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Sample records for enhances astrocyte proliferation

  1. Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse

    PubMed Central

    Sourial, Mary; Doering, Laurie C.

    2016-01-01

    An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. Highlights: • We studied the proliferation of neural stem and progenitor cells in Fragile X. • We examined the role of astrocyte-secreted factors in neural precursor cell biology. • Astrocyte-secreted factors with differential expression in Fragile X identified. PMID:27242437

  2. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    SciTech Connect

    Kang, Kyungjun; Song, Mi-Ryoung

    2010-05-07

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  3. The expression of CAP1 after traumatic brain injury and its role in astrocyte proliferation.

    PubMed

    Zhang, Haiyan; Liu, Yonghua; Li, Yao; Zhou, Ying; Chen, Dongjian; Shen, Jianhong; Yan, Yaohua; Yan, Song; Wu, Xinmin; Li, Aihong; Guo, Aisong; Cheng, Chun

    2014-12-01

    Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins involved in the regulation of actin filaments, was recently reported to play a role in the pathology of sciatic nerves injury. However, the distribution and function of CAP1 in the central nervous system (CNS) remain unclear. To investigate whether CAP1 is involved in CNS injury and repair, we used an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of CAP1 in ipsilateral peritrauma cortex compared with the contralateral and sham-operated ones. Double immunofluorescence staining showed that CAP1 was co-expressed with glial fibrillary acidic protein (GFAP). In addition, we detected that Ki-67 had colocalization with GFAP and CAP1 after TBI. In vitro, during the process of lipopolysaccharide (LPS)-induced primary astrocyte proliferation, we observed enhanced expression of CAP1. Specially, CAP1-specific siRNA-transfected primary astrocytes show significantly decreased ability for proliferation. Together, all these data indicated that the change of CAP1 protein expression was associated with astrocyte proliferation after the trauma of the central nervous system (CNS).

  4. Increased proliferation and gliogenesis of cultured rat neural progenitor cells by lipopolysaccharide-stimulated astrocytes.

    PubMed

    Go, Hyo Sang; Shin, Chan Young; Lee, Sung Hoon; Jeon, Se-Jin; Kim, Ki Chan; Choi, Chang Soon; Ko, Kwang Ho

    2009-01-01

    Neural progenitor cells (NPC) are self-renewing multipotent cells that generate neurons and glial cells in the brain. NPCs generate neurons and glia not only during development but also after neural injury. Recent studies have shown that endogenous NPCs are activated after brain injury and migrate toward damaged areas where astrocyte activation occurs. Considering the massive proliferation of astrocytes as well as the production of several kinds of cytoactive molecules after brain injury, such as NO, growth factors and cytokines, it is tempting to think that cytoactive molecules released by activated glial cells regulate neural progenitor differentiation and proliferation through inflammatory mediators. To test this hypothesis, we stimulated rat primary astrocytes with lipopolysaccharide (LPS) to induce the activation of astrocytes. After addition of the conditioned media from LPS-stimulated astrocytes to NPC culture, proliferation was examined by MTT assay and bromodeoxyuridine (BrdU) incorporation. The differentiation of NPC into neurons and astrocytes was examined by Western blot, ELISA and immunocytochemical staining with cell-type-specific markers. Conditioned media from LPS-stimulated astrocytes increased NPC proliferation as well as gliogenesis as compared with control conditioned media from astrocytes without LPS stimulation. In contrast, neurogenesis was decreased by LPS-conditioned media. To investigate the molecular mechanism mediating glial differentiation and proliferation of NPC by reactive astrocytes, we added inhibitors of the Erk and JNK pathways during LPS stimulation. These inhibitors - except for a p38 inhibitor - decreased NPC proliferation and glial differentiation. These results suggest that LPS stimulated astrocytes generate factors regulating NPC proliferation and gliogenesis via the Erk and JNK pathways. PMID:19609085

  5. ROCK inhibition with Y27632 promotes the proliferation and cell cycle progression of cultured astrocyte from spinal cord.

    PubMed

    Yu, Zhiyuan; Liu, Miao; Fu, Peicai; Xie, Minjie; Wang, Wei; Luo, Xiang

    2012-12-01

    Rho-associated Kinase (ROCK) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. Although its effects on astrocyte proliferation have not been well characterized, ROCK has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. In the present study, our aim was to investigate the effect of ROCK inhibition by [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (Y27632) on proliferation and DNA synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. Western blots showed that treatment of astrocytes with Y27632 increased their expression of cyclin D1, CDK4, and cyclin E, thereby causing cell cycle progression. Furthermore, Y27632-induced astrocyte proliferation was mediated through the extracellular-signal-regulated kinase signaling cascade. These results indicate the importance of ROCK in astrocyte proliferation.

  6. Opposite influence of the metabotropic glutamate receptor subtypes mGlu3 and -5 on astrocyte proliferation in culture.

    PubMed

    Ciccarelli, R; Sureda, F X; Casabona, G; Di Iorio, P; Caruso, A; Spinella, F; Condorelli, D F; Nicoletti, F; Caciagli, F

    1997-12-01

    In non-synchronized, subconfluent secondary cultures of rat cortical astrocytes, the selective group-I metabotropic glutamate (mGlu) receptor agonist 3,5-dihydroxyphenylglycine (DHPG) increased [methyl-3H]-thymidine incorporation. This effect was mediated by the activation of the mGlu5 receptor, which was shown to be present by either RT-PCR or Western blot analysis. The mixed mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine reduced the increase in both intracellular Ca2+ and [methyl-3H]-thymidine incorporation produced by DHPG. In contrast, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), a potent and selective agonist of group-II mGlu receptors, reduced [methyl-3H]-thymidine incorporation in non-synchronized astrocyte cultures. The antiproliferative effect of DCG-IV was prevented by the selective group-II mGlu receptor antagonist (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV). The opposite effect of DHPG and DCG-IV on astrocyte proliferation was confirmed in cultures deprived of serum for 48 hours and then stimulated to proliferate with either epidermal growth factor (EGF) or the metabolically stable ATP analogue adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP). We conclude that activation of mGlu5 receptors enhances proliferation in cultured astrocytes, whereas activation of a receptor with pharmacological characteristics similar to those of mGlu2/3 receptors reduces proliferation. PMID:9419014

  7. Astrocytes.

    ERIC Educational Resources Information Center

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  8. Acetylcholine as a mitogen: muscarinic receptor-mediated proliferation of rat astrocytes and human astrocytoma cells.

    PubMed

    Guizzetti, M; Costa, P; Peters, J; Costa, L G

    1996-02-22

    The mitogenic effect of muscarinic receptor agonists in glial cells has been characterized in rat cortical astrocytes and human 132 1N1 astrocytoma cells. The muscarinic receptor agonist carbachol caused a dose- and time-dependent increase in proliferation, as measured by [3H]thymidine incorporation. The mitogenic effect was mimicked by several muscarinic, but not nicotinic receptor agonists, and was blocked by muscarinic receptor antagonists. Reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated the presence of m2, m3 and to a lesser degree, m5 muscarinic receptor mRNA in both astrocytes and astrocytoma cells. Proliferation experiments with subtype-specific muscarinic receptor antagonists suggest that carbachol-induced proliferation is due to activation of muscarinic M3 receptors. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) also stimulated glial cell proliferation. Down-regulation of protein kinase C, or the protein kinase C antagonist 1,5-(isoquinolynsulfanyl)-2-methylpiperazine dihydrochloride (H7) blocked proliferation induced by either TPA or carbachol. Of other neurotransmitters tested, histamine caused glial cell proliferation, norepinephrine and gamma-aminobutyric acid were ineffective, while serotonin and glutamate inhibited basal or serum-stimulated proliferation. PMID:8666059

  9. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  10. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

    PubMed Central

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K.; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  11. Changes in the morphology and proliferation of astrocytes induced by two modalities of chemically functionalized single-walled carbon nanotubes are differentially mediated by glial fibrillary acidic protein.

    PubMed

    Gottipati, Manoj K; Bekyarova, Elena; Brenner, Michael; Haddon, Robert C; Parpura, Vladimir

    2014-07-01

    Alterations in glial fibrillary acidic protein (GFAP) levels accompany the changes in the morphology and proliferation of astrocytes induced by colloidal solutes and films of carbon nanotubes (CNTs). To determine if GFAP is required for the effects of CNTs on astrocytes, we used astrocytes isolated from GFAP null mice. We find that selected astrocytic changes induced by CNTs are mediated by GFAP, i.e., perimeter, shape, and cell death for solutes, and proliferation for films.

  12. mGlu3 receptor blockade inhibits proliferation and promotes astrocytic phenotype in glioma stem cells.

    PubMed

    Zhou, Kun; Song, Yechun; Zhou, Wei; Zhang, Chunqing; Shu, Haifeng; Yang, Hui; Wang, Bin

    2014-04-01

    We have characterised, using both in vivo and in vitro methods, the effects of the metabotropic glutamate receptor subtype 3 (mGlu3) antagonist (LY341495) and agonist (LY379268) on the proliferation and differentiation of glioma stem cells (GSC). For in vitro studies, a CCK-8 assay was used to determine the cell proliferation, flow cytometry was performed to determine cell cycle phases, and immunohistochemistry and laser confocal microscopy were employed to detect CD133 expression. For in vivo studies, GSCs were injected into nude mice treated with either LY379268 or LY341495 and the growth of the tumours was measured after 3 weeks. When compared with controls, the proliferation rates and proportion of cells in S phase within the LY341495 treated group decreased in a time-dependent manner. In the presence of differentiation medium containing LY341495, GSC differentiation was diverted into an astrocyte rather than neuronal phenotype. The growth rate and volume of tumours injected into nude mice was reduced in LY341495 treated mice compared with controls. Thus pharmacological blockade of mGlu3 receptor signalling pathway significantly inhibits the growth and proliferation of GSCs both in vitro and in vivo while promoting differentiation to astrocytes. These results further implicate mGlu3 in the biology of glioma and as a target for continued research. PMID:24482010

  13. Maternal obesity leads to increased proliferation and numbers of astrocytes in the developing fetal and neonatal mouse hypothalamus.

    PubMed

    Kim, Dong Won; Glendining, Kelly A; Grattan, David R; Jasoni, Christine L

    2016-10-01

    Maternal obesity during pregnancy is associated with chronic maternal, placental, and fetal inflammation; and it elevates the risk for offspring obesity. Changes in the development of the hypothalamus, a brain region that regulates body weight and energy balance, are emerging as important determinants of offspring risk, but such changes are only beginning to be defined. Here we focused on the hypothesis that the pathological exposure of developing hypothalamic astrocytes to cytokines would alter their development. A maternal high-fat diet (mHFD) mouse model was used to investigate changes in hypothalamic astrocytes in the fetus during late gestation and in early neonates by using immunochemistry, confocal microscopy, and qPCR. The number of astrocytes and the proportion of proliferating astrocytes was significantly higher in the arcuate nucleus (ARC) and the supraoptic nucleus (SON) of the hypothalamus at both ages compared to control offspring from normal weight pregnancies. Supplemental to this we found that cultured fetal hypothalamic astrocytes proliferated significantly in response to IL6 (10ng/ml), one of the cytokines significantly elevated in fetuses of obese dams, via the JAK/STAT3 signaling pathway. Thus, maternal obesity during pregnancy stimulated the proliferation and thereby increased numbers of astrocytes in the fetal as well as early neonatal hypothalamus, which may be driven, during fetal life, by IL6. PMID:27326907

  14. Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury.

