Publications by authors named "Gilda Cobellis"

75 Publications

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells.

Int J Mol Sci 2021 Mar 3;22(5). Epub 2021 Mar 3.

Department of Experimental Medicine, Sez. Bottazzi, Università degli Studi della Campania "L. Vanvitelli", Via Costantinopoli 16, 80138 Napoli, Italy.

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3β-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and β-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.
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http://dx.doi.org/10.3390/ijms22052540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961766PMC
March 2021

CircRNA Role and circRNA-Dependent Network (ceRNET) in Asthenozoospermia.

Front Endocrinol (Lausanne) 2020 10;11:395. Epub 2020 Jul 10.

Dipartimento di Medicina Sperimentale, Università degli Studi della Campania Luigi Vanvitelli, Naples, Italy.

The role of circRNA in reproduction is under investigation. CircRNAs are expressed in human testis, spermatozoa (SPZ), and seminal plasma. Their involvement in embryo development has also been suggested. Asthenozoospermia, a common cause of male infertility, is characterized by reduced or absent sperm motility in fresh ejaculate. While abnormal mitochondrial function, altered sperm tail, and genomic causes have been deeply investigated, the epigenetic signature of asthenozoospermic derived SPZ still remains unexplored. CircRNAs may take part in the repertoire of differentially expressed molecules in infertile men. Considering this background, we carried out a circRNA microarray, identifying a total of 9,138 transcripts, 22% of them novel based and 83.5% with an exonic structure. Using KEGG analysis, we evaluated the circRNA contribution in pathways related to mitochondrial function and sperm motility. In order to discriminate circRNAs with a differential expression in SPZ with differential morphological parameters, we separated sperm cells by Percoll gradient and analyzed their differential circRNA payload. A bioinformatic approach was then utilized to build a circRNA/miRNA/mRNA network. With the aim to demonstrate a dynamic contribution of circRNAs to the sperm epigenetic signature, we verified their modulation as a consequence of an oral amino acid supplementation, efficacious in improving SPZ motility.
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http://dx.doi.org/10.3389/fendo.2020.00395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366322PMC
May 2021

The Pan-Sirtuin Inhibitor MC2494 Regulates Mitochondrial Function in a Leukemia Cell Line.

Front Oncol 2020 21;10:820. Epub 2020 May 21.

Dipartimento di Medicina di Precisione, Università degli Studi della Campania "Luigi Vanvitelli", Naples, Italy.

The involvement of sirtuins (SIRTs) in modulating metabolic and stress response pathways is attracting growing scientific interest. Some SIRT family members are located in mitochondria, dynamic organelles that perform several crucial functions essential for eukaryotic life. Mitochondrial dysfunction has emerged as having a key role in a number of human diseases, including cancer. Here, we investigated mitochondrial damage resulting from treatment with a recently characterized pan-SIRT inhibitor, MC2494. MC2494 was able to block mitochondrial biogenesis and function in terms of ATP synthesis and energy metabolism, suggesting that it might orchestrate cell response to metabolic stress and thereby interfere with cancer promotion and progression. Targeting mitochondrial function could thus be considered a potential anticancer strategy for use in clinical therapy.
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http://dx.doi.org/10.3389/fonc.2020.00820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255067PMC
May 2020

The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation.

Int J Mol Sci 2020 Apr 28;21(9). Epub 2020 Apr 28.

Department of Experimental Medicine, Sez. Bottazzi, Università degli Studi della Campania "L. Vanvitelli", Via Costantinopoli 16, 80138 Napoli, Italy.

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from epididymis of -knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from and epididymis of wild-type and -knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of -gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that -gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of and mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms.
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http://dx.doi.org/10.3390/ijms21093117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247701PMC
April 2020

Author Correction: Pro-inflammatory cytokines activate hypoxia-inducible factor 3α via epigenetic changes in mesenchymal stromal/stem cells.

Sci Rep 2020 Apr 17;10(1):6776. Epub 2020 Apr 17.

Department of Biochemistry, Biophysics and General Pathology, Università degli Studi della Campania L. Vanvitelli, Via L. De Crecchio, 7, 80138, Naples, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-62861-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165168PMC
April 2020

Fetal-Perinatal Exposure to Bisphenol-A Affects Quality of Spermatozoa in Adulthood Mouse.

