences in amplitude and spatial organization of ROS production. (C) 2004 Elsevier Inc. All rights reserved.

2004 - Effect of different ROS scavengers on glucose transport activity of a human megakaryocytic cell line [Articolo su rivista]
Prata, C.; Fiorentini, D.; Maraldi, Tullia; Hakim, G.; Landi, L.


2004 - Green tea modulates a-1-adrenergic stimulated glucose transport in cultured cardiomyocytes via protein kinase C beta and delta isoforms [Articolo su rivista]
Angeloni, C.; Maraldi, Tullia; Ghelli, A.; Rugolo, M.; Bordoni, A.; Biagi, P. L.; Hakim, G.; Hrelia, S.


2004 - Plasma membrane electron transport (tPMET) in two human megakaryocytic cell lines. [Abstract in Atti di Convegno]
Prata, C.; Maraldi, Tullia; Fiorentini, D.; Zambonin, L.; Hakim, G.; Landi, L.


2004 - Reactive oxygen species as mediators of the activation of glucose transport in M07e cells: role of intracellular calcium [Abstract in Atti di Convegno]
Maraldi, Tullia; Fiorentini, D.; Prata, C.; Landi, L.; Hakim, G.


2004 - ROS production and Glut1 activity in two human megakaryocytic cell lines [Articolo su rivista]
C., Prata; Maraldi, Tullia; L., Zambonin; D., Fiorentini; G., Hakim; L., Landi

Reactive oxygen species (ROS) has been increasingly recognised as intracellular messengers in signal transduction following receptor activation by a variety of bioactive peptides including growth factors, cytokines and hormones. In this study ROS production and glucose transport activity were evaluated in the growth factor dependent M07e cells and in B1647 cells. not requiring additional hematopoietic cytokines for growth: the aim was to investigate whether ROS could be involved in the regulation of Glut1-mediated glucose uptake in both cell lines. The effect of the synthetic superoxide and hydrogen peroxide scavenger EUK-134 on DOG uptake activity and intracellular ROS formation supports the concept of reactive oxygen species as signalling molecules. In order to investigate ROS generation sources, diphenyleneiodonium, an inhibitor of flavoprotein centres and apocynin, an inhibitor of NAD(P)H oxidase, were used: they inhibit both ROS production and glucose uptake activation. All these data support the hypothesis that ROS can contribute to the regulation of glucose transport, not only in M07e cells but also in B1647 cells; we could speculate that one possible source of ROS, linked somehow with Glut I activity, can be a NAD(P)H oxidase similar to that one present in phagocytic cells.

2004 - Stem Cell Factor and H2O2 induce GLUT1 translocation in M07e cells [Articolo su rivista]
Maraldi, Tullia; D., Fiorentini; C., Prata; L., Landi; G., Hakim

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase V-max for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.

2003 - Enhancement of glucose transport in rat thymocytes by different radical sources [Articolo su rivista]
G., Hakim; D., Fiorentini; Maraldi, Tullia; L., Landi

This study demonstrates that oxidative stress induced in rat thymocytes by the hydrophilic 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), the lipophilic cumene hydroperoxide (CumOOH) and the freely diffusible H2O2 is associated with an activation of facilitative glucose transport rate, mediated by GLUT1, the major transporter in this cell type. We compared the effects of the three tested radical sources on the kinetic transport parameters, showing that the transport rate enhancement in the treated cells can be ascribed to an increase in the V-max value, apart from the site of generation of the oxidative stress. The enhancement of glucose transport by the three oxidants in thymocytes was significantly attenuated both by protein tyrosine kinase inhibitors as genistein and tyrphostin A23 and by U73122, a phospholipase C inhibitor. Genistein and U73122 reversed also the cited increase of V-max values. It is concluded that the stimulation of glucose transport in response to different oxidants is mediated, at least in part, through reactive oxygen species (ROS)-induced stimulation of protein tyrosine kinase and phospholipase C pathways.

2002 - Doxorubicin induces early lipid peroxidation associated with changes in glucose transport in cultured cardiomyocytes [Articolo su rivista]
S., Hrelia; D., Fiorentini; Maraldi, Tullia; C., Angeloni; A., Bordoni; Pl, Biagi; G., Hakim

Doxorubicin (DOX) has not only chronic, but also acute toxic effects in the heart, ascribed to the generation of reactive oxygen species (ROS). Focusing on the DOX-induced early biochemical changes in rat cardiomyocytes, we demonstrated that lipid peroxidation is an early event, in fact conjugated diene production increased after 1-h DOX exposure, while cell damage, evaluated as lactate dehydrogenase (LDH) release, was observed only later, when at least one third of the cell antioxidant defences were consumed. Cell pre-treatment with alpha-tocopherol (TC) inhibited both conjugated diene production and LDH release. In cardiomyocytes, DOX treatment caused a maximal increase in glucose uptake at 1 h, demonstrating that glucose transport may represent an early target for DOX. At longer times, as the cell damage become significant, the glucose uptake stimulation diminished. Immunoblotting of glucose transporter isoform GLUT I in membranes after 1-h DOX exposure revealed an increase in GLUT1 amount similar to the increase in transport activity; both effects were inhibited by alphaTC. Early lipid peroxidation evokes an adaptive response resulting in an increased glucose uptake, presumably to restore cellular energy. The regulation of nutrient transport mechanisms in cardiomyocytes may be considered an early event in the development of the cardiotoxic effects of the anthracycline.

2001 - Acute regulation of glucose transport in a human megakaryocytic cell line: Difference between growth factors and H2O2 [Articolo su rivista]
D., Fiorentini; G., Hakim; L., Bonsi; Gp, Bagnara; Maraldi, Tullia; L., Landi

The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H2O2 on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H2O2; (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H2O2: cytokines increased predominantly the affinity for glucose, while H2O2 raised both the V-max and K-m values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H2O2-induced stimulation of hexose transport, reversing the variations of K-m and V-max but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H2O2 on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor. (C) 2001 Elsevier Science Inc.