    PubMed

    Gu, Jun; Ni, Yingjie; Xu, Lin; Xu, Hongliang; Cai, Zhengdong

    2016-01-22

    Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in the spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.

  15. PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1.

    PubMed

    Lv, Youyou; Zhang, Bo; Zhai, Chunchun; Qiu, Jin; Zhang, Yue; Yao, Wenlong; Zhang, Chuanhan

    2015-12-01

    Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

  16. Tricyclic Antidepressant Amitriptyline Indirectly Increases the Proliferation of Adult Dentate Gyrus-Derived Neural Precursors: An Involvement of Astrocytes

    PubMed Central

    Boku, Shuken; Hisaoka-Nakashima, Kazue; Nakagawa, Shin; Kato, Akiko; Kajitani, Naoto; Inoue, Takeshi; Kusumi, Ichiro; Takebayashi, Minoru

    2013-01-01

    Antidepressants increase the proliferation of neural precursors in adult dentate gyrus (DG), which is considered to be involved in the therapeutic action of antidepressants. However, the mechanism underlying it remains unclear. By using cultured adult rat DG-derived neural precursors (ADP), we have already shown that antidepressants have no direct effects on ADP. Therefore, antidepressants may increase the proliferation of neural precursors in adult DG via unknown indirect mechanism. We have also shown that amitriptyline (AMI), a tricyclic antidepressant, induces the expressions of GDNF, BDNF, FGF2 and VEGF, common neurogenic factors, in primary cultured astrocytes (PCA). These suggest that AMI-induced factors in astrocytes may increase the proliferation of neural precursors in adult DG. To test this hypothesis, we examined the effects of AMI-induced factors and conditioned medium (CM) from PCA treated with AMI on ADP proliferation. The effects of CM and factors on ADP proliferation were examined with BrdU immunocytochemistry. AMI had no effect on ADP proliferation, but AMI-treated CM increased it. The receptors of GDNF, BDNF and FGF2, but not VEGF, were expressed in ADP. FGF2 significantly increased ADP proliferation, but not BDNF and GDNF. In addition, both of a specific inhibitor of FGF receptors and anti-FGF2 antibody significantly counteracted the increasing effect of CM on ADP proliferation. In addition, FGF2 in brain is mainly derived from astrocytes that are key components of the neurogenic niches in adult DG. These suggest that AMI may increase ADP proliferation indirectly via PCA and that FGF2 may a potential candidate to mediate such an indirect effect of AMI on ADP proliferation via astrocytes. PMID:24260208

  17. Notch1–STAT3–ETBR signaling axis controls reactive astrocyte proliferation after brain injury

    PubMed Central

    LeComte, Matthew D.; Shimada, Issei S.; Sherwin, Casey; Spees, Jeffrey L.

    2015-01-01

    Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP+) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETBR) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETBR expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETBR-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1–STAT3–ETBR axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury. PMID:26124113

  18. Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2.

    PubMed

    Kirby, Elizabeth D; Muroy, Sandra E; Sun, Wayne G; Covarrubias, David; Leong, Megan J; Barchas, Laurel A; Kaufer, Daniela

    2013-04-16

    Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain. DOI:http://dx.doi.org/10.7554/eLife.00362.001.

  19. Lactate flux in astrocytes is enhanced by a non-catalytic action of carbonic anhydrase II

    PubMed Central

    Stridh, Malin H; Alt, Marco D; Wittmann, Sarah; Heidtmann, Hella; Aggarwal, Mayank; Riederer, Brigitte; Seidler, Ursula; Wennemuth, Gunther; McKenna, Robert; Deitmer, Joachim W; Becker, Holger M

    2012-01-01

    Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a ‘proton collecting antenna’ for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate. PMID:22451434

  20. Morphological transformation and proliferation of rat astrocytes as induced by sulfated polysaccharides from the sea cucumber Stichopus japonicus.

    PubMed

    Sheng, Xiehuang; Zhang, Nannan; Song, Shuliang; Li, Min; Liang, Hao; Zhang, Yuejie; Wang, Yunshan; Ji, Aiguo

    2011-09-26

    In this report, we demonstrate that the sulfated polysaccharide, Haishen (HS), which was isolated from the body wall of the sea cucumber Stichopus japonicus can induce morphological transformation and proliferation of astrocytes in vitro when combined with basic fibroblast growth factor 2 (FGF-2). Cell morphology showed no change when induced by HS or FGF-2 alone. However, combinational treatment of HS and FGF-2 promoted transformation of normal astrocyte into a stella morphology (stellation), along with an increase in the expression and rearrangement of glial fibrillary acidic protein (GFAP). Further analysis of HS- and FGF-2-treated cells indicated a reduced percentage of cells in the G0/G1 phase, whereas the cell proliferation index (S phase) was increased. The proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells increased in response to the combination of HS and FGF-2. With respect to cell cycle signaling, immunoblotting assay demonstrated an accumulation of Cyclin D1. These observations suggest that HS may play a role in astrocyte morphological transformation and proliferation, and this activation requires a synergism with FGF-2.

  1. Astrocytic TIMP-1 Promotes Oligodendrocyte Differentiation and Enhances CNS Myelination

    PubMed Central

    Moore, Craig S.; Milner, Richard; Nishiyama, Akiko; Frausto, Ricardo F.; Serwanski, David R.; Pagarigan, Roberto R.; Whitton, J. Lindsay; Miller, Robert H.; Crocker, Stephen J.

    2011-01-01

    Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1KO mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1KO mice which coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1KO mice revealed a specific deficit of NG2+ oligodendrocyte progenitor cells. Application of rmTIMP-1 to TIMP-1KO neurosphere cultures evoked a dose-dependent increase in NG2+ cell numbers, while treatment with GM6001, a potent broad spectrum MMP inhibitor did not. Similarly, administration of recombinant murine TIMP-1 (rmTIMP-1) to A2B5+ immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1+ oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5+ oligodendrocyte progenitors grown in conditioned media derived from TIMP-1KO primary glial cultures resulted in reduced differentiation of mature O1+ oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS. PMID:21508247

  2. Nuclear translocation of PKM2 modulates astrocyte proliferation via p27 and -catenin pathway after spinal cord injury.

    PubMed

    Zhang, Jinlong; Feng, Guijuan; Bao, Guofeng; Xu, Guanhua; Sun, Yuyu; Li, Weidong; Wang, Lingling; Chen, Jiajia; Jin, Huricha; Cui, Zhiming

    2015-01-01

    Aberrant functionality of the cell cycle has been implicated in the pathology of traumatic SCI. Although it has been reported that the expressions of various cell cycle related proteins were altered significantly following SCI, detailed information on the subject remains largely unclear. The embryonic pyruvate kinase M2 (PKM2) is an important metabolic kinase in aerobic glycolysis or the warburg effect, however, its functions in central nervous system (CNS) injury remains elusive. Here we demonstrate that PKM2 was not only significantly upregulated by western blot and immunohistochemistry but certain traumatic stimuli also induced translocation of PKM2 into the nucleus in astrocytes following spinal cord injury (SCI). Furthermore, the expression levels and localization of p-β-catenin, p27, cyclin D1 and PCNA were correlated with PKM2 after SCI. In vitro, we also found that PKM2 co-immunoprecipitation with p-β-catenin and p27 respectively. Knockdown of PKM2 apparently decreased the level of PCNA, cyclinD1, p27 in primary astrocyte cells. Taken together, our findings indicate that nuclear translocation of PKM2 promotes astrocytes proliferation after SCI through modulating cell cycle signaling. These discoveries firstly uncovered the role of PKM2 in spinal cord injury and provided a potential therapeutic target for CNS injury and repair.

  3. IL-17 enhancement of the IL-6 signaling cascade in astrocytes.

    PubMed

    Ma, Xiangyu; Reynolds, Stephanie L; Baker, Brandi J; Li, Xingang; Benveniste, Etty N; Qin, Hongwei

    2010-05-01

    Astrocytes have important physiological roles in CNS homeostasis and serve as a bridge between the CNS and immune system. IL-17 and IL-6 are important in many CNS disorders characterized by neuroinflammation. We examined the role of IL-17 on the IL-6 signaling cascade in primary astrocytes. IL-17 functioned in a synergistic manner with IL-6 to induce IL-6 expression in astrocytes. The synergistic effect involved numerous signaling pathways including NF-kappaB, JNK MAPK, and p38 MAPK. The NF-kappaB pathway inhibitor BAY-11, JNK inhibitor JNKi II, and p38 inhibitor SB203580 suppressed the synergistic effect of IL-6 and IL-17 on IL-6 expression. IL-17 synergized with IL-6 to enhance the recruitment of activated NF-kappaB p65, c-Fos, c-Jun, and the histone acetyltransferases CREB-binding protein and p300 to the IL-6 promoter in vivo to induce IL-6 transcription. This was accompanied by enhanced acetylation of histones H3 and H4 on the IL-6 promoter. Moreover, we elucidated an important role for suppressor of cytokine signaling (SOCS) 3 in IL-17 enhancement of IL-6 signaling in astrocytes. SOCS3 small interfering RNA knockdown and SOCS3 deletion in astrocytes augmented the synergistic effect of IL-6 and IL-17 due to an enhancement of activation of the NF-kappaB and MAPK pathways. These results indicate that astrocytes can serve as a target of Th17 cells and IL-17 in the CNS, and SOCS3 participates in IL-17 functions in the CNS as a negative feedback regulator.

  4. Diclofenac enhances proinflammatory cytokine-induced aquaporin-4 expression in cultured astrocyte.

    PubMed

    Asai, Hayato; Kakita, Hiroki; Aoyama, Mineyoshi; Nagaya, Yoshiaki; Saitoh, Shinji; Asai, Kiyofumi

    2013-04-01

    Acute encephalopathy is a generic term for acute brain dysfunction occurring after infection. Acute encephalopathy induced by influenza virus results in high mortality, and most cases of influenza-associated encephalopathy (IAE) result in brain edema. Administration of diclofenac sodium (DCF), a non-steroidal anti-inflammatory drug (NSAID), is associated with a significant increased mortality rate of IAE. These previous clinical findings proposed further investigation of DCF administration and brain edema to clarify how DCF aggravates IAE. Aquaporin-4 (AQP4) is the predominant water channel protein in the mammalian brain, and is mainly expressed in astrocytes. AQP4 plays an important role in brain water homeostasis. Therefore, we investigated a possible association between DCF and AQP4 production in astrocytes. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor α, and interferon γ, and then treated with DCF. DCF enhanced proinflammatory cytokine-induced AQP4 gene and protein expression in astrocytes, whereas DCF alone did not change the AQP4 gene expression. The addition of nuclear factor-kappa B (NF-κB) inhibitor abrogated AQP4 gene and protein expression completely in astrocytes treated with cytokines alone and in those also treated with DCF. In conclusion, this study demonstrated that AQP4 is upregulated in astrocyte by proinflammatory cytokines, and that the addition of DCF further augments AQP4 production. This effect is mediated via NF-κB signaling. The enhancement of AQP4 production by DCF may explain the significantly increased mortality rates in IAE patients treated with DCF.