Int J Endocrinol 2020 20;2020:2750501. Epub 2020 Mar 20.

Department of Experimental Medicine, Sez. Bottazzi, Università degli Studi della Campania "L. Vanvitelli", Via Costantinopoli 16, 80138 Napoli, Italy.

Bisphenol-A (BPA) is considered an endocrine disruptor with estrogenic activity. It is described as an environment-polluting industrial chemical whose adverse effects on the male reproductive system depend on the period of exposure (i.e., fetal, prepubertal, or adult life). We exposed male mice to BPA during the fetal-perinatal period (from 10 days post to 31 days post ) and investigated the impact of this early-life exposure on gamete health in adulthood animals at 78 days of age. Both in control and BPA-exposed mice, viability and motility of spermatozoa, as well as sperm motility acquisition and chromatin condensation of spermatozoa, have been evaluated. Results reveal harmful effect of BPA on viability and motility of sperm cells as well as on chromatin condensation status during epididymal maturation of spermatozoa. In particular, BPA exposure interferes with biochemical mechanism useful to stabilize sperm chromatin condensation, as it interferes with oxidation of thiol groups associated to chromatin.
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http://dx.doi.org/10.1155/2020/2750501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109585PMC
March 2020

Histone Post-Translational Modifications and CircRNAs in Mouse and Human Spermatozoa: Potential Epigenetic Marks to Assess Human Sperm Quality.

J Clin Med 2020 Feb 27;9(3). Epub 2020 Feb 27.

Department of Experimental Medicine, University of Campania "L. Vanvitelli", Via Costantinopoli 16, 80138 Napoli, Italy.

Spermatozoa (SPZ) are motile cells, characterized by a cargo of epigenetic information including histone post-translational modifications (histone PTMs) and non-coding RNAs. Specific histone PTMs are present in developing germ cells, with a key role in spermatogenic events such as self-renewal and commitment of spermatogonia (SPG), meiotic recombination, nuclear condensation in spermatids (SPT). Nuclear condensation is related to chromatin remodeling events and requires a massive histone-to-protamine exchange. After this event a small percentage of chromatin is condensed by histones and SPZ contain nucleoprotamines and a small fraction of nucleohistone chromatin carrying a landascape of histone PTMs. Circular RNAs (circRNAs), a new class of non-coding RNAs, characterized by a nonlinear back-spliced junction, able to play as microRNA (miRNA) sponges, protein scaffolds and translation templates, have been recently characterized in both human and mouse SPZ. Since their abundance in eukaryote tissues, it is challenging to deepen their biological function, especially in the field of reproduction. Here we review the critical role of histone PTMs in male germ cells and the profile of circRNAs in mouse and human SPZ. Furthermore, we discuss their suggested role as novel epigenetic biomarkers to assess sperm quality and improve artificial insemination procedure.
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http://dx.doi.org/10.3390/jcm9030640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141194PMC
February 2020

A New LC-MS/MS Method for Simultaneous and Quantitative Detection of Bisphenol-A and Steroids in Target Tissues: A Power Tool to Characterize the Interference of Bisphenol-A Exposure on Steroid Levels.

Molecules 2019 Dec 21;25(1). Epub 2019 Dec 21.

Department of Experimental Medicine, University of Campania "L. Vanvitelli",Via S. M. di Costantinopoli, 16, 80138 Naples, Italy.

Bisphenol A (BPA), an endocrine disruptor, may affect in situ steroidogenesis and alter steroids levels. The present work proposes a liquid chromatography tandem mass spectrometry method to simultaneously quantify BPA, 17β-Estradiol and testosterone in two target tissues: testis and visceral fat mass. Analytes were isolated and lipophilic impurities removed by two serial steps: liquid-liquid and solid phase extraction. All compounds were separated in a single gradient run by Kinetex F5 column and detected via multiple reaction monitoring using a triple quadrupole with a TurboIon electrospray source in both negative and positive modes. The method is selective and very sensitive. In the investigated concentration range, the linearity of the detector response is verified in both tissues. The use of specific SPE cartridges for affinity chromatography purification allows obtaining high percentages of process efficiency (68.0-83.3% for testicular tissue; 63.7-70.7% for visceral fat mass). Good repeatability and reproducibility was observed. The validated method can be efficiently applied for direct biological monitoring in testis and visceral fat mass from mice exposed to BPA. The quantification of compounds in a single assay could be achieved without a loss of sensitivity.
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http://dx.doi.org/10.3390/molecules25010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983012PMC
December 2019

Autophagy Function and Dysfunction: Potential Drugs as Anti-Cancer Therapy.

