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Background: Dietary supplements of Carica papaya Linn fermented with yeast using a biotechnological process have well recognized positive effects on immunological, hematological, inflammatory, and oxidative stress parameters, utilized as biomarkers of chronic and degenerative diseases. Although many natural products fermented with lactic acid bacteria (LAB) have shown beneficial effects on the immune system and on antioxidant defenses, formulations of papaya fermented with LAB have not yet been studied. Aims: The aims of this study were to investigate the immunomodulatory activity linked to the type of papaya fermentation (yeast vs LAB) in macrophages and to evaluate whether the type of fermentation differently modulates oxidative stress both in cell free system and in a model of embryonic brain cells. Methods: Cytotoxicity was evaluated through cell proliferation kinetic and lactate dehydrogenase release assays; immunomodulatory activity through the transcriptional activation of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor a (TNF alpha) by qRT- PCR in RAW 264.7 macrophages; antioxidant capacity was assessed, in cell free system and in pheochromocytoma cells embryonic brain cells, by measuring the intracellular ROS levels through a fluorescent dye. Results: Our data showed that all the formulations studied are safe at low concentrations (3-6 mg/ml); the LAB-fermented formulations promoted the expression of iNOS and TNFa more efficiently than yeast-fermented papaya preparation (p < 0.001). In a cell free system, the LABfermented formulation acted as mild scavengers of ROS while, in cells, both formulations didn't show any significant effect. Conclusions: This study corroborates previous reports showing the efficacy of yeast fermented papaya as a potent immunostimulant and highlights the beneficial contribution of lactic bacteria fermentation.

Despite the currently available pharmacotherapies, today, thirty percent of worldwide deaths are due to cardiovascular diseases (CVDs), whose primary cause is atherosclerosis, an inflammatory disorder characterized by the buildup of lipid deposits on the inside of arteries. Multiple cellular signaling pathways have been shown to be involved in the processes underlying atherosclerosis, and evidence has been accumulating for the crucial role of Notch receptors in regulating the functions of the diverse cell types involved in atherosclerosis onset and progression. Several classes of nutraceuticals have potential benefits for the prevention and treatment of atherosclerosis and CVDs, some of which could in part be due to their ability to modulate the Notch pathway. In this review, we summarize the current state of knowledge on the role of Notch in vascular health and its modulation by nutraceuticals for the prevention of atherosclerosis and/or treatment of related CVDs.

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652-2663, 2016. © 2016 Wiley Periodicals, Inc.

Additional file 1: Figure S1. Characterization of primary cultures of rat aortic smooth muscle cells by immunofluorescent staining. Figure S2. Oil-red staining of cholesterol loaded rat aortic smooth muscle cells. Table S1. Primers used for quantitative RT-PCR. Table S2. Semiquantitative evaluation of histological characteristics and Notch receptors immunostainings. Table S3. P values associated to the results of correlations analyses among the expression levels of mRNAs and miRs analyzed. Table S4. Clinical follow up and molecular analysis.

Cardiovascular diseases are one of the leading causes of morbidity and mortality in women after menopause and 56% of all causes of death in Western European countries. Nowadays, with increasing life span, women spend approximately one-third of their life-time in postmenopausal state; therefore, the development of new strategies to improve the prevention and treatment of menopause-associated pathologies is important topic in clinical practice. The studies to assess the safety of hormone replacement therapy in women with estrogen deficiency have not been conclusive due to the relative contraindications; therefore, hormone replacement therapy is prescribed only in selected cases and for a limited time. For this reason, today women are encouraged to use naturally available compounds to prevent or to attenuate menopausal symptoms and correlated pathologies, with fewer side effects. Among these compounds, berberine, an isoquinoline alkaloid derived from plants of the generis Berberis, has been recognized as being capable of decreasing oxidative stress, LDL, triglycerides, and insulin resistance and of improving the mood. This review describes the cellular and clinical effects associated with the use of berberine, which suggest that this molecule could be an effective natural supplement to ensure a smooth peri- and postmenopausal transition.

Heart failure (HF) is characterized by inflammatory conditions which cause endothelium dysfunctions and promote a worsening of the disease. We have previously shown that the serum of HF patients has a pro-apoptotic effect on human umbilical vein endothelial cells (HUVECs) which correlates with circulating levels of TNF-α. TNF-α dysregulates Notch signalling in HUVECs causing apoptosis. To establish whether the pro-apoptotic effect of HF serum in HUVECs could be due to dysregulation of Notch signalling, HUVECs were treated with serum of patients with mild and moderate/severe HF (New York Heart Association (NYHA) class I/II and III/IV respectively) or healthy controls and the effects on Notch signalling were evaluated. Western blot analyses showed reduced levels of Notch4 receptor in cells treated with serum from NYHA class I/II patients (3/12) and NYHA class III/IV (7/12) compared to control. Similarly, in serum-treated cells the mature form of Notch ligand Delta Like-4 (Dll4) was absent (2/12 class NYHA I/II and 7/12 class III/IV). Only with serum from class III/IV patients we found both reduced levels of Notch 4 and absence of Dll4 (4/12). Notch 1, 2 as well as Jagged1 ligand protein levels were unchanged by treatment. qRT-PCR showed increased Hey2 and reduced Hes1 and Hey1 mRNAs in treated cells compared to controls. These differences in mRNAs didn’t correlate with the severity of disease. In conclusion Notch signalling in HUVECs is modulated by inflammatory cytokines in serum of HF patients indicating that dysregulation of this pathway could be part of a mechanism by which these cytokines induce endothelial dysfunctions in HF patients.