  5. Enhancement of FGF-1 release along with cytosolic proteins from rat astrocytes by hydrogen peroxide.

    PubMed

    Ito, Jin-Ichi; Nagayasu, Yuko; Hoshikawa, Mariko; Kato, Koichi H; Miura, Yutaka; Asai, Kiyofumi; Hayashi, Hideki; Yokoyama, Shinji; Michikawa, Makoto

    2013-07-19

    We previously observed that the production and release of fibroblast growth factor (FGF-1) are increased in rat astrocytes during in vitro long-term culture, that FGF-1 enhances the generation of apoE-containing high density lipoproteins (apoE/HDL), and that the wound healing of brain cryoinjury delays in apoE-deficient mouse. The detail mechanism underlying these phenomena remains unknown. In this study, we examined effects of oxidative stress on release of FGF-1 from cultured rat astrocytes. The treatment of rat astrocytes with 100µM hydrogen peroxide (H2O2) for 10min enhanced FGF-1 release without inducing apoptosis. The conditioned medium prepared from the cells cultured in a fresh medium after the treatment with H2O2 had the FGF-1-like activities, which enhanced cholesterol synthesis, signalings to phosphorylate Akt and ERK, and apoE secretion. The oxidative stress induced by H2O2 enhanced the release of cytosolic proteins such as HSP70 and HSP90 in addition to FGF-1. Antioxidants such as ascorbic acid and ebselen suppressed the release of cytosolic proteins induced by H2O2 treatment. The addition of lipoproteins such as low density lipoproteins (LDL), furthermore, canceled H2O2-induced release of FGF-1 and cytosolic proteins. Proteolysis of cytosolic proteins in the H2O2-treated rat astrocytes was enhanced in the presence of exogenous trypsin, which was attenuated by the pretreatment with LDL, suggesting that H2O2 increases the permeability of the membrane of cells, which was prevented by the addition of lipoproteins. These findings suggest that oxidative stress is one of the candidates which triggers FGF-1 release from astrocytes in the brain, and that the lipid homeostasis in the cell membrane may regulate H2O2-induced release of FGF-1. PMID:23727614

  6. Mdivi-1 Inhibits Astrocyte Activation and Astroglial Scar Formation and Enhances Axonal Regeneration after Spinal Cord Injury in Rats

    PubMed Central

    Li, Gang; Cao, Yang; Shen, Feifei; Wang, Yangsong; Bai, Liangjie; Guo, Weidong; Bi, Yunlong; Lv, Gang; Fan, Zhongkai

    2016-01-01

    After spinal cord injury (SCI), astrocytes become hypertrophic, and proliferative, forming a dense network of astroglial processes at the site of the lesion. This constitutes a physical and biochemical barrier to axonal regeneration. Mitochondrial fission regulates cell cycle progression; inhibiting the cell cycle of astrocytes can reduce expression levels of axon growth-inhibitory molecules as well as astroglial scar formation after SCI. We therefore investigated how an inhibitor of mitochondrial fission, Mdivi-1, would affect astrocyte proliferation, astroglial scar formation, and axonal regeneration following SCI in rats. Western blot and immunofluorescent double-labeling showed that Mdivi-1 markedly reduced the expression of the astrocyte marker glial fibrillary acidic protein (GFAP), and a cell proliferation marker, proliferating cell nuclear antigen, in astrocytes 3 days after SCI. Moreover, Mdivi-1 decreased the expression of GFAP and neurocan, a chondroitin sulfate proteoglycan. Notably, immunofluorescent labeling and Nissl staining showed that Mdivi-1 elevated the production of growth-associated protein-43 and increased neuronal survival at 4 weeks after SCI. Finally, hematoxylin-eosin staining, and behavioral evaluation of motor function indicated that Mdivi-1 also reduced cavity formation and improved motor function 4 weeks after SCI. Our results confirm that Mdivi-1 promotes motor function after SCI, and indicate that inhibiting mitochondrial fission using Mdivi-1 can inhibit astrocyte activation and astroglial scar formation and contribute to axonal regeneration after SCI in rats. PMID:27807407

  7. Characterization of the BAC Id3-enhanced green fluorescent protein transgenic mouse line for in vivo imaging of astrocytes

    PubMed Central

    Lamantia, Cassandra; Tremblay, Marie-Eve; Majewska, Ania

    2014-01-01

    Abstract. Astrocytes are highly ramified glial cells with critical roles in brain physiology and pathology. Recently, breakthroughs in imaging technology have expanded our understanding of astrocyte function in vivo. The in vivo study of astrocytic dynamics, however, is limited by the tools available to label astrocytes and their processes. Here, we characterize the bacterial artificial chromosome transgenic Id3-EGFP knock-in mouse to establish its usefulness for in vivo imaging of astrocyte processes. Using fixed brain sections, we observed enhanced green fluorescent protein expression in astrocytes and blood vessel walls throughout the brain, although the extent and cell type specificity of expression depended on the brain area and developmental age. Using in vivo two-photon imaging, we visualized astrocytes in cortical layers 1–3 in both thin skull and window preparations. In adult animals, astrocytic cell bodies and fine processes could be followed over many hours. Our results suggest that Id3 mice could be used for in vivo imaging of astrocytes and blood vessels in development and adulthood. PMID:26157970

  8. Increased astrocytic ATP release results in enhanced excitability of the hippocampus.

    PubMed

    Lee, Hae Ung; Yamazaki, Yoshihiko; Tanaka, Kenji F; Furuya, Kishio; Sokabe, Masahiro; Hida, Hideki; Takao, Keizo; Miyakawa, Tsuyoshi; Fujii, Satoshi; Ikenaka, Kazuhiro

    2013-02-01

    Astrocytes, a major subtype of glia, interact with neurons as a supportive partner supplying energy sources and growth factors. Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters (gliotransmitters). However, the precise role of gilotransmitters in regulating neuronal activity is still under debate. Here, we report that a subtle enhancement in the release of one gliotransmitter, ATP, affects synaptic potentiation from an analysis of mice containing an astrocyte-selective (GFAP) mutation. We found that, relative to normal mice, weaker stimulation induced long-term potentiation (LTP) in mutant mice, indicating that the threshold to induce LTP was lowered in the mutant. While excitatory transmission was normal in the mutant, inhibitory GABAergic transmission was suppressed. We found that a low concentration of adenosine selectively attenuated inhibitory neuronal activity and lowered the threshold to induce LTP in wild type mice. In comparison, adenosine A(1) receptor antagonism reversed the lowered LTP threshold back to normal in the mutant mouse. We verified that adenosine levels in the cerebrospinal fluid of mutant mice were slightly elevated compared to wild type mice. This was apparently caused by an increase in ATP release from mutant astrocytes that could provide a source of augmented adenosine levels in the mutant. ATP is thought to suppress the excitability of neuronal circuits; however, a small increase in ATP release can result in a suppressed inhibitory tone and enhanced excitability of neuronal circuitry. These findings demonstrate that ATP released from astrocytes acts in a bidirectional fashion to regulate neuronal excitability depending on concentration. PMID:23018918

  9. Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration.

    PubMed

    Merzak, A; Koochekpour, S; Fillion, M P; Fillion, G; Pilkington, G J

    1996-09-01

    Expression of seven serotonin or 5-hydroxytryptamine (5-HT) receptors (5-HT1D alpha, 5-HT1E, 5-HT2, 5-HT1A, 5-HT1C, 5-HT1D beta, and 5-HT6) was investigated in human normal fetal astrocytes and eight glioma cell lines by reverse transcription and polymerase chain reaction (RT-PCR). No expression of 5-HT1D beta and 5-HT6 was observed in any of the cell lines studied. The 5-HT1D alpha receptor was found to be expressed in two human glioma cell lines but not in normal astrocytes. In addition, only three glioma cell lines expressed the 5-HT1E receptor. The 5-HT1C receptor was expressed in six glioma cell lines but not in normal astrocytes while the 5-HT1A was found to be expressed in normal astrocytes from the left hemisphere and in six glioma cell lines but not in normal astrocytes from the cerebellum. Interestingly, the 5-HT2 receptor was expressed in all cells studied but very weakly in normal astrocytes. The effect of 5-HT on glioma cell proliferation, migration, and invasion was also investigated. Serotonin was found to positively modulate these three processes in vitro. These results suggest that 5-HT may play an important role in the control of the biological properties of human glioma cells. PMID:8883928

  10. Acute effects of neonatal dexamethasone treatment on proliferation and astrocyte immunoreactivity in hippocampus and corpus callosum: towards a rescue strategy.

    PubMed

    Claessens, Sanne E F; Belanoff, Joseph K; Kanatsou, Sofia; Lucassen, Paul J; Champagne, Danielle L; de Kloet, E Ronald

    2012-10-30

    Dexamethasone (DEX), a synthetic glucocorticoid, has been used to treat respiratory distress syndrome in prematurely born infants. Despite the important short-term benefit on lung function, there is growing concern about the long-term outcome of this treatment, since follow-up studies of prematurely born infants have shown lasting adverse neurodevelopmental effects. Since the mechanism underlying these neurodevelopmental impairments is largely unknown, the aim of the present study was (i) to investigate the acute effects of neonatal DEX treatment on the developing brain; and (ii) to block specifically the effects of DEX on the brain by central administration of the glucocorticoid receptor (GR) antagonist mifepristone. Long Evans rat pups were injected subcutaneously with tapering doses of DEX or saline (SAL) on postnatal days (pnd) 1, 2 and 3. Separate groups received intracerebroventricular injections with mifepristone prior to DEX treatment. On pnd 4 and 10, pups were sacrificed and brains collected for analysis of cell proliferation (Ki-67) and astrogliosis (GFAP). We report that neonatal DEX treatment reduced hippocampal cell proliferation on pnd 4, an effect that was normalized by pnd 10. Although on pnd 4, GFAP expression was not affected, DEX treatment caused a significant reduction in the number and density of astrocytes in hippocampus and corpus callosum on pnd 10, which was normalized by mifepristone pre-treatment. These acute alterations in the neonate brain might underlie later functional impairments reported in DEX-treated animals and humans and further illustrate the impact of early GR activation on brain development.

  11. Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-{kappa}B signaling in cultured astrocytes

    SciTech Connect

    Kakita, Hiroki; Aoyama, Mineyoshi Hussein, Mohamed Hamed; Kato, Shin; Suzuki, Satoshi; Ito, Tetsuya; Togari, Hajime; Asai, Kiyofumi

    2009-07-01

    Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1{beta}, tumor necrosis factor-{alpha} and interferon-{gamma}, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-{kappa}B inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-{kappa}B p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, L-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-{kappa}B signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.

  12. Neuroprotective Effect of Uncaria rhynchophylla in Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression

    PubMed Central

    Liu, Chung-Hsiang; Lin, Yi-Wen; Tang, Nou-Ying; Liu, Hsu-Jan; Hsieh, Ching-Liang

    2012-01-01

    Uncaria rhynchophylla (UR), which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA-) induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABAA) receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus PMID:21837247

  13. Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF.