Cancers (Basel) 2019 Sep 29;11(10). Epub 2019 Sep 29.

Department of Precision Medicine, University of Campania "L. Vanvitelli", via L. De Crecchio, 7, 80138 Naples, Italy.

Autophagy is a highly conserved catabolic and energy-generating process that facilitates the degradation of damaged organelles or intracellular components, providing cells with components for the synthesis of new ones. Autophagy acts as a quality control system, and has a pro-survival role. The imbalance of this process is associated with apoptosis, which is a "positive" and desired biological choice in some circumstances. Autophagy dysfunction is associated with several diseases, including neurodegenerative disorders, cardiomyopathy, diabetes, liver disease, autoimmune diseases, and cancer. Here, we provide an overview of the regulatory mechanisms underlying autophagy, with a particular focus on cancer and the autophagy-targeting drugs currently approved for use in the treatment of solid and non-solid malignancies.
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http://dx.doi.org/10.3390/cancers11101465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826381PMC
September 2019

Expression Patterns of Circular RNAs in High Quality and Poor Quality Human Spermatozoa.

Front Endocrinol (Lausanne) 2019 3;10:435. Epub 2019 Jul 3.

Dipartimento di Medicina Sperimentale, Università degli Studi della Campania L. Vanvitelli, Naples, Italy.

Circular RNAs (circRNAs) are expressed in human testis and seminal plasma. Until today, there is missing information about a possible payload of circRNAs in human spermatozoa (SPZ). With this in mind, we carried out a circRNA microarray identifying a total of 10.726 transcripts, 28% novel based and 84.6% with exonic structure; their potential contribution in molecular pathways was evaluated by KEGG analysis. Whether circRNAs may be related to SPZ quality was speculated evaluating two different populations of SPZ (A SPZ = good quality, B SPZ = low quality), separated on the basis of morphology and motility parameters, by Percoll gradient. Thus, 148 differentially expressed (DE)-circRNAs were identified and the expression of selected specific SPZ-derived circRNAs was evaluated in SPZ head/tail-enriched preparations, to check the preservation of these molecules during SPZ maturation and their transfer into oocyte during fertilization. Lastly, circRNA/miRNA/mRNA network was built by bioinformatics approach.
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http://dx.doi.org/10.3389/fendo.2019.00435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626923PMC
July 2019

CircNAPEPLD is expressed in human and murine spermatozoa and physically interacts with oocyte miRNAs.

RNA Biol 2019 09 14;16(9):1237-1248. Epub 2019 Jun 14.

c Dipartimento di Medicina Sperimentale, sez "F. Bottazzi", Università della Campania "Luigi Vanvitelli" , Napoli , Italy.

Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan® Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centred on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein. Interestingly, the expression analysis in murine-unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant. Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.
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http://dx.doi.org/10.1080/15476286.2019.1624469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693540PMC
September 2019

Hypoxia-Regulated miRNAs in Human Mesenchymal Stem Cells: Exploring the Regulatory Effects in Ischemic Disorders.

Int J Mol Sci 2019 Mar 16;20(6). Epub 2019 Mar 16.

Department of Precision Medicine, Università degli Studi della Campania L. Vanvitelli, Via L. De Crecchio, 7, 80138 Naples, Italy.

Human mesenchymal/stromal stem cells (hMSC) are the most promising cell source for adult cell therapies in regenerative medicine. Many clinical trials have reported the use of autologous transplantation of hMSCs in several disorders, but with limited results. To exert their potential, hMSCs could exhibit efficient homing and migration toward lesion sites among other effects, but the underlying process is not clear enough. To further increase the knowledge, we studied the co-regulation between hypoxia-regulated genes and miRNAs. To this end, we investigated the miRNA expression profile of healthy hMSCs in low oxygen/nutrient conditions to mimic ischemia and compared with cells of patients suffering from critical limb ischemia (CLI). miRNAs are small, highly conserved, non-coding RNAs, skilled in the control of the target's expression level in a fine-tuned way. After analyzing the miRNOme in CLI-derived hMSC cells and healthy controls, and intersecting the results with the mRNA expression dataset under hypoxic conditions, we identified two miRNAs potentially relevant to the disease: miR-29b as a pathological marker of the disease and miR-638 as a therapeutic target. This study yielded a deeper understanding of stem cell biology and ischemic disorders, opening new potential treatments in the future.
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http://dx.doi.org/10.3390/ijms20061340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471025PMC
March 2019

