Gabriella Teti

Neuroprotective effects of cyanidin 3-O-glucopyranoside on amyloid beta (25-35) oligomer-induced toxicity.

Recent studies suggest that the oligomers of short amyloid beta (Abeta) peptides such as Abeta(25-35) as well as full-length Abeta peptides (i.e. Abeta(1-40) and Abeta(1-42) peptides) are responsible for synaptic dysfunction and/or neuronal loss in Alzheimers disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, cyanidin 3-O-glucoside (Cy-3G) has recently gained attention for its neuroprotective properties. In this in vitro study, we demonstrated that Cy-3G can inhibit Abeta(25-35) spontaneous aggregation into oligomers and their neurotoxicity in human neuronal SH-SY5Y cells. In particular, the pre- and co-treatment of SH-SY5Y cells with Cy-3G reduced the neuronal death, in terms of apoptosis and necrosis, elicited by Abeta(25-35) oligomers. Cy-3G also shows the interesting ability to prevent the early events leading to neuronal death such as the Abeta(25-35) oligomer binding to plasma membrane and the subsequent membrane integrity loss. Taken together, these findings suggest that Cy-3G may be considered a phytochemical with neuroprotective properties useful in finding potential drug or food supplements for the therapy of AD.

Involvement of nuclear PLCβ1 in lamin B1 phosphorylation and G2/M cell cycle progression

Inositide‐specific phospholipase Cβ1 (PLCβ1) signaling in cell proliferation has been investigated thoroughly in the G1 cell cycle phase. However, little is known about its involvement in G2/M progression. We used murine erythroleukemia cells to investigate the role of PLCβ1 in G2/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen‐activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G2/M progression. We report that PLCβ1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si‐RNA mediated silencing showed a role of JNK, PKCα, PKCβI, and the β1 isoform of PI‐PLC in cell accumulation in G2/M [as observed by fluorescence‐activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCα,orPLCβ1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCβ1, and, foremost, the finding that the PLCβ1‐mediated pathway represents a functional nuclear inositide signaling in the G2/M transition.— Fiume, R., Ramazzotti, G., Teti, G., Chiarini, F., Faenza, I., Mazzotti, G., Billi, A. M., Cocco, L., Involvement of nuclear PLCβl in lamin B1 phosphorylation and G2/M cell cycle progression. FASEB J. 23, 957–966 (2009)

Sulforaphane treatment protects skeletal muscle against damage induced by exhaustive exercise in rats

Sulforaphane (SF), one of the most important isothiocyanates in the human diet, present in cruciferous vegetables, is known to have chemopreventive activities in different tissues. No data are available on its effects in the prevention of skeletal muscle damage. In this study, we investigated the potential protective effects of SF treatment on muscle damage and oxidative stress induced by an acute bout of exhaustive exercise in rats. Male Wistar rats were treated with SF (25 mg/kg body wt ip) for 3 days before undergoing an acute exhaustive exercise protocol in a treadmill (+7% slope and 24 m/min). Acute exercise resulted in a significant increase in plasma lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities. It also resulted in a significant increase in thiobarbituric acid-reactive substances, in a significant decrease in tissue total antioxidant capacity, and in a significant decrease in NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in vastus lateralis muscle. SF treatment significantly increased muscle NQO1, glutathione-S-transferase, and glutathione reductase expression and activity, with no effect on glutathione peroxidase and thioredoxin reductase. The observed SF-induced upregulation of phase II enzymes was accompanied by a significant increase in nuclear erythroid 2 p45-related factor 2 expression and correlated with a significant increase in total antioxidant capacity and a decrease in plasma LDH and CPK activities. Our data demonstrate that SF acts as an indirect antioxidant in skeletal muscle and could play a critical role in the modulation of the muscle redox environment, leading to the prevention of exhaustive exercise-induced muscle damage.