    PubMed

    Yang, Fan; Liu, Yunhui; Tu, Jie; Wan, Jun; Zhang, Jie; Wu, Bifeng; Chen, Shanping; Zhou, Jiawei; Mu, Yangling; Wang, Liping

    2014-12-17

    Astrocytes provide neuroprotective effects against degeneration of dopaminergic (DA) neurons and play a fundamental role in DA differentiation of neural stem cells. Here we show that light illumination of astrocytes expressing engineered channelrhodopsin variant (ChETA) can remarkably enhance the release of basic fibroblast growth factor (bFGF) and significantly promote the DA differentiation of human embryonic stem cells (hESCs) in vitro. Light activation of transplanted astrocytes in the substantia nigra (SN) also upregulates bFGF levels in vivo and promotes the regenerative effects of co-transplanted stem cells. Importantly, upregulation of bFGF levels, by specific light activation of endogenous astrocytes in the SN, enhances the DA differentiation of transplanted stem cells and promotes brain repair in a mouse model of Parkinson's disease (PD). Our study indicates that astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and may provide a strategy targeting astrocytes for treatment of PD.

  14. Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1

    PubMed Central

    Yao, Jie; Qin, Li; Miao, Sen; Wang, Xiangshan; Wu, Xuejian

    2016-01-01

    There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, β-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3′-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/β-catenin signaling pathway, and inhibition of this pathway by β-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/β-catenin signaling pathway.

  15. Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1

    PubMed Central

    Yao, Jie; Qin, Li; Miao, Sen; Wang, Xiangshan; Wu, Xuejian

    2016-01-01

    There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, β-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3′-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/β-catenin signaling pathway, and inhibition of this pathway by β-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/β-catenin signaling pathway. PMID:27602115

  16. PKC signaling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes.

    PubMed

    Hama, Hiroshi; Hara, Chikako; Yamaguchi, Kazuhiko; Miyawaki, Atsushi

    2004-02-01

    Here we provide evidence that astrocytes affect neuronal synaptogenesis by the process of adhesion. Local contact with astrocytes via integrin receptors elicited protein kinase C (PKC) activation in individual dissociated neurons cultured in astrocyte-conditioned medium. This activation, initially focal, soon spread throughout the entire neuron. We then demonstrated pharmacologically that the arachidonic acid cascade, triggered by the integrin reception, is responsible for the global activation of PKC. Local astrocytic contact also facilitated excitatory synaptogenesis throughout the neuron, a process which could be blocked by inhibitors of both integrins and PKC. Thus, propagation of PKC signaling represents an underlying mechanism for global neuronal maturation following local astrocyte adhesion.

  17. Pro-inflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes

    PubMed Central

    Williams, Rachel; Dhillon, Navneet K.; Hegde, Sonia T.; Yao, Honghong; Peng, Fuwang; Callen, Shannon; Chebloune, Yahia; Davis, Randall L.; Buch, Shilpa J.

    2009-01-01

    HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-γ inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the pro-inflammatory cytokines, IFN-γ and TNF-α, are also markedly increased in CNS tissues during HIV-1 infection. In the present study we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the pro-inflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-κB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD. PMID:18985732

  18. [Enhanced control of proliferation in telomerized cells].

    PubMed

    Egorov, E E; Moldaver, M V; Vishniakova, Kh S; Terekhov, S M; Dashinimaev, E B; Cheglakov, I B; Toropygin, I Iu; Iarygin, K N; Chumakov, P M; Korochkin, L I; Antonova, G A; Rybalkina, E Iu; Saburina, I N; Burnaevskiĭ, N S; Zelenin, A V

    2007-01-01

    Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people

  19. Progranulin enhances neural progenitor cell proliferation through glycogen synthase kinase 3β phosphorylation.

    PubMed

    Nedachi, T; Kawai, T; Matsuwaki, T; Yamanouchi, K; Nishihara, M

    2011-06-30

    Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3β), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3β-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3β phosphorylation. PMID:21540081

  20. Physical Exercise Enhances Cognitive Flexibility as Well as Astrocytic and Synaptic Markers in the Medial Prefrontal Cortex

    PubMed Central

    Brockett, Adam T.; LaMarca, Elizabeth A.; Gould, Elizabeth

    2015-01-01

    Physical exercise enhances a wide range of cognitive functions in humans. Running-induced cognitive enhancement has also been demonstrated in rodents but with a strong emphasis on tasks that require the hippocampus. Additionally, studies designed to identify mechanisms that underlie cognitive enhancement with physical exercise have focused on running-induced changes in neurons with little attention paid to such changes in astrocytes. To further our understanding of how the brain changes with physical exercise, we investigated whether running alters performance on cognitive tasks that require the prefrontal cortex and whether any such changes are associated with astrocytic, as well as neuronal, plasticity. We found that running enhances performance on cognitive tasks known to rely on the prefrontal cortex. By contrast, we found no such improvement on a cognitive task known to rely on the perirhinal cortex. Moreover, we found that running enhances synaptic, dendritic and astrocytic measures in several brain regions involved in cognition but that changes in the latter measures were more specific to brain regions associated with cognitive improvements. These findings suggest that physical exercise induces widespread plasticity in both neuronal and nonneuronal elements and that both types of changes may be involved in running-induced cognitive enhancement. PMID:25938418

  1. Differentiation of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; de Vellis, J.

    1981-01-01

    Homogeneous cultures of astrocytes and oligodendrocytes provide an excellent model system for studying the regulation of glial structure and function. Recently, a chemically defined (CD) medium was developed for purified cultures of astrocytes, thus eliminating the requirement for serum and providing a controlled system for the study of astroglial properties. Due to the widespread use of astrocyte cultures and the potential benefits to be gained from using a defined medium, astrocyte cultures raised in CD medium were analyzed for purity as well as morphological and biochemical properties. Purity was assessed using immunocytochemical staining for glial fibrillary acidic protein (GFAP) and fibronectin. Astrocytes raised in CD medium are 95% pure using the expression of GFAP as a criterion. Fewer than 1% of the cells in CD medium stained positive for fibronectin eliminating the possibility that CD medium is selective for meningeal or endothelial cells. Astrocytes raised in CD medium exhibit a striking degree of morphological differentiation as seen in scanning electron micrographs. They also exhibit a high degree of biochemical differentiation illustrated by increases in the specific activity of S-100 protein and the induction of glutamine synthetase by glucocorticoids. A defined medium that supports the proliferation of rat astrocytes and enhances numerous morphological and biochemical properties should greatly facilitate the study of factors controlling glial proliferation and differentiation.

  2. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

    PubMed

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

  3. The Modulation of Neurotrophin and Epigenetic Regulators: Implication for Astrocyte Proliferation and Neuronal Cell Apoptosis After Spinal Cord Injury

    PubMed Central

    2016-01-01

    Objective To investigate alterations in the expression of the main regulators of neuronal survival and death related to astrocytes and neuronal cells in the brain in a mouse model of spinal cord injury (SCI). Methods Eight-week-old male imprinting control region mice (n=36; 30–35 g) were used in this study and randomly assigned to two groups: the naïve control group (n=18) and SCI group (n=18). The mice in both groups were randomly allocated to the following three time points: 3 days, 1 week, and 2 weeks (n=6 each). The expression levels of regulators such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), histone deacetylase 1 (HDAC1), and methyl-CpG-binding protein 2 (MeCP 2) in the brain were evaluated following thoracic contusive SCI. In addition, the number of neuronal cells in the motor cortex (M1 and M2 areas) and the number of astrocytes in the hippocampus were determined by immunohistochemistry. Results BDNF expression was significantly elevated at 2 weeks after injury (p=0.024). The GDNF level was significantly elevated at 3 days (p=0.042). The expression of HDAC1 was significantly elevated at 1 week (p=0.026). Following SCI, compared with the control the number of NeuN-positive cells in the M1 and M2 areas gradually and consistently decreased at 2 weeks after injury. In contrast, the number of astrocytes was significantly increased at 1 week (p=0.029). Conclusion These results demonstrate that the upregulation of BDNF, GDNF and HDAC1 might play on important role in brain reorganization after SCI. PMID:27606261

  4. The Modulation of Neurotrophin and Epigenetic Regulators: Implication for Astrocyte Proliferation and Neuronal Cell Apoptosis After Spinal Cord Injury

    PubMed Central

    2016-01-01

    Objective To investigate alterations in the expression of the main regulators of neuronal survival and death related to astrocytes and neuronal cells in the brain in a mouse model of spinal cord injury (SCI). Methods Eight-week-old male imprinting control region mice (n=36; 30–35 g) were used in this study and randomly assigned to two groups: the naïve control group (n=18) and SCI group (n=18). The mice in both groups were randomly allocated to the following three time points: 3 days, 1 week, and 2 weeks (n=6 each). The expression levels of regulators such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), histone deacetylase 1 (HDAC1), and methyl-CpG-binding protein 2 (MeCP 2) in the brain were evaluated following thoracic contusive SCI. In addition, the number of neuronal cells in the motor cortex (M1 and M2 areas) and the number of astrocytes in the hippocampus were determined by immunohistochemistry. Results BDNF expression was significantly elevated at 2 weeks after injury (p=0.024). The GDNF level was significantly elevated at 3 days (p=0.042). The expression of HDAC1 was significantly elevated at 1 week (p=0.026). Following SCI, compared with the control the number of NeuN-positive cells in the M1 and M2 areas gradually and consistently decreased at 2 weeks after injury. In contrast, the number of astrocytes was significantly increased at 1 week (p=0.029). Conclusion These results demonstrate that the upregulation of BDNF, GDNF and HDAC1 might play on important role in brain reorganization after SCI.

  5. Assessment of C-phycocyanin effect on astrocytes-mediated neuroprotection against oxidative brain injury using 2D and 3D astrocyte tissue model

    PubMed Central

    Min, Seul Ki; Park, Jun Sang; Luo, Lidan; Kwon, Yeo Seon; Lee, Hoo Cheol; Jung Shim, Hyun; Kim, Il-Doo; Lee, Ja-Kyeong; Shin, Hwa Sung

    2015-01-01

    Drugs are currently being developed to attenuate oxidative stress as a treatment for brain injuries. C-phycocyanin (C-Pc) is an antioxidant protein of green microalgae known to exert neuroprotective effects against oxidative brain injury. Astrocytes, which compose many portions of the brain, exert various functions to overcome oxidative stress; however, little is known about how C-Pc mediates the antioxidative effects of astrocytes. In this study, we revealed that C-Pc intranasal administration to the middle cerebral artery occlusion (MCAO) rats ensures neuroprotection of ischemic brain by reducing infarct size and improving behavioral deficits. C-Pc also enhanced viability and proliferation but attenuated apoptosis and reactive oxygen species (ROS) of oxidized astrocytes, without cytotoxicity to normal astrocytes and neurons. To elucidate how C-Pc leads astrocytes to enhance neuroprotection and repair of ischemia brain, we firstly developed 3D oxidized astrocyte model. C-Pc had astrocytes upregulate antioxidant enzymes such as SOD and catalase and neurotrophic factors BDNF and NGF, while alleviating inflammatory factors IL-6 and IL-1β and glial scar. Additionally, C-Pc improved viability of 3D oxidized neurons. In summary, C-Pc was concluded to activate oxidized astrocytes to protect and repair the ischemic brain with the combinatorial effects of improved antioxidative, neurotrophic, and anti-inflammatory mechanisms. PMID:26399322

  6. Assessment of C-phycocyanin effect on astrocytes-mediated neuroprotection against oxidative brain injury using 2D and 3D astrocyte tissue model.