Transcriptional landscape of mouse-aged ovaries reveals a unique set of non-coding RNAs associated with physiological and environmental ovarian dysfunctions.

Cell Death Discov 2018 5;4:112. Epub 2018 Dec 5.

1Department of Science and Technology, University of Sannio, Via Port'Arsa 11, 82100 Benevento, Italy.

The progressive and physiological decline in ovarian function depends on the rate of follicular loss by atresia, contributing to the reduction in ovarian reserve. Genetics and environmental factors play important roles in ovarian senescence and in the onset of ovarian dysfunctions such as diminished ovarian reserve. A better understanding of the mechanisms underlying ovarian aging and their regulation by genetic and environmental factors is needed to evaluate ovarian reserve and to predict fertility potential by identification of more accurate and less invasive markers. We report transcriptomic data (i) implicating novel (e.g. EIF2 signalling) and well-known pathways (e.g. TGFβ signalling), and (ii) defining a unique set of non-coding RNA (ncRNA), both associated with ovarian function. The latter includes miRNAs (e.g. and ), snoRNAs (e.g. and ), and one lncRNA (), which are differentially expressed in middle-aged ovaries (12 months) young-aged (3 months) from CD1 mice. Experimental analysis confirms that ovary lifespan varies across genetic backgrounds in mice and, genetics influences the response to environmental perturbations such as diet. Moreover, the identified ncRNAs were verified in a model of reproductive dysfunction promoted by the environmental toxicant ethylenthiourea. We also report the increase of miRNA143 and miRNA145 in follicular fluid of women with diminished ovarian reserve. Their levels inversely correlate with the hormonal profile and with the number of the oocytes recruited upon hormonal stimulation. Overall, we report a transcriptomic signature for ovarian dysfunction in vivo that provides a valuable resource for translational research in human reproductive aging.
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http://dx.doi.org/10.1038/s41420-018-0121-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281605PMC
December 2018

Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave.

Front Endocrinol (Lausanne) 2018 29;9:269. Epub 2018 May 29.

Department of Experimental Medicine, Sez. Bottazzi, Università degli Studi della Campania "L. Vanvitelli", Naples, Italy.

Endocannabinoids are lipid mediators, enzymatically synthesized and hydrolyzed, that bind cannabinoid receptors. Together with their receptors and metabolic enzymes, they form the "endocannabinoid system" (ECS). Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the main endocannabinoids studied in testis. In this study, using the first wave of spermatogenesis as an model to verify the progressive appearance of germ cells in seminiferous tubules [i.e., spermatogonia, spermatocytes, and spermatids], we analyzed the expression of the main enzymes and receptors of ECS in rat testis. In particular, the expression profile of the main enzymes metabolizing AEA and 2-AG as well as the expression of cannabinoid receptors, such as CB1 and CB2, and specific markers of mitotic, meiotic, and post-meiotic germ cell appearance or activities have been analyzed by RT-PCR and appropriately correlated. Our aim was to envisage a relationship between expression of ECS components and temporal profile of germ cell appearance or activity as well as among ECS components. Results show that expression of ECS components is related to germ cell progression. In particular, CB2 and 2-AG appear to be related to mitotic/meiotic stages, while CB1 and AEA appear to be related to spermatogonia stem cells activity and spermatids appearance, respectively. Our data also suggest that a functional interaction among ECS components occurs in the testis. Indeed, -incubated testis show that AEA-CB2 activity affects negatively monoacylglycerol-lipase levels upregulation of CB1 suggesting a CB1/CB2-mediated relationship between AEA and 2-AG. Finally, we provide the first evidence that CB1 is present in fetal gonocytes, during mitotic arrest.
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http://dx.doi.org/10.3389/fendo.2018.00269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986923PMC
May 2018

Pro-inflammatory cytokines activate hypoxia-inducible factor 3α via epigenetic changes in mesenchymal stromal/stem cells.

