Vittoria D’Esposito

Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) Significantly Improve Prostate Cancer Detection at Initial Biopsy in a Total PSA Range of 2–10 ng/ml

Many efforts to reduce prostate specific antigen (PSA) overdiagnosis and overtreatment have been made. To this aim, Prostate Health Index (Phi) and Prostate Cancer Antigen 3 (PCA3) have been proposed as new more specific biomarkers. We evaluated the ability of phi and PCA3 to identify prostate cancer (PCa) at initial prostate biopsy in men with total PSA range of 2–10 ng/ml. The performance of phi and PCA3 were evaluated in 300 patients undergoing first prostate biopsy. ROC curve analyses tested the accuracy (AUC) of phi and PCA3 in predicting PCa. Decision curve analyses (DCA) were used to compare the clinical benefit of the two biomarkers. We found that the AUC value of phi (0.77) was comparable to those of %p2PSA (0.76) and PCA3 (0.73) with no significant differences in pairwise comparison (%p2PSA vs phi p = 0.673, %p2PSA vs. PCA3 p = 0.417 and phi vs. PCA3 p = 0.247). These three biomarkers significantly outperformed fPSA (AUC = 0.60), % fPSA (AUC = 0.62) and p2PSA (AUC = 0.63). At DCA, phi and PCA3 exhibited a very close net benefit profile until the threshold probability of 25%, then phi index showed higher net benefit than PCA3. Multivariable analysis showed that the addition of phi and PCA3 to the base multivariable model (age, PSA, %fPSA, DRE, prostate volume) increased predictive accuracy, whereas no model improved single biomarker performance. Finally we showed that subjects with active surveillance (AS) compatible cancer had significantly lower phi and PCA3 values (p<0.001 and p = 0.01, respectively). In conclusion, both phi and PCA3 comparably increase the accuracy in predicting the presence of PCa in total PSA range 2–10 ng/ml at initial biopsy, outperforming currently used %fPSA.

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.

Alginate-hyaluronan composite hydrogels accelerate wound healing process.

In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

Growth-promoting action and growth factor release by different platelet derivatives.

Abstract Platelet derivatives are commonly used in wound healing and tissue regeneration. Different procedures of platelet preparation may differentially affect growth factor release and cell growth. Preparation of platelet-rich fibrin (PRF) is accompanied by release of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1), and several cytokines. When compared with the standard procedure for platelet-rich plasma (PRP), PRF released 2-fold less PDGF, but >15-fold and >2-fold VEGF and TGFβ1, respectively. Also, the release of several cytokines (IL-4, IL-6, IL-8, IL-10, IFNγ, MIP-1α, MIP-1β and TNFα) was significantly increased in PRF-conditioned medium (CM), compared to PRP-CM. Incubation of both human skin fibroblasts and human umbilical vein endothelial cells (HUVECs) with PRF-derived membrane (mPRF) or with PRF-CM enhanced cell proliferation by >2-fold (p < 0.05). Interestingly, PRP elicited fibroblast growth at a higher extent compared to PRF. At variance, PRF effect on HUVEC growth was significantly greater than that of PRP, consistent with a higher concentration of VEGF in the PRF-CM. Thus, the procedure of PRP preparation leads to a larger release of PDGF, as a possible result of platelet degranulation, while PRF enhances the release of proangiogenic factors.

Bisphenol A environmental exposure and the detrimental effects on human metabolic health: is it necessary to revise the risk assessment in vulnerable population?

In the last decades, many reports have focused the attention on deleterious effects of novel environmental chemical compounds, including bisphenol A (BPA), on human health. BPA, a common and widely chemical contaminant acting as endocrine disruptor, accumulates in adipose tissue and may affect adipocyte metabolic and inflammatory functions. BPA, at low chronic doses, is now considered as an obesogen compound, and might contribute to the rise of metabolic syndrome, visceral adiposity and diabetes epidemics. The BPA worldwide presence in the environment is responsible for chronic exposure during vulnerable periods, such as foetal and neonatal life. The BPA source of contamination can occur via food, beverage, wastewater, air, dust and soil. BPA, as lipophilic compound, may accumulate into the adipose tissue already during foetal life and may affect adulthood health, through adverse effects on the growth and development of organs and tissues. Thus, based on several studies, it would be crucial to consider further actions aimed to refine risk assessment at least in vulnerable population, such as foetuses, infants and young children, to prevent metabolic diseases and obesity.

Comparison between fibroblast wound healing and cell random migration assays in vitro

Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques.

Ultrasmall silver nanoparticles loaded in alginate–hyaluronic acid hybrid hydrogels for treating infected wounds

ABSTRACT Nowadays, silver nanoparticles are in the limelight to control infection during wound healing process, and a vast variety of antimicrobial dressings based on colloidal silver have been marketed to fight wound invasion of pathogen bacteria, which represents one of the main adverse effects limiting the repair process. Here we propose a biofunctional hydrogel based on alginate (ALG) and hyaluronic acid (HA) embedding ultrasmall silver nanoparticles (usSN, <1 nm) as antimicrobial component. The hydrogels were fabricated in different size by a straightforward internal gelation method using CaCO3 and glucono-δ-lactone. To follow usSN release from the hydrogels in aqueous media, catalytic activity of usSN-loaded hydrogels was evaluated. Results suggested that catalytic activity was low in intact hydrogels and high when hydrogels dissolved, which suggests that usSN firmly interact with polymer chains and are available in the medium depending on the extent of hydrogel degradation. HA-containing hydrogels showed faster dissolution in simulated physiological conditions and higher antibiofouling properties as compared to hydrogels made only of ALG. Free usSN were not toxic toward human mesenchymal stem cells (Ad-MSCs), previously isolated from subcutaneous adipose tissue biopsies, up to 50 µg/mL. At this concentration, viability of Ad-MSCs was unaffected whereas their motility was significantly higher as compared to control (p<0.01) for both free usSN and hydrogel integrating. Antimicrobial activity on clinical isolates of both Gram-positive and Gram-negative bacteria demonstrated that usSN at 50 µg/mL were able to kill all the bacteria tested after 24 and 48 h of contact time. In the case of hydrogels, a matrix effect was found and bacterial killing was significant only at 24 h and dependent on bacterial strain, being Gram-negative bacteria more susceptible. These results clearly indicate that usSN interaction with polymer network and exposure time can strongly affect usSN antimicrobial profile in the hydrogel and, in turn, timing of hydrogel change at injured site in a clinical setting. On the whole, ALG/HA hydrogels integrating usSN can be considered a suitable option to fabricate biofunctional dressings for hospitalized patients and worth of further in vivo investigation. GRAPHICAL ABSTRACT

An Integrated Approach Based on Multiplexed Protein Array and iTRAQ Labeling for In-Depth Identification of Pathways Associated to IVF Outcome

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.

Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.