    PubMed

    Min, Seul Ki; Park, Jun Sang; Luo, Lidan; Kwon, Yeo Seon; Lee, Hoo Cheol; Shim, Hyun Jung; Kim, Il-Doo; Lee, Ja-Kyeong; Shin, Hwa Sung

    2015-01-01

    Drugs are currently being developed to attenuate oxidative stress as a treatment for brain injuries. C-phycocyanin (C-Pc) is an antioxidant protein of green microalgae known to exert neuroprotective effects against oxidative brain injury. Astrocytes, which compose many portions of the brain, exert various functions to overcome oxidative stress; however, little is known about how C-Pc mediates the antioxidative effects of astrocytes. In this study, we revealed that C-Pc intranasal administration to the middle cerebral artery occlusion (MCAO) rats ensures neuroprotection of ischemic brain by reducing infarct size and improving behavioral deficits. C-Pc also enhanced viability and proliferation but attenuated apoptosis and reactive oxygen species (ROS) of oxidized astrocytes, without cytotoxicity to normal astrocytes and neurons. To elucidate how C-Pc leads astrocytes to enhance neuroprotection and repair of ischemia brain, we firstly developed 3D oxidized astrocyte model. C-Pc had astrocytes upregulate antioxidant enzymes such as SOD and catalase and neurotrophic factors BDNF and NGF, while alleviating inflammatory factors IL-6 and IL-1β and glial scar. Additionally, C-Pc improved viability of 3D oxidized neurons. In summary, C-Pc was concluded to activate oxidized astrocytes to protect and repair the ischemic brain with the combinatorial effects of improved antioxidative, neurotrophic, and anti-inflammatory mechanisms. PMID:26399322

  7. Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements.

    PubMed

    Chatterjee, Sreejata; Sikdar, Sujit K

    2013-12-01

    While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress. PMID:24123181

  8. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  9. Graphene Enhances Cellular Proliferation through Activating the Epidermal Growth Factor Receptor.

    PubMed

    Liu, Wei; Sun, Cheng; Liao, Chunyang; Cui, Lin; Li, Haishan; Qu, Guangbo; Yu, Wenlian; Song, Naining; Cui, Yuan; Wang, Zheng; Xie, Wenping; Chen, Huiming; Zhou, Qunfang

    2016-07-27

    Graphene has promising applications in food packaging, water purification, and detective sensors for contamination monitoring. However, the biological effects of graphene are not fully understood. It is necessary to clarify the potential risks of graphene exposure to humans through diverse routes, such as foods. In the present study, graphene, as the model nanomaterial, was used to test its potential effects on the cell proliferation based on multiple representative cell lines, including HepG2, A549, MCF-7, and HeLa cells. Graphene was characterized by Raman spectroscopy, particle size analysis, atomic force microscopy, and transmission electron microscopy. The cellular responses to graphene exposure were evaluated using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and alamarBlue assays. Rat cerebral astrocyte cultures, as the non-cancer cells, were used to assess the potential cytotoxicity of graphene as well. The results showed that graphene stimulation enhanced cell proliferation in all tested cell cultures and the highest elevation in cell growth was up to 60%. A western blot assay showed that the expression of epidermal growth factor (EGF) was upregulated upon graphene treatment. The phosphorylation of EGF receptor (EGFR) and the downstream proteins, ShC and extracellular regulating kinase (ERK), were remarkably induced, indicating that the activation of the mitogen-activated protein kinase (MAPK)/ERK signaling pathway was triggered. The activation of PI3 kinase p85 and AKT showed that the PI3K/AKT signaling pathway was also involved in graphene-induced cell proliferation, causing the increase of cell ratios in the G2/M phase. No influences on cell apoptosis were observed in graphene-treated cells when compared to the negative controls, proving the low cytotoxicity of this emerging nanomaterial. The findings in this study revealed the potential cellular biological effect of graphene, which may give useful hints on its biosafety

  10. ENHANCING ADVANCED CANDU PROLIFERATION RESISTANCE FUEL WITH MINOR ACTINIDES

    SciTech Connect

    Gray S. Chang

    2010-05-01

    The advanced nuclear system will significantly advance the science and technology of nuclear energy systems and to enhance the spent fuel proliferation resistance. Minor actinides (MA) are viewed more as a resource to be recycled, and transmuted to less hazardous and possibly more useful forms, rather than simply disposed of as a waste stream in an expensive repository facility. MAs can play a much larger part in the design of advanced systems and fuel cycles, not only as additional sources of useful energy, but also as direct contributors to the reactivity control of the systems into which they are incorporated. In this work, an Advanced CANDU Reactor (ACR) fuel unit lattice cell model with 43 UO2 fuel rods will be used to investigate the effectiveness of a Minor Actinide Reduction Approach (MARA) for enhancing proliferation resistance and improving the fuel cycle performance. The main MARA objective is to increase the 238Pu / Pu isotope ratio by using the transuranic nuclides (237Np and 241Am) in the high burnup fuel and thereby increase the proliferation resistance even for a very low fuel burnup. As a result, MARA is a very effective approach to enhance the proliferation resistance for the on power refueling ACR system nuclear fuel. The MA transmutation characteristics at different MA loadings were compared and their impact on neutronics criticality assessed. The concept of MARA, significantly increases the 238Pu/Pu ratio for proliferation resistance, as well as serves as a burnable absorber to hold-down the initial excess reactivity. It is believed that MARA can play an important role in atoms for peace and the intermediate term of nuclear energy reconnaissance.

  11. Early-life environmental intervention may increase the number of neurons, astrocytes, and cellular proliferation in the hippocampus of rats.

    PubMed

    Winkelmann-Duarte, Elisa C; Padilha-Hoffmann, Camila B; Martins, Daniel F; Schuh, Artur F S; Fernandes, Marilda C; Santin, Ricardo; Merlo, Suelen; Sanvitto, Gilberto L; Lucion, Aldo B

    2011-11-01

    Neonatal handling reduces the stress response in adulthood due to a feedback mechanism. The present study analyzed the effects of repeated neonatal environmental intervention (daily handling during the first 10 days after birth) on neuron-, astroglial cell density, and cellular proliferation of the hippocampal (CA1, CA2, and CA3) pyramidal cell layers in female rats. Pups were divided into two groups, nonhandled and handled, which were submitted to repeated handling sessions between postnatal days 1 and 10. Histological and immunohistochemical procedures were used to determine changes in neuron density, astroglial cell density, and cellular proliferation. We found an increase in neuron density in each pyramidal cell layer of the hippocampus (CA1, CA2, and CA3) in female rats (11 and 90 day old) that were handled during the neonatal period. Furthermore, we found an increase in astroglial cell density in both hemispheres of the brain in the handled group. Finally, we observed an increase in cellular proliferation in both hippocampi (CA1, CA2, and CA3) of the brain in female pups (11 days old) handled during the neonatal period. This study demonstrates that an early-life environmental intervention may induce morphological changes in a structure involved with several functions, including the stress response. The results of the current study suggest that neonatal handling may influence the animals' responses to environmental adversities later in life.

  12. Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells

    PubMed Central

    1986-01-01

    Using primary culture methods, we show that purified astrocytes from embryonic mouse or rat central nervous system (CNS) can be induced to produce interferon (IFN) activity when pretreated with a standard IFN- superinducing regimen of polyribonucleotide, cycloheximide, and actinomycin D, whereas IFN activity was not inducible in neuronal cultures derived from mouse CNS. Astrocyte IFN displays inductive, kinetic, physicochemical, and antigenic properties similar to those of IFN-alpha/beta, but is dissimilar to lymphocyte IFN (IFN-gamma). Treatment of pure astrocytic cultures or astrocytes cultured with neurons with astrocyte IFN or IFN-alpha/beta induced a dramatic increase in the expression of H-2 antigens on a subpopulation of astrocytes. Neither neurons nor oligodendroglia expressed detectable levels of H-2 antigens when exposed to astrocyte IFN, IFN-alpha/beta, or to IFN-beta. Injection of astrocyte IFN or IFN-alpha/beta directly into brains of newborn mice indicated that H-2 antigens were also induced in vivo. None of the IFNs (astrocyte, alpha/beta, or beta) tested induced Ia antigens on CNS cells in vitro or in vivo. Since H-2 antigens have a critical role in immune responses, astrocyte IFN may initiate and participate in immune reactions that contribute to immunoprotective and immunopathological responses in the CNS. PMID:2423537

  13. Secreted miR-34a in astrocytic shedding vesicles enhanced the vulnerability of dopaminergic neurons to neurotoxins by targeting Bcl-2.

    PubMed

    Mao, Susu; Sun, Qi; Xiao, Hui; Zhang, Chenyu; Li, Liang

    2015-07-01

    MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.

  14. Enhancing BWR proliferation resistance fuel with minor actinides

    NASA Astrophysics Data System (ADS)

    Chang, Gray S.

    2009-03-01

    To reduce spent fuel for storage and enhance the proliferation resistance for the intermediate-term, there are two major approaches (a) increase the discharged spent fuel burnup in the advanced light water reactor- LWR (Gen-III Plus), which not only can reduce the spent fuel for storage, but also increase the 238Pu isotopes ratio to enhance the proliferation resistance, and (b) use of transuranic nuclides ( 237Np and 241Am) in the high burnup fuel, which can drastically increase the proliferation resistance isotope ratio of 238Pu/Pu. For future advanced nuclear systems, minor actinides (MA) are viewed more as a resource to be recycled, and transmuted to less hazardous and possibly more useful forms, rather than simply disposed of as a waste stream in an expensive repository facility. As a result, MAs play a much larger part in the design of advanced systems and fuel cycles, not only as additional sources of useful energy, but also as direct contributors to the reactivity control of the systems into which they are incorporated. In the study, a typical boiling water reactor (BWR) fuel unit lattice cell model with UO 2 fuel pins will be used to investigate the effectiveness of minor actinide reduction approach (MARA) for enhancing proliferation resistance and improving the fuel cycle performance in the intermediate-term goal for future nuclear energy systems. To account for the water coolant density variation from the bottom (0.76 g/cm 3) to the top (0.35 g/cm 3) of the core, the axial coolant channel and fuel pin were divided to 24 nodes. The MA transmutation characteristics at different elevations were compared and their impact on neutronics criticality discussed. The concept of MARA, which involves the use of transuranic nuclides ( 237Np and/or 241Am), significantly increases the 238Pu/Pu ratio for proliferation resistance, as well as serves as a burnable absorber to hold-down the initial excess reactivity. It is believed that MARA can play an important role in