Sci Rep 2018 04 11;8(1):5842. Epub 2018 Apr 11.

Department of Biochemistry, Biophysics and General Pathology, Università degli Studi della Campania L. Vanvitelli, Via L. De Crecchio, 7, 80138, Naples, Italy.

Human mesenchymal stromal/stem cells (hMSCs) emerged as a promising therapeutic tool for ischemic disorders, due to their ability to regenerate damaged tissues, promote angiogenesis and reduce inflammation, leading to encouraging, but still limited results. The outcomes in clinical trials exploring hMSC therapy are influenced by low cell retention and survival in affected tissues, partially influenced by lesion's microenvironment, where low oxygen conditions (i.e. hypoxia) and inflammation coexist. Hypoxia and inflammation are pathophysiological stresses, sharing common activators, such as hypoxia-inducible factors (HIFs) and NF-κB. HIF1α and HIF2α respond essentially to hypoxia, activating pathways involved in tissue repair. Little is known about the regulation of HIF3α. Here we investigated the role of HIF3α in vitro and in vivo. Human MSCs expressed HIF3α, differentially regulated by pro-inflammatory cytokines in an oxygen-independent manner, a novel and still uncharacterized mechanism, where NF-κB is critical for its expression. We investigated if epigenetic modifications are involved in HIF3α expression by methylation-specific PCR and histone modifications. Robust hypermethylation of histone H3 was observed across HIF3A locus driven by pro-inflammatory cytokines. Experiments in a murine model of arteriotomy highlighted the activation of Hif3α expression in infiltrated inflammatory cells, suggesting a new role for Hif3α in inflammation in vivo.
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http://dx.doi.org/10.1038/s41598-018-24221-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895792PMC
April 2018

RIP1-HAT1-SIRT Complex Identification and Targeting in Treatment and Prevention of Cancer.

Clin Cancer Res 2018 06 13;24(12):2886-2900. Epub 2018 Mar 13.

Dipartimento di Medicina di Precisione, Università degli Studi della Campania "Luigi Vanvitelli", Napoli, Italy.

Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes. We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and studies with different mice models. Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3-SIRT1/2-HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential , in leukemic blasts and in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8-mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene-induced mammary gland hyperproliferation These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3081DOI Listing
June 2018

A Possible Early Biomarker for Bicuspid Aortopathy: Circulating Transforming Growth Factor β-1 to Soluble Endoglin Ratio.

Circ Res 2017 May 18;120(11):1800-1811. Epub 2017 Apr 18.

From the Experimental Medicine (A.F., M.C.), Cardiothoracic Sciences (C.B., M.B., M.D.F., A.D.C.), and Biophysics, Biochemistry, and General Pathology (G.C.), University of Campania "L.Vanvitelli", Naples Italy; and Department of Cardiac Surgery, Cardiovascular Center, Paracelsus Medical University, Nürnberg, Germany (G.S., T.J.M.F.).

Rationale: The pathogenesis of bicuspid aortic valve (BAV)-associated aortopathy is poorly understood, and no prognostic biomarker is currently available.

Objective: We aimed to identify putative circulating biomarkers pathogenetically and prognostically linked to bicuspid aortopathy.

Methods And Results: By reverse transcription polymerase chain reaction, we evaluated gene expression variations (versus normal aorta) of transforming growth factor-β1 (TGF-β1), connective tissue growth factor, matrix metalloproteinase-2 (MMP-2), MMP-14, endoglin (ENG), and superoxide dismutase 3 in ascending aorta samples from 50 tricuspid and 70 patients with BAV undergoing surgery for aortic stenosis (aorta diameter ≤45 mm: BAV or >45 mm: BAV). Expression changes of the TGF-β1 active dimer and ENG were analyzed also by Western blot in ascending aorta samples from other 10 tricuspid aortic valve, 10 BAV, and 10 BAV patients. The serum concentration of study targets was assessed through ELISA and the ratio of serum TGF-β1/ENG (T/E) was evaluated. All BAV patients underwent follow-up echocardiography to assess aortic growth rate. In BAV patients, TGF-β1 and MMP-2 gene expression increased significantly, whereas MMP-14 and ENG expression decreased versus controls. Expression changes were confirmed at protein level for TGF-β1 and ENG. TGF-β1 serum concentration significantly decreased in tricuspid aortic valve and BAV patients versus healthy subjects. ENG serum concentration decreased in all patients, more markedly in BAV. A significant increase of the T/E ratio versus healthy subjects was unique of patients with BAV. In BAV patients, a T/E ≥9 was independently associated in multivariable analysis with higher MMP-2 and lower superoxide dismutase 3 gene expression, independent of age and aortic diameter. A significant correlation was observed between baseline T/E ratio and aortic diameter growth rate in BAV patients (=0.66, <0.001).

