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Damen R.,Yale University | Haugen M.,Yale University | Svejda B.,Yale University | Alaimo D.,Yale University | And 4 more authors.
PLoS ONE | Year: 2013

Objective:We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.Design:The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.Results:HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (∼90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5′-ASA treatment was confirmed in a TNBS-model.Conclusion:Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD. © 2013 Damen et al.

Bengesser S.A.,Medical University of Graz | Lackner N.,Medical University of Graz | Birner A.,Medical University of Graz | Fellendorf F.T.,Medical University of Graz | And 11 more authors.
Journal of Affective Disorders | Year: 2015

Results The major finding was a significantly decreased TAC in BD compared to the CG (p<0.01; BD: M 1.18, SD 0.47; CG: M 1.39, SD 0.49). MDA was significantly and TBARS by trend higher in the CG compared to the euthymic bipolar test persons (MDA: p<0.01, BD: M 0.70, SD 0.18; CG: M 0.81, SD 0.25; TBARS: p<0.1, BD: M 0.78, SD 0.28; CG: M 0.76, SD 0.30). The antioxidative enzyme GST was significantly elevated in both patients and controls (BD: M 298.24, SD 133.02; CG: M 307.27 SD 118.18). Subgroup analysis revealed that the CG with concurrent MetS and obesity had significantly elevated TAC when compared to CG without concurrent MetS (p<0.05, no MetS: M 1.33, SD 0.50; MetS: M 1.67, SD 0.32), as well as persons with BD with or without current MetS (no MetS: M 1.18, SD 0.44; MetS: M 1.15, SD 0.49). Significant correlations between GST and anthropometric variables were found in male study participants. Multivariate analysis indicated a significant gender effect concerning TBARS values in all patients and CG (p<0.01, females: M 0.73, SD 0.29; males: M 0.83, SD 0.28).Methods Peripheral markers of oxidative stress [i.e. thiobarbituric acid reactive substance, (TBARS), malondialdehyde (MDA), and carbonyl proteins] and antioxidant markers [e.g. total antioxidative capacity (TAC), superoxide dismutase (SOD), glutathione S-transferase (GST)] were obtained in a cohort of euthymic adults with BD (N=113) and compared to healthy controls (CG) (N=78). Additionally, anthropometric measures included the body mass index (BMI) [kg/m2], waist and hip circumference [cm], waist-to-hip-ratio (WHR), waist to height ratio (WtHR) as well as the IDF-defined MetS.Introduction Oxidative and nitrosative stress are implicated in the pathogenesis of uni- and bipolar disorder. Herein we primarily sought to characterize markers of oxidative/nitrosative stress during euthymia in adults with bipolar disorder (BD). Oxidative markers were further evaluated in this BD sample in synopsis with excess overweight or obesity and/or comorbid metabolic syndrome (MetS).Conclusion Euthymic bipolar adults exhibit peripheral evidence of a disturbed biosignature of oxidative stress and antioxidative defense. Male test persons showed significantly higher peripheral markers of oxidative stress than women- female sex may exert protective effects. Furthermore, the biosignature of oxidative stress obtained herein was more pronounced in males with concurrent metabolic disorders. Our results further extend knowledge by introducing the moderating influence of gender and obesity on oxidative stress and BD. © 2014 Elsevier B.V. All rights reserved.

Haugen M.,Yale University | Dammen R.,Yale University | Svejda B.,Yale University | Gustafsson B.I.,Norwegian University of Science and Technology | And 3 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2012

The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O2. Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O2 would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O2 levels (0.5%). Reducing O2 also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O2 to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (~75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/ cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O2 levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing. © 2012 the American Physiological Society.

Svejda B.,Institute of Pathophysiology and Immunology | Svejda B.,Yale University | Kidd M.,Yale University | Giovinazzo F.,Yale University | And 4 more authors.
Cancer | Year: 2010

BACKGROUND: Fibrosis is a cardinal feature of small intestinal neuroendocrine tumors (SI-NETs) both in local peritumoral tissue and systemic sites (cardiac). 5-HT, a commonly secreted NET amine, is a known inducer of fibrosis, although the mechanistic basis for it and growth factors regulating fibrosis and proliferation in the tumor microenvironment are unclear. We hypothesized that targeting 5-HT2B receptors on tumor cells would inhibit SI-NET 5-HT release and, thereby, fibroblast activation in the tumor microenvironment. METHODS: We studied the 5-HT2B receptor antagonist PRX-08066 in NET cell lines (KRJ-I, H720) and in the coculture system (KRJ-I cells: fibroblastic HEK293 cells) using real time polymerase chain reaction, ELISA, Ki67 immunostaining, and flow cytometry-based caspase 3 assays to assess antiproliferative and profibrotic signaling pathways. RESULTS: In the 5-HT 2B expressing SI-NET cell line, KRJ-I, PRX-08066 inhibited proliferation (IC50 4.6×10-9M) and 5-HT secretion (6.9 × 10-9M) and decreased ERK1/2 phosphorylation and profibrotic growth factor synthesis and secretion (transforming growth factor beta 1 [TGFβ1], connective tissue growth factor [CTGF] and fibroblast growth factor [FGF2]). In the KRJ-I:HEK293 coculture system, PRX-08066 significantly decreased 5-HT release (>60%), Ki67 (transcript and immunostaining: 20%-80%), TGFβ1, CTGF, and FGF2 transcription (20%-50%) in the KRJ-I cell line. 5-HT itself stimulated HEK293 proliferation (25%) and synthesis of TGFβ1, CTGF and FGF2. PRX-08066 inhibition of KRJ-I function reversed these effects in the coculture system. CONCLUSIONS: Targeting the 5-HT2B receptor may be an effective antiproliferative and antifibrotic strategy for SI-NETs because it inhibits tumor microenvironment fibroblasts as well as NET cells. Fibrosis and proliferation appear to be biologically interfaced neuroendocrine neoplasia domains. © 2010 American Cancer Society.

Rokstad A.M.,Norwegian University of Science and Technology | Gustafsson B.I.,Norwegian University of Science and Technology | Espevik T.,Norwegian University of Science and Technology | Bakke I.,Norwegian University of Science and Technology | And 4 more authors.
Cancer Science | Year: 2012

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5-HT) producing small intestinal neuroendocrine tumor cell line KRJ-1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno-barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ-1 cells to achieve long-term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca2+/Ba2+) and microcapsule core properties, and variations in KRJ-1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5-HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200-350 μm. Long-term survival (>30 days) was seen with solid Ca2+/Ba2+ microbeads and hollow microcapsules. Basal 5-HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca2+/Ba2+ microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ-1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5-HT secretion, while Ca2+/Ba2+ microbeads provide a more stable bioreactor similar to non-encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies. © 2012 Japanese Cancer Association.

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