  15. Enhancing BWR Proliferation Resistance Fuel with Minor Actinides

    SciTech Connect

    Gray S. Chang

    2008-07-01

    Key aspects of the Global Nuclear Energy Partnership (GNEP) are to significantly advance the science and technology of nuclear energy systems and the Advanced Fuel Cycle (AFC) program. It consists of both innovative nuclear reactors and innovative research in separation and transmutation. To accomplish these goals, international cooperation is very important and public acceptance is crucial. The merits of nuclear energy are high-density energy, with low environmental impacts (i.e. almost zero greenhouse gas emission). Planned efforts involve near-term and intermediate-term improvements in fuel utilization and recycling in current light water reactors (LWRs) as well as the longer-term development of new nuclear energy systems that offer much improved fuel utilization and proliferation resistance, along with continued advances in operational safety. The challenges are solving the energy needs of the world, protection against nuclear proliferation, the problem of nuclear waste, and the global environmental problem. To reduce spent fuel for storage and enhance the proliferation resistance for the intermediate-term, there are two major approaches (a) increase the discharged spent fuel burnup in the advanced LWR (Gen-III Plus), which not only can reduce the spent fuel for storage, but also increase the 238Pu and 240Pu isotopes ratio to enhance the proliferation resistance, and (b) use of transuranic nuclides (237Np and 241Am) in the high burnup fuel, which can drastically increase the proliferation resistance isotope ratio of 238Pu /Pu. For future advanced nuclear systems, the minor actinides (MA) are viewed more as a resource to be recycled, or transmuted to less hazardous and possibly more useful forms, rather than simply as a waste stream to be disposed of in expensive repository facilities. As a result, MAs play a much larger part in the design of advanced systems and fuel cycles, not only as additional sources of useful energy, but also as direct contributors to the

  16. Enhanced osteoblast proliferation and collagen gene expression by estradiol

    SciTech Connect

    Ernest, M.; Schmid, Ch.; Froesch, E.R. )

    1988-04-01

    Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17{beta}-estradiol on bone-forming cells, the authors used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphorlogy. 17{beta}-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17{alpha}-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17{beta}-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro{alpha}1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17{beta}-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by ({sup 3}H)proline pulse) that was digestible by collagenase was increased, indicating that 17{beta}-estradiol acts as pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17{beta}-estradiol.

  17. Role of Astrocytes in Epilepsy

    PubMed Central

    Coulter, Douglas A.; Steinhäuser, Christian

    2016-01-01

    Astrocytes express ion channels, transmitter receptors, and transporters and, thus, are endowed with the machinery to sense and respond to neuronal activity. Recent studies have implicated that astrocytes play important roles in physiology, but these cells also emerge as crucial actors in epilepsy. Astrocytes are abundantly coupled through gap junctions allowing them to redistribute elevated K+ and transmitter concentrations from sites of enhanced neuronal activity. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial K+ and water channels. In addition, malfunction of glutamate transporters and the astrocytic glutamate-converting enzyme, glutamine synthetase, has been observed in epileptic tissue. These findings suggest that dysfunctional astrocytes are crucial players in epilepsy and should be considered as promising targets for new therapeutic strategies. PMID:25732035

  18. Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

    PubMed Central

    Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Engqvist, Håkan; Karlsson Ott, Marjam

    2015-01-01

    Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70% ±0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ±7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts. PMID:26023912

  19. Effects of diet-induced obesity and voluntary exercise in a tauopathy mouse model: implications of persistent hyperleptinemia and enhanced astrocytic leptin receptor expression.

    PubMed

    Koga, Shunsuke; Kojima, Ayako; Ishikawa, Chieko; Kuwabara, Satoshi; Arai, Kimihito; Yoshiyama, Yasumasa

    2014-11-01

    The number of patients with Alzheimer's disease (AD) is increasing worldwide, and available drugs have shown limited efficacy. Hence, preventive interventions and treatments for presymptomatic AD are currently considered very important. Obesity rates have also been increasing dramatically and it is an independent risk factor of AD. Therefore, for the prevention of AD, it is important to elucidate the pathomechanism between obesity and AD. We generated high calorie diet (HCD)-induced obese tauopathy model mice (PS19), which showed hyperleptinemia but limited insulin resistance. HCD enhanced tau pathology and glial activation. Conversely, voluntary exercise with a running wheel normalized the serum leptin concentration without reducing body weight, and restored the pathological changes induced by HCD. Thus, we speculated that persistent hyperleptinemia played an important role in accelerating pathological changes in PS19 mice. Leptin primarily regulates food intake and body weight via leptin receptor b (LepRb). Interestingly, the nuclear staining for p-STAT3, which was activated by LepRb, was decreased in hippocampal neurons in HCD PS19 mice, indicating leptin resistance. Meanwhile, astroglial activation and the astrocytic expression of a short LepR isoform, LepRa, were enhanced in the hippocampus of HCD PS19 mice. Real-time PCR analysis demonstrated that leptin increased mRNA levels for pro-inflammatory cytokines including IL-1β and TNF-α in primary cultured astrocytes from wild type and LepRb-deficient mice. These observations suggest that persistent hyperleptinemia caused by obesity induces astrocytic activation, astrocytic leptin hypersensitivity with enhanced LepRa expression, and enhanced inflammation, consequently accelerating tau pathology in PS19 mice. PMID:25132556

  20. Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

    PubMed Central

    Ryu, Sun; Lee, Seung-Hoon; Kim, Seung U.; Yoon, Byung-Woo

    2016-01-01

    Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen NeuN, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ⁄ anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke. PMID:27073384

  1. Activation of A(1) adenosine or mGlu3 metabotropic glutamate receptors enhances the release of nerve growth factor and S-100beta protein from cultured astrocytes.

    PubMed

    Ciccarelli, R; Di Iorio, P; Bruno, V; Battaglia, G; D'Alimonte, I; D'Onofrio, M; Nicoletti, F; Caciagli, F

    1999-09-01

    Pharmacological activation of A(1) adenosine receptor with 2-chloro-N6-cyclopentyladenosine (CCPA) or mGlu3 metabotropic glutamate receptors with (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV) or aminopyrrolidine-2R, 4R-dicarboxylate (2R,4R-APDC) enhanced the release of nerve growth factor (NGF) or S-100beta protein from rat cultured astrocytes. Stimulation of release by CCPA and DCG-IV or 2R,4R-APDC was inhibited by the A(1) adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine and by the mGlu2/3 receptor antagonist (2S,1'S, 2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-4), respectively. Time-course studies revealed a profound difference between the release of S-100beta protein and the release of NGF in response to extracellular signals. Stimulation of S-100beta protein exhibited rapid kinetics, peaking after 1 h of drug treatment, whereas the enhancement of NGF release was much slower, requiring at least 6 h of A(1) adenosine or mGlu3 receptor activation. In addition, stimulation of NGF but not S-100beta release was substantially reduced in cultures treated with the protein synthesis inhibitor cycloheximide. In addition, a 6-8 h treatment of cultured astrocytes with A(1) or mGlu3 receptor agonists increased the levels of both NGF mRNA and NGF-like immunoreactive proteins, including NGF prohormone. We conclude that activation of A(1) adenosine or mGlu3 receptors produces pleiotropic effects in astrocytes, stimulating the synthesis and/or the release of protein factors. Astrocytes may therefore become targets for drugs that stimulate the local production of neurotrophic factors in the CNS, and this may provide the basis for a novel therapeutic strategy in chronic neurodegenerative disorders. PMID:10457374

  2. Disentangling the Role of Astrocytes in Alcohol Use Disorder.

    PubMed

    Adermark, Louise; Bowers, M Scott

    2016-09-01

    Several laboratories recently identified that astrocytes are critical regulators of addiction machinery. It is now known that astrocyte pathology is a common feature of ethanol (EtOH) exposure in both humans and animal models, as even brief EtOH exposure is sufficient to elicit long-lasting perturbations in astrocyte gene expression, activity, and proliferation. Astrocytes were also recently shown to modulate the motivational properties of EtOH and other strongly reinforcing stimuli. Given the role of astrocytes in regulating glutamate homeostasis, a crucial component of alcohol use disorder (AUD), astrocytes might be an important target for the development of next-generation alcoholism treatments. This review will outline some of the more prominent features displayed by astrocytes, how these properties are influenced by acute and long-term EtOH exposure, and future directions that may help to disentangle astrocytic from neuronal functions in the etiology of AUD. PMID:27476876

  3. Disentangling the Role of Astrocytes in Alcohol Use Disorder.

    PubMed

    Adermark, Louise; Bowers, M Scott

    2016-09-01

    Several laboratories recently identified that astrocytes are critical regulators of addiction machinery. It is now known that astrocyte pathology is a common feature of ethanol (EtOH) exposure in both humans and animal models, as even brief EtOH exposure is sufficient to elicit long-lasting perturbations in astrocyte gene expression, activity, and proliferation. Astrocytes were also recently shown to modulate the motivational properties of EtOH and other strongly reinforcing stimuli. Given the role of astrocytes in regulating glutamate homeostasis, a crucial component of alcohol use disorder (AUD), astrocytes might be an important target for the development of next-generation alcoholism treatments. This review will outline some of the more prominent features displayed by astrocytes, how these properties are influenced by acute and long-term EtOH exposure, and future directions that may help to disentangle astrocytic from neuronal functions in the etiology of AUD.

  4. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  5. Nerve growth factor enhances Clara cell proliferation after lung injury.

    PubMed

    Sonar, S S; Schwinge, D; Kilic, A; Yildirim, A O; Conrad, M L; Seidler, K; Müller, B; Renz, H; Nockher, W A

    2010-07-01

    The lung epithelia facilitate wound closure by secretion of various cytokines and growth factors. Nerve growth factor (NGF) has been well described in airway inflammation; however, its likely role in lung repair has not been examined thus far. To investigate the repair function of NGF, experiments were performed in vitro using cultured alveolar epithelial cells and in vivo using a naphthalene-induced model of Clara epithelial cell injury. Both in vitro and in vivo experiments revealed airway epithelial cell proliferation following injury to be dependent on NGF and the expression of its receptor, tropomyosin-receptor-kinase A. Additionally, NGF also augmented in vitro migration of alveolar type II cells. In vivo, transgenic mice over-expressing NGF in Clara cells (NGFtg) did not reveal any proliferation or alteration in Clara cell phenotype. However, following Clara cell specific injury, proliferation was increased in NGFtg and impaired upon inhibition of NGF. Furthermore, NGF also promoted the expression of collagen I and fibronectin in vitro and in vivo during repair, where significantly higher levels were measured in re-epithelialising NGFtg mice. Our study demonstrates that NGF promotes the proliferation of lung epithelium in vitro and the renewal of Clara cells following lung injury in vivo.

  6. Glucocorticoids decrease astrocyte numbers by reducing glucocorticoid receptor expression in vitro and in vivo.

    PubMed

    Unemura, Kazuhiro; Kume, Toshiaki; Kondo, Minami; Maeda, Yuki; Izumi, Yasuhiko; Akaike, Akinori

    2012-01-01

    Glucocorticoids are stress hormones released from the adrenal cortex and their concentration is controlled by the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of glucocorticoids on the number of astrocytes and glucocorticoid receptor (GR) expression in vitro and in vivo. Proliferation of cultured astrocytes was reduced following treatment with corticosterone and dexamethasone for 72 h. Corticosterone and dexamethasone also reduced GR expression in astrocytes. RU486, a GR antagonist, inhibited the reduction in both astrocyte proliferation and GR expression. Furthermore, GR knockdown by siRNA inhibited astrocyte proliferation. We also examined the effect of excessive glucocorticoid release on GR expression and the number of astrocytes in vivo by administering adrenocorticotropic hormone to rats for 14 days. GR expression was reduced in the prefrontal cortex and hippocampus and the number of astrocytes was reduced in the frontal cortex. Overall, our results suggest that glucocorticoids decrease the number of astrocytes by reducing GR expression.