Conclusions: The novel evidence of a possible value of the T/E ratio as a biomarker of BAV aortopathy was presented: further validation studies are warranted.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.310833DOI Listing
May 2017

Carcinogenic risk and Bisphenol A exposure: A focus on molecular aspects in endoderm derived glands.

Mol Cell Endocrinol 2017 Dec 19;457:20-34. Epub 2017 Jan 19.

Department of Science and Technology, University of Sannio, via Port'Arsa 11, 82100 Benevento, Italy. Electronic address:

Epidemiological and experimental evidence associates the exposure to Bisphenol A with the increase of cancer risk in several organs, including prostate. BPA targets different pathways involved in carcinogenicity including the Nuclear Receptors (i.e. estrogen and androgen receptors), stress regulated proteins and, finally, epigenetic changes. Here, we analyse BPA-dependent carcinogenesis in endoderm-derived glands, thyroid, liver, pancreas and prostate focusing on cell signalling, DNA damage repair pathways and epigenetic modifications. Mainly, we gather molecular data evidencing harmful effects at doses relevant for human risk (low-doses). Since few molecular data are available, above all for the pancreas, we analysed transcriptomic data generated in our laboratory to suggest possible mechanisms of BPA carcinogenicity in endoderm-derived glands, discussing the role of nuclear receptors and stress/NF-kB pathways. We evidence that an in vitro toxicogenomic approach might suggest mechanisms of toxicity applicable to cells having the same developmental origin. Although we cannot draw firm conclusions, published data summarized in this review suggest that exposure to BPA, primarily during the developmental stages, represents a risk for carcinogenesis of endoderm-derived glands.
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http://dx.doi.org/10.1016/j.mce.2017.01.027DOI Listing
December 2017

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

Stem Cell Reports 2017 01 22;8(1):1-10. Epub 2016 Dec 22.

Stem Cell Fate Laboratory, Institute of Genetics and Biophysics, 'A. Buzzati-Traverso', CNR, 80131 Naples, Italy. Electronic address:

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.
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http://dx.doi.org/10.1016/j.stemcr.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233408PMC
January 2017

Bisphenol A induces hypothalamic down-regulation of the the cannabinoid receptor 1 and anorexigenic effects in male mice.

Pharmacol Res 2016 11 15;113(Pt A):376-383. Epub 2016 Sep 15.

Department of Experimental Medicine, Sez. Bottazzi, II University of Naples, 80138, Napoli, Italy.

Bisphenol A is an environment-polluting industrial chemical able to interfere with the endocrine system. An obesogenic effect in perinatally exposed rodents has been described as estrogenic activity. We exposed male mice to Bisphenol A during fetal-perinatal period (from 10 days post coitum to 31 days post partum) and investigated the effects of this early-life exposure at 78 days of age. Body weight, food intake, fat mass, and hypothalamic signals related to anorexigenic control of food intake were analyzed. Results show that Bisphenol A exposure reduced body weight and food intake. In addition, the exposure decreased epididymal fat mass and adiposity, acting negatively on adipocyte volume. At hypothalamic level, Bisphenol A exposure reduced the expression of the cannabinoid receptor 1 and induced gene expression of cocaine and amphetamine-regulated transcript-1. This observation suggests that Bisphenol A induces activation of anorexigenic signals via down-regulation of the hypothalamic cannabinoid receptor 1 with negative impact on food intake.
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http://dx.doi.org/10.1016/j.phrs.2016.09.005DOI Listing
November 2016

Kisspeptins, Estrogens and Male Fertility.

Curr Med Chem 2016 ;23(36):4070-4091

Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope, Via Medina 40, 80133 Napoli, Italy.