Membrane lipid rafts are detergent-resistant microdomains containing glycosphingolipids, cholesterol and glycosylphosphatidylinositol-linked proteins; they seem to be actively involved in many cellular processes including signal transduction, apoptosis, cell adhesion and migration. Lipid rafts may represent important functional platforms where redox signals are produced and transmitted in response to various agonists or stimuli. In addition, a new concept is emerging that could be used to define the interactions or amplification of both redox signalling and lipid raft-associated signalling. This concept is characterized by redox-mediated feed forward amplification in lipid platforms. It is proposed that lipid rafts are formed in response to various stimuli; for instance, NAD(P)H oxidase (Nox) subunits are aggregated or recruited in these platforms, increasing Nox activity. Superoxide and hydrogen peroxide generation could induce various regulatory activities, such as the induction of glucose transport activity and proliferation in leukaemia cells. The aim of our study is to probe: i) the involvement of lipid rafts in the modulation of the glucose transporter Glut1 in human acute leukemia cells; ii) the involvement of plasma membrane caveolae/lipid rafts in VEGF-mediated redox signaling via Nox activation in human leukemic cells; iii) the role of p66shc, an adaptor protein, in VEGF signaling and ROS production in endothelial cells (ECs); iv) the role of Sindecan-2, a transmembrane heparan sulphate proteoglycan, in VEGF signaling and physiological response in ECs and v) the antioxidant and pro-apoptotic activities of simple dietary phenolic acids, i. e. caffeic, syringic and protocatechuic acids in leukemia cells, characterized by a very high ROS content. Our results suggest that the role played by NAD(P)H oxidase-derived ROS in the regulation of glucose uptake, proliferation and migration of leukaemia and endothelial cells could likely occur through the control of lipid raft-associated signalling.

Cholesterol depletion modifies plasma membrane lipid raft integrity and affects glucose uptake in a human leukemic cell line C. Caliceti, L. Zambonin, C. Prata, F. Vieceli Dalla Sega, G. Hakim and D. Fiorentini Dipartimento di Biochimica “G. Moruzzi”, Università di Bologna - Italy In hemopoietic M07e cells, stem cell factor (SCF) activates glucose transport through GLUT1 translocation from intracellular stores. Recently, it has been reported that in many cell types GLUT1 is localized in part in detergent-resistant membrane (DRM) domains and a role for lipid rafts in GLUT1 stimulation has been suggested. We investigated whether a plasma membrane cholesterol depletion might play a role in GLUT1 activity. Cell exposure to 10 mM methyl--cyclodextrin (MBCD) for 20 min resulted in a loss of cholesterol from plasma membrane, without affecting viability. MBCD-treated cells exhibited a rise in glucose uptake, higher than that observed upon SCF treatment. The combined cell treatment with SCF and MBCD determined a further increase in glucose uptake, showing an additive effect. Membrane fractionation by flotation on sucrose density gradient showed that in untreated cells most of GLUT1 is distributed in the high-density region, but a little amount is colocalized with lipid raft marker proteins in low-density regions, corresponding to DRM. Upon MBCD treatment, GLUT1 resulted totally confined to the high-density region. DRM exhibited also the highest cholesterol content. The cholesterol distribution profile of the MBCD-treated samples shows that the cholesterol preferentially decreased in DRM, confirming its selective depletion. Cell membrane isolation by biotinylation and immunofluorescence staining indicated that MBCD treatment, like SCF stimulation, greatly enhanced the amount of GLUT1 at cell surface, promoting a translocation from intracellular stores. MBCD increases Akt-pS473, while SCF positively regulates PLCγ signaling cascade, both involved in GLUT translocation and in glucose uptake control. Therefore, these two stimuli could proceed through distinct signalling pathways. In summary, a mild lipid environment alteration in lipid rafts enhances GLUT1 activity in M07e cells via a cholesterol-dependent mechanism, possibly involving Akt.

Role of aquaporins in the cell signalling mediated by hydrogen peroxide in leukemic cells F. Vieceli Dalla Sega, C. Prata, C. Caliceti, L. Zambonin, D. Fiorentini, G. Hakim, L. Landi Department of Biochemistry “G. Moruzzi”, University of Bologna, Bologna, Italy Aquaporins (AQP) constitute a large family of integral membrane proteins, present in all domains of life, that form pores appearing to be designed for the selective passage of water and glycerol [1]. Hydrogen peroxide has been long thought to be freely diffusible but recent evidence suggest that specific mammalian aquaporin homologues (AQP8) possess the capacity to channel H2O2 across membranes [2,3]. Moreover, it has been recently shown that human cells AQP3 expression can modulate the intracellular level of H2O2 and is required for NOX-derived H2O2 signalling [3]. Redox deregulation of proliferative pathways involving ROS (particularly H2O2) in cancer initiation and progression is now firmly established. Here we tested the hypothesis that specific isoforms of aquaporins regulate the level of intracellular H2O2 produced by NOX affecting downstream pathways linked to cell proliferation. In the present study, we show that in different leukemic cell lines NOX2 and/or NOX4 are expressed and are necessary to produce ROS that, in turn, are able to sustain glucose uptake and cell proliferation. Furthermore, we report that inhibition of aquaporins diminishes intracellular ROS accumulation either when H2O2 is produced by Nox enzymes or when is added exogenously to the medium. Finally, we observe a decrease in glucose transport following aquaporins inhibition. Taken together data suggest that aquaporins can regulate hydrogen peroxide uptake and downstream pathways mediated by H2O2.