  7. Effects of fractionated radiation on the brain vasculature in a murine model: Blood-brain barrier permeability, astrocyte proliferation, and ultrastructural changes

    SciTech Connect

    Yuan Hong; Gaber, M. Waleed . E-mail: wgaber@utmem.edu; Boyd, Kelli; Wilson, Christy M.; Kiani, Mohammad F.; Merchant, Thomas E.

    2006-11-01

    Purpose: Radiation therapy of CNS tumors damages the blood-brain barrier (BBB) and normal brain tissue. Our aims were to characterize the short- and long-term effects of fractionated radiotherapy (FRT) on cerebral microvasculature in mice and to investigate the mechanism of change in BBB permeability in mice. Methods and Materials: Intravital microscopy and a cranial window technique were used to measure BBB permeability to fluorescein isothiocyanate (FITC)-dextran and leukocyte endothelial interactions before and after cranial irradiation. Daily doses of 2 Gy were delivered 5 days/week (total, 40 Gy). We immunostained the molecules to detect the expression of glial fibrillary acidic protein and to demonstrate astrocyte activity in brain parenchyma. To relate the permeability changes to endothelial ultrastructural changes, we used electron microscopy. Results: Blood-brain barrier permeability did not increase significantly until 90 days after FRT, at which point it increased continuously until 180 days post-FRT. The number of adherent leukocytes did not increase during the study. The number of astrocytes in the cerebral cortex increased significantly; vesicular activity in endothelial cells increased beginning 90 days after irradiation, and most tight junctions stayed intact, although some were shorter and less dense at 120 and 180 days. Conclusions: The cellular and microvasculature response of the brain to FRT is mediated through astrogliosis and ultrastructural changes, accompanied by an increase in BBB permeability. The response to FRT is delayed as compared with single-dose irradiation treatment, and does not involve leukocyte adhesion. However, FRT induces an increase in the BBB permeability, as in the case of single-dose irradiation.

  8. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells.

    PubMed

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N

    2016-06-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5-CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion.

  9. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells

    PubMed Central

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N.

    2016-01-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5–CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion. PMID:27335323

  10. Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation.

    PubMed

    Smeriglio, Piera; Dhulipala, Lakshmi; Lai, Janice H; Goodman, Stuart B; Dragoo, Jason L; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-02-01

    Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

  11. MECHANISMS INVOLVED IN THE ENHANCED SUSCEPTIBILITY OF SENESCENT RATS TO THE HEPATOCARCINOGENIC EFFECT OF PEROXISOME PROLIFERATORS: ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA (PPARA), CELL PROLIFERATION AND OXIDATIVE STRESS

    EPA Science Inventory

    Mechanisms involved in the ENHANCED SUSCEPTIBILITY of SENESCENT Rats TO THE HEPATOCARCINOGENIC EFFECT OF PEROXISOME PROLIFERATORS: Role of peroxisome proliferator-activated receptor alpha (PPARa), cell proliferation and oxidative stress

    Jihan A. Youssef1, Pierre Ammann2, B...

  12. Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

    PubMed Central

    Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

    2016-01-01

    Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

  13. Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals.

    PubMed

    Bose, Chhanda; Udupa, Kodetthoor B

    2008-08-01

    Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH(2) terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells.

  14. Clopidogrel Enhances Mesenchymal Stem Cell Proliferation Following Periodontitis.

    PubMed

    Coimbra, L S; Steffens, J P; Alsadun, S; Albiero, M L; Rossa, C; Pignolo, R J; Spolidorio, L C; Graves, D T

    2015-12-01

    Bone formation is dependent on the differentiation of osteoblasts from mesenchymal stem cells (MSCs). In addition to serving as progenitors, MSCs reduce inflammation and produce factors that stimulate tissue formation. Upon injury, MSCs migrate to the periodontium, where they contribute to regeneration. We examined the effect of clopidogrel and aspirin on MSCs following induction of periodontitis in rats by placement of ligatures. We showed that after the removal of ligatures, which induces resolution of periodontal inflammation, clopidogrel had a significant effect on reducing the inflammatory infiltrate. It also increased the number of osteoblasts and MSCs. Mechanistically, the latter was linked to increased proliferation of MSCs in vivo and in vitro. When given prior to inducing periodontitis, clopidogrel had little effect on MSC or osteoblasts numbers. Applying aspirin before or after induction of periodontitis did not have a significant effect on the parameters measured. These results suggest that clopidogrel may have a positive effect on MSCs in conditions where a reparative process has been initiated.

  15. Astrocyte growth is regulated by neuropeptides through Tis 8 and basic fibroblast growth factor.

    PubMed Central

    Hu, R M; Levin, E R

    1994-01-01

    The important intracellular mechanisms of astrocyte growth are not well defined. Using an inhibitor of astrocyte proliferation, atrial natriuretic peptide (ANP), and the glial mitogen endothelin (ET-3), we sought a common pathway for growth regulation in these neural cells. In cultured fetal rat diencephalic astrocytes, ANP selectively and rapidly inhibited the Tis 8 immediate early gene and protein. After 4 h, ANP selectively inhibited the basic fibroblast growth factor (bFGF) gene and protein. ET-3 significantly stimulated both Tis 8 and bFGF mRNAs and protein, but also stimulated several other immediate early and growth factor/receptor genes. An antisense oligonucleotide to Tis 8 strongly prevented ET-stimulated thymidine incorporation, while the inhibitory action of ANP was enhanced. The Tis 8 antisense oligonucleotide also significantly reversed ET-stimulated bFGF transcription and enhanced the bFGF inhibition caused by ANP. In addition, an antisense oligonucleotide to bFGF significantly reversed the ET-stimulated thymidine incorporation and enhanced the ANP inhibition of DNA synthesis. The sequential modulation of Tis 8, followed by bFGF, provides a novel mechanism for both positive and negative regulation of astrocyte growth by endogenous neuropeptides. Images PMID:8163680

  16. [Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment].

    PubMed

    Hao, Tong; Li, Jun-Jie; Du, Zhi-Yan; Duan, Cui-Mi; Wang, Yan-Meng; Wang, Chang-Yong; Song, Jing-Ping; Wang, Lin-Jie; Li, Ying-Hui; Wang, Yan

    2012-10-01

    This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity. PMID:23114150

  17. Enhanced lymphocyte proliferation in patients with adrenoleukodystrophy treated with erucic acid (22:1)-rich triglycerides.

    PubMed

    Pour, R B; Stöckler-Ipsiroglu, S; Hunneman, D H; Gahr, M; Korenke, G C; Pabst, W; Hanefeld, F; Peters, A

    2000-03-01

    Lymphocytopenia and depression of natural killer cells have been observed in patients with adrenoleukodystrophy (ALD) treated with glycerol trioleate and glycerol trierucate ('Lorenzo's oil'). To investigate possible alterations of cellular immunoreactivity, we measured lymphocyte proliferation in response to mitogens (PHA, Con A, PWM, OKT3) in 27 patients on treatment and in 14 patients without treatment. In patients on treatment, lymphocyte proliferation in response to the mitogens PHA and Con A was significantly higher than in patients without treatment. Lymphocyte proliferation in patients without treatment was comparable to that of normal control lymphocytes. Additionally, we found increased concentrations of erucic acid, C22:1, in lymphocytes from patients with treatment. The enhanced proliferation of lymphocytes in response to mitogens is an indication of increased reactivity of cellular immunity to unspecific immunological stimuli. Long-term side-effects on cellular immunoreactivity have to be considered in ALD patients treated with Lorenzo's oil.

  18. Aberrant cell proliferation by enhanced mitochondrial biogenesis via mtTFA in arsenical skin cancers.

    PubMed

    Lee, Chih-Hung; Wu, Shi-Bei; Hong, Chien-Hui; Liao, Wei-Ting; Wu, Ching-Ying; Chen, Gwo-Shing; Wei, Yau-Huei; Yu, Hsin-Su

    2011-05-01

    Arsenic-induced Bowen's disease (As-BD), a cutaneous carcinoma in situ, is thought to arise from gene mutation and uncontrolled proliferation. However, how mitochondria regulate the arsenic-induced cell proliferation remains unclear. The aim of this study was to clarify whether arsenic interfered with mitochondrial biogenesis and function, leading to aberrant cell proliferation in As-BD. Skin biopsy samples from patients with As-BD and controls were stained for cytochrome c oxidase (Complex IV), measured for mitochondrial DNA (mtDNA) copy number and the expression levels of mitochondrial biogenesis-related genes, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA). The results showed that expression of cytochrome c oxidase, mtTFA, NRF-1, and PGC-1α was increased in As-BD compared with in healthy subjects. Treatment of primary keratinocytes with arsenic at concentrations lower than 1.0 μmol/L induced cell proliferation, along with enhanced mitochondrial biogenesis. Furthermore, we observed that the mitochondrial oxygen consumption rate and intracellular ATP level were increased in arsenic-treated keratinocytes. Blocking of mitochondrial function by oligomycin A (Complex V inhibitor) or knockdown of mtTFA by RNA interference abrogated arsenic-induced cell proliferation without affecting cyclin D1 expression. We concluded that mtTFA up-regulation, augmented mitochondrial biogenesis, and enhanced mitochondrial functions may contribute to arsenic-induced cell proliferation. Targeting mitochondrial biogenesis may help treat arsenical cancers at the stage of cell proliferation.

  19. Nitrite circumvents canonical cGMP signaling to enhance proliferation of myocyte precursor cells.

    PubMed

    Totzeck, Matthias; Schicho, Andreas; Stock, Pia; Kelm, Malte; Rassaf, Tienush; Hendgen-Cotta, Ulrike B

    2015-03-01

    Skeletal muscle tissue has a remarkable high regenerative capacity. The underlying cellular events are governed by complex signaling processes, and the proliferation of skeletal myoblasts is a key initial event. The role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, an NO oxidation product, is a stable source for NO-like bioactivity particularly in cases when oxygen shortage compromises NO-synthases activity. Although numerous studies suggest that nitrite effects are largely related to NO-dependent signaling, emerging evidence also implicates that nitrite itself can activate protein pathways albeit under physiological, normoxic conditions. This includes a recently demonstrated cyclic guanosine monophosphate-(cGMP)-independent enhancement of endothelial cell proliferation. Whether nitrite itself has the potential to affect myoblast proliferation and metabolism with or without activation of the canonical NO/cGMP pathway to subsequently support muscle cell regeneration is not known. Here we show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3 oxide and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. PMID:25501648

  20. Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

    PubMed

    Chapouly, Candice; Tadesse Argaw, Azeb; Horng, Sam; Castro, Kamilah; Zhang, Jingya; Asp, Linnea; Loo, Hannah; Laitman, Benjamin M; Mariani, John N; Straus Farber, Rebecca; Zaslavsky, Elena; Nudelman, German; Raine, Cedric S; John, Gareth R

    2015-06-01

    In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an

  1. High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury

    NASA Astrophysics Data System (ADS)

    Khaing, Zin Z.; Milman, Brian D.; Vanscoy, Jennifer E.; Seidlits, Stephanie K.; Grill, Raymond J.; Schmidt, Christine E.