Background: The control of male fertility requires accurate endocrine, paracrine and autocrine communications along the hypothalamus-pituitary-gonad (HPG) axis. In this respect, the possible interplay between upcoming/classical modulators of reproductive functions deserves attention in that may be a successful tool for the future exploitation of new potential therapeutic targets in the treatment of fertility disorders.

Methods: In this review we will discuss upcoming data concerning the role of kisspeptins, the products of the Kiss1 gene, and estrogens - classically considered as female hormones - as well as their possible interplay in testis.

Results: Kisspeptins, via the activation of kisspeptin receptor Gpr54 represent the main gatekeeper of the hypothalamic Gonadotropin Releasing Hormone (GnRH) centrally modulating the onset and maintaining reproductive functions. As a consequence, the loss of kisspeptin signalling causes hypogonadotrophic hypogonadism in humans and animal models. In spite of the well recognized functions at hypothalamic levels, recent data strongly support direct production and activity of kisspeptin in testis and its involvement in the control of Leydig cells, germ cells progression and sperm functions. Similarly, estrogens exhibit high impact on proliferative/apoptotic/differentiative events in testis, thus resulting as local key modulators for the production - but also for the release, transport and maturation - of high quality spermatozoa.

Conclusion: This review summarizes the upcoming data from experimental models and humans concerning the testicular activity of kisspeptins and estrogens to preserve male fertility. Mutual enhancement of kisspeptin and estradiol signalling for the progression of spermatogenesis has also been discussed.
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http://dx.doi.org/10.2174/0929867323666160902155434DOI Listing
February 2017

Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells.

Front Endocrinol (Lausanne) 2016 3;7:47. Epub 2016 Jun 3.

Dipartimento di Medicina Sperimentale, Seconda Università di Napoli , Napoli , Italy.

Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis.
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http://dx.doi.org/10.3389/fendo.2016.00047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891325PMC
July 2016

BPA-Induced Deregulation Of Epigenetic Patterns: Effects On Female Zebrafish Reproduction.

Sci Rep 2016 Feb 25;6:21982. Epub 2016 Feb 25.

Dipartimento Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy.

Bisphenol A (BPA) is one of the commonest Endocrine Disruptor Compounds worldwide. It interferes with vertebrate reproduction, possibly by inducing deregulation of epigenetic mechanisms. To determine its effects on female reproductive physiology and investigate whether changes in the expression levels of genes related to reproduction are caused by histone modifications, BPA concentrations consistent with environmental exposure were administered to zebrafish for three weeks. Effects on oocyte growth and maturation, autophagy and apoptosis processes, histone modifications, and DNA methylation were assessed by Real-Time PCR (qPCR), histology, and chromatin immunoprecipitation combined with qPCR analysis (ChIP-qPCR). The results showed that 5 μg/L BPA down-regulated oocyte maturation-promoting signals, likely through changes in the chromatin structure mediated by histone modifications, and promoted apoptosis in mature follicles. These data indicate that the negative effects of BPA on the female reproductive system may be due to its upstream ability to deregulate epigenetic mechanism.
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http://dx.doi.org/10.1038/srep21982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766405PMC
February 2016

Pluripotent Stem Cells: Current Understanding and Future Directions.

Stem Cells Int 2016 20;2016:9451492. Epub 2015 Dec 20.

Department of Biophysics, Biochemistry and General Pathology, Second University of Naples, Via L. de Crecchio 7, 80131 Napoli, Italy.

Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells.
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http://dx.doi.org/10.1155/2016/9451492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699068PMC
January 2016

HSA21 Single-Minded 2 (Sim2) Binding Sites Co-Localize with Super-Enhancers and Pioneer Transcription Factors in Pluripotent Mouse ES Cells.

PLoS One 2015 8;10(5):e0126475. Epub 2015 May 8.

Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland; University Hospitals of Geneva, Geneva, Switzerland; iGE3 Institute of Genetics and Genomics of Geneva, Geneva, Switzerland.

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126475PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425456PMC
April 2016

Corrigendum to "SIRT1 inhibition affects angiogenic properties of human MSCs".

Biomed Res Int 2015 25;2015:132086. Epub 2015 Mar 25.

Department of Biochemistry, Biophysics and General Pathology, Second University of Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy.