Effects of HEMA on type I collagen protein in human gingival fibroblasts

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations on specific proteins such as type I collagen has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen protein. Primary lines of human gingival fibroblasts were exposed to 3 mmol/L HEMA for different periods of time (24 h, 72 h, 96 h). The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. The presence and localization of type I collagen was determined by immunofluorescence in HGFs treated with HEMA for the same period of time. The vitality of the cells decreased after 72 h of exposure. The HGFs grown in monolayer and observed by field emission in-lens scanning electron microscopy demonstrated a preserved surface morphology after 24 h of treatment, while they showed an altered morphology after 96 h of treatment. Immunofluorescence demonstrated a reduction of type I collagen due to HEMA exposure after 96 h. From these results, we conclude that low concentrations of HEMA can significantly alter the morphology of human gingival fibroblasts and interfere with the presence of type I collagen protein.

Cyanidin 3-O-glucopyranoside protects and rescues SH-SY5Y cells against amyloid-beta peptide-induced toxicity.

The amyloid-&bgr; (A&bgr;) peptide (1–42) aggregation into oligomeric and fibrillar species affects neuronal viability, having a causal role in the development of Alzheimers disease. Among dietary anthocyanins, cyanidin 3-O-glucoside (Cy-3G) and its metabolites, such as protocatechuic acid (PA), have gained attention as potential neuroprotective agents. In this in-vitro study, we demonstrated that Cy-3G, but not PA, can inhibit A&bgr;1–42 spontaneous aggregation using thioflavin T fluorescence assay and transmission electron microscopy. Furthermore, treatment of human neuronal SH-SY5Y cells with Cy-3G during oligomeric and fibrillar A&bgr;1−42 treatment prevents neuronal viability loss. These protective effects were still evident when Cy-3G treatment was initiated after the appearance of oligomeric A&bgr;1−42 neurotoxicity. Taken together, these results suggest that Cy-3G may protect and rescue the neuronal cells from toxicity induced by A&bgr;1−42.

HEMA down-regulates procollagen α1 type I in human gingival fibroblasts

2-Hydroxyethyl methacrylate (HEMA) can be released from restorative materials and diffused into the tooth pulp over long periods of time. Although cytotoxicity due to high concentrations of monomers has been well studied, little is known about the risk of chronic toxicity resulting from low concentrations. The purpose of the study was to evaluate the effects of a minor toxic concentration of HEMA in the synthesis and expression of procollagen alpha1 type I produced by human gingival fibroblasts (HGF). HGF were exposed to 3 mM HEMA from 24 to 96 h. An MTT assay was performed to evaluate cell viability while reverse-transcriptase polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), and Western-blot analysis were carried out to evaluate the variability in the expression and synthesis of procollagen alpha1. Immunofluorescence was performed to detect the protein inside the cells. The results showed that there was a strong reduction of procollagen alpha 1 type I expression at 72 and 96 h. These findings demonstrate that, even if it does not reduce cell viability, 3 mM HEMA interferes both with the synthesis of the procollagen alpha 1 type I protein and its mRNA expression, suggesting that normal cell production and activity are modified by HEMA at concentrations below those which cause acute cytotoxicity.

Stem cell origin differently affects bone tissue engineering strategies

Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches.

In vitro reparative dentin: a biochemical and morphological study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.

Dentin matrix protein 1 and dentin sialophosphoprotein in human sound and carious teeth: an immunohistochemical and colorimetric assay

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are extracellular matrix proteins produced by odontoblasts involved in the dentin mineralization. The aim this study was to compare the distribution of DMP1 and DSPP in human sound dentin vs human sclerotic dentin. Sixteen sound and sixteen carious human molars were selected, fixed in paraformaldehyde and processed for immunohistochemical detection of DMP1 and DSPP by means of light microscopy, transmission electron microscopy (TEM) and high-resolution field emission in-lens scanning electron microscopy (FEI-SEM). Specimens were submitted to a pre-embedding or a post-embedding immunolabeling technique using primary antibodies anti DMP1 and anti-DSPP and gold-conjugated secondary antibodies. Other samples were processed for the detection of DMP1 and DSPP levels. Dentin from these samples was mechanically fractured to powder, then a protein extraction and a protein level detection assay were performed. DMP1 and DSPP were more abundant in carious than in sound samples. Immunohistochemical analyses in sclerotic dentin disclosed a high expression of DMP1 and DSPP inside the tubules, suggesting an active biomineralization of dentin by odontoblasts. Furthermore, the detection of small amounts of these proteins inside the tubules far from the carious lesion, as shown in the present study, is consistent with the hypothesis of a preventive defense of all dentin after a noxious stimulus has undermined the tooth.

Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.