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http://dx.doi.org/10.1155/2015/132086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389819PMC
July 2015

SIRT1 inhibition affects angiogenic properties of human MSCs.

Biomed Res Int 2014 27;2014:783459. Epub 2014 Aug 27.

Department of Biochemistry, Biophysics and General Pathology, Second University of Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy ; Istituto Nazionale Tumori, Struttura Complessa Oncologia Medica Melanoma Immunoterapia Oncologica e Terapia Innovativa, Via Mariano Semmola, 80131 Napoli, Italy.

Human mesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewal and of differentiating into several cell types. Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesis in ischemic disorders. However, the molecular mechanisms underlying these effects remain unclear. A recent report has connected MSCs multipotency to sirtuin families, showing that SIRT1 can regulate MSCs function. Furthermore, SIRT1 is a critical modulator of endothelial angiogenic functions. Here, we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic and pharmacological level. We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA and protein level. Inhibition of SIRT1 by sirtinol, a cell-permeable inhibitor, or by specific sh-RNA resulted in an increase of premature-senescence phenotype, a reduction of proliferation rate with increased apoptosis. Furthermore, we observed a consistent reduction of tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1α protein and its transcriptional activity in hMSCs. Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCs and may contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cells in ischemic disorders through SIRT1 modulation.
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http://dx.doi.org/10.1155/2014/783459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163475PMC
June 2015

Modulators of hypothalamic-pituitary-gonadal axis for the control of spermatogenesis and sperm quality in vertebrates.

Front Endocrinol (Lausanne) 2014 18;5:135. Epub 2014 Aug 18.

Department of Experimental Medicine, Second University of Naples , Naples , Italy.

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http://dx.doi.org/10.3389/fendo.2014.00135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135230PMC
September 2014

Intra-testicular signals regulate germ cell progression and production of qualitatively mature spermatozoa in vertebrates.

Front Endocrinol (Lausanne) 2014 8;5:69. Epub 2014 May 8.

Dipartimento di Medicina Sperimentale sez "F. Bottazzi", Seconda Università degli Studi di Napoli , Naples , Italy.

Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic-pituitary control, being regulated by the secretion of pituitary gonadotropins, follicle stimulating hormone, and luteinizing hormone, in response to stimulation exerted by gonadotropin releasing hormone from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intra-gonadal signals sustains the production of high quality mature spermatozoa. In this review, we focus on the recent advances in the area of the intra-gonadal signals supporting sperm development.
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http://dx.doi.org/10.3389/fendo.2014.00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021137PMC
June 2014

Endocannabinoids are Involved in Male Vertebrate Reproduction: Regulatory Mechanisms at Central and Gonadal Level.

Front Endocrinol (Lausanne) 2014 15;5:54. Epub 2014 Apr 15.

Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope , Naples , Italy.

Endocannabinoids (eCBs) are natural lipids regulating a large array of physiological functions and behaviors in vertebrates. The eCB system is highly conserved in evolution and comprises several specific receptors (type-1 and type-2 cannabinoid receptors), their endogenous ligands (e.g., anandamide and 2-arachidonoylglycerol), and a number of biosynthetic and degradative enzymes. In the last few years, eCBs have been described as critical signals in the control of male and female reproduction at multiple levels: centrally, by targeting hypothalamic gonadotropin-releasing-hormone-secreting neurons and pituitary, and locally, with direct effects on the gonads. These functions are supported by the extensive localization of cannabinoid receptors and eCB metabolic enzymes at different levels of the hypothalamic-pituitary-gonadal axis in mammals, as well as bonyfish and amphibians. In vivo and in vitro studies indicate that eCBs centrally regulate gonadal functions by modulating the gonadotropin-releasing hormone-gonadotropin-steroid network through direct and indirect mechanisms. Several proofs of local eCB regulation have been found in the testis and male genital tracts, since eCBs control Sertoli and Leydig cells activity, germ cell progression, as well as the acquisition of sperm functions. A comparative approach usually is a key step in the study of physiological events leading to the building of a general model. Thus, in this review, we summarize the action of eCBs at different levels of the male reproductive axis, with special emphasis, where appropriate, on data from non-mammalian vertebrates.
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http://dx.doi.org/10.3389/fendo.2014.00054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995072PMC
June 2014