    2011-08-01

    A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

  2. High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury.

    PubMed

    Khaing, Zin Z; Milman, Brian D; Vanscoy, Jennifer E; Seidlits, Stephanie K; Grill, Raymond J; Schmidt, Christine E

    2011-08-01

    A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

  3. High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury.

    PubMed

    Khaing, Zin Z; Milman, Brian D; Vanscoy, Jennifer E; Seidlits, Stephanie K; Grill, Raymond J; Schmidt, Christine E

    2011-08-01

    A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI. PMID:21753237

  4. The immobilization of hepatocytes on 24 nm-sized gold colloid for enhanced hepatocytes proliferation.

    PubMed

    Gu, Hai-Ying; Chen, Zhong; Sa, Rong-Xiao; Yuan, Su-Su; Chen, Hong-Yuan; Ding, Yi-Tao; Yu, Ai-Min

    2004-08-01

    Bioartificial liver and hepatocyte transplantation is anticipated to supply a temporary metabolic support for candidates of liver transplantation or for patients with fulminant liver failure. An essential restriction of this form is the inability to acquire an enough amount of hepatocytes. Enhancement of the proliferation and differentiated function of hepatocytes is becoming a pursued target. Here, porcine hepatocytes were successfully immobilized on nano-sized gold colloid particles to construct a "hepatocyte/gold colloid" interface at which hepatocytes can be quickly proliferated. The properties of this resulting interface were characterized and confirmed by scanning electron microscopy and atomic force microscopy. The proliferative mechanism of hepatocytes was also discussed. The proliferated hepatocytes could be applied to the clinic based on their excellent functions for the synthesis of protein, glucose and urea as well as lower lactate dehydrogenase release. PMID:15020118

  5. SIRT2 activates G6PD to enhance NADPH production and promote leukaemia cell proliferation.

    PubMed

    Xu, Shuang-Nian; Wang, Tian-Shi; Li, Xi; Wang, Yi-Ping

    2016-01-01

    Like most other types of cancer cells, leukaemia cells undergo metabolic reprogramming to support rapid proliferation through enhancing biosynthetic processes. Pentose phosphate pathway (PPP) plays a pivotal role in meeting the anabolic demands for cancer cells. However, the molecular mechanism by which PPP contributes to leukaemia remains elusive. Here, we report that leukaemia cell proliferation is dependent on the oxidative branch of PPP, in particular the first and rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Knockdown of G6PD reduces NADPH level in acute myeloid leukaemia (AML) cell lines. Exogenous lipid supplements partially restore the proliferation of G6PD-depleted cells. Deacetylase SIRT2 promotes NADPH production through deacetylating G6PD at lysine 403 (K403). Activation of G6PD by SIRT2 supports the proliferation and clonogenic activity of leukaemia cells. Chemical inhibitors against SIRT2 suppress G6PD activity, leading to reduced cell proliferation of leukaemia cells, but not normal hematopoietic stem and progenitor cells. Importantly, SIRT2 is overexpressed in clinical AML samples, while K403 acetylation is downregulated and G6PD catalytic activity is increased comparing to that of normal control. Together, our study reveals that acetylation regulation of G6PD is involved in the metabolic reprogramming of AML, and SIRT2 serves as a promising target for further therapeutic investigations. PMID:27586085

  6. SIRT2 activates G6PD to enhance NADPH production and promote leukaemia cell proliferation

    PubMed Central

    Xu, Shuang-Nian; Wang, Tian-Shi; Li, Xi; Wang, Yi-Ping

    2016-01-01

    Like most other types of cancer cells, leukaemia cells undergo metabolic reprogramming to support rapid proliferation through enhancing biosynthetic processes. Pentose phosphate pathway (PPP) plays a pivotal role in meeting the anabolic demands for cancer cells. However, the molecular mechanism by which PPP contributes to leukaemia remains elusive. Here, we report that leukaemia cell proliferation is dependent on the oxidative branch of PPP, in particular the first and rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Knockdown of G6PD reduces NADPH level in acute myeloid leukaemia (AML) cell lines. Exogenous lipid supplements partially restore the proliferation of G6PD-depleted cells. Deacetylase SIRT2 promotes NADPH production through deacetylating G6PD at lysine 403 (K403). Activation of G6PD by SIRT2 supports the proliferation and clonogenic activity of leukaemia cells. Chemical inhibitors against SIRT2 suppress G6PD activity, leading to reduced cell proliferation of leukaemia cells, but not normal hematopoietic stem and progenitor cells. Importantly, SIRT2 is overexpressed in clinical AML samples, while K403 acetylation is downregulated and G6PD catalytic activity is increased comparing to that of normal control. Together, our study reveals that acetylation regulation of G6PD is involved in the metabolic reprogramming of AML, and SIRT2 serves as a promising target for further therapeutic investigations. PMID:27586085

  7. Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

    PubMed

    Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

    2016-05-20

    RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation. PMID:26917381

  8. Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

    PubMed

    Patrulea, V; Hirt-Burri, N; Jeannerat, A; Applegate, L A; Ostafe, V; Jordan, O; Borchard, G

    2016-05-20

    RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

  9. Increased in vitro glial fibrillary acidic protein expression, telomerase activity, and telomere length after productive human immunodeficiency virus-1 infection in murine astrocytes.

    PubMed

    Ojeda, Diego; López-Costa, Juan José; Sede, Mariano; López, Ester María; Berria, María Isabel; Quarleri, Jorge

    2014-02-01

    Although HIV-associated neurocognitive disorders (HAND) result from injury and loss of neurons, productive infection routinely takes place in cells of macrophage lineage. In such a complex context, astrocytosis induced by local chemokines/cytokines is one of the hallmarks of HIV neuropathology. Whether this sustained astrocyte activation is able to alter telomere-aging process is unknown. We hypothesized that interaction of HIV with astrocytes may impact astrocyte telomerase activity (TA) and telomere length in a scenario of astrocytic activation measured by expression of glial fibrillary acidic protein (GFAP). To test this hypothesis, cultured murine astrocytes were challenged with pseudotyped HIV/vesicular stomatitis virus (HIV/VSV) to circumvent the absence of viral receptors; and GFAP, telomerase activity, and telomere length were quantified. As an early and transient event after HIV infection, both TA activity and telomere length were significantly augmented (P < 0.001). Later, a strong negative correlation (-0.8616, P < 0.0001) between virus production and telomerase activity was demonstrated. Once HIV production had reached a peak (7 dpi), the TA decreased, showing levels similar to those of noninfected cells. In contrast, the astrocyte became activated, exhibiting significantly increased levels of GFAP expression directly related to the level of HIV/VSV replication (P < 0.0001). Our results suggest that HIV-infected astrocytes exhibit early disturbance in their cellular functions, such as telomerase activity and telomere length, that may attenuate cell proliferation and enhance the astrocyte dysregulation, contributing to HIV neuropathogenesis. Understanding the mechanisms involved in HIV-mediated persistence by altering the telomere-related aging processes could aid in the development of therapeutic modalities for neurological complications of HIV infection.

  10. CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

    PubMed Central

    Timchenko, N A; Harris, T E; Wilde, M; Bilyeu, T A; Burgess-Beusse, B L; Finegold, M J; Darlington, G J

    1997-01-01

    CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels. PMID:9372966

  11. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    PubMed Central

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  12. Impaired Autophagy in APOE4 Astrocytes.

    PubMed

    Simonovitch, Shira; Schmukler, Eran; Bespalko, Alina; Iram, Tal; Frenkel, Dan; Holtzman, David M; Masliah, Eliezer; Michaelson, Danny M; Pinkas-Kramarski, Ronit

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aβ plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aβ plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aβ plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aβ plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aβ plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

  13. Boiling Method-Based Zinc Oxide Nanorods for Enhancement of Adipose-Derived Stem Cell Proliferation.

    PubMed

    Jin, Su-Eon; Ahn, Hyo-Sun; Kim, Ji Hye; Arai, Yoshie; Lee, Soo-Hong; Yoon, Tae-Jong; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2016-09-01

    Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant. The physicochemical properties of the nanorods such as their crystallinity, morphology, size, and solvent compatibility were evaluated, and then, the ability of the synthesized ZnO nanorods to enhance ASC proliferation was investigated. Scanning electron microscopy images of all of the ZnO powders showed rod-shaped nanoflakes with lengths of 200-500 nm. Notably, although ZnO-G produced by the grinding method was well dispersed in ethanol, atomic force microscopy images of dispersions of both ZnO-B from boiling methods and ZnO-G indicated the presence of clusters of ZnO nanorods. In contrast, ZnO-B was freely dispersible in 5% dextrose of water and dimethyl sulfoxide, whereas ZnO-G and ZnO-M, produced by boiling with ethanolamine, were not. All three types of ZnO nanorods increased the proliferation of ASCs in a dose-dependent manner. These results collectively suggest that ZnO nanorods have promising potential for use as an agent for the enhancement of ASC proliferation. PMID:27464704

  14. Boiling Method-Based Zinc Oxide Nanorods for Enhancement of Adipose-Derived Stem Cell Proliferation.

    PubMed

    Jin, Su-Eon; Ahn, Hyo-Sun; Kim, Ji Hye; Arai, Yoshie; Lee, Soo-Hong; Yoon, Tae-Jong; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2016-09-01

    Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant. The physicochemical properties of the nanorods such as their crystallinity, morphology, size, and solvent compatibility were evaluated, and then, the ability of the synthesized ZnO nanorods to enhance ASC proliferation was investigated. Scanning electron microscopy images of all of the ZnO powders showed rod-shaped nanoflakes with lengths of 200-500 nm. Notably, although ZnO-G produced by the grinding method was well dispersed in ethanol, atomic force microscopy images of dispersions of both ZnO-B from boiling methods and ZnO-G indicated the presence of clusters of ZnO nanorods. In contrast, ZnO-B was freely dispersible in 5% dextrose of water and dimethyl sulfoxide, whereas ZnO-G and ZnO-M, produced by boiling with ethanolamine, were not. All three types of ZnO nanorods increased the proliferation of ASCs in a dose-dependent manner. These results collectively suggest that ZnO nanorods have promising potential for use as an agent for the enhancement of ASC proliferation.

  15. Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation.

    PubMed

    Ueno, Nobuhiro; Shimizu, Akio; Kanai, Michiyuki; Iwaya, Yugo; Ueda, Shugo; Nakayama, Jun; Seo, Misuzu Kurokawa

    2016-01-01

    Deregulated expression of fibroblast growth factor receptors (FGFRs) and their ligands plays critical roles in tumorigenesis. The gene expression of an alternatively spliced isoforms of FGFR3, FGFR3IIIc, was analyzed by RT-PCR in samples from patients with esophageal carcinoma (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). The incidence of FGFR3IIIc was higher in EC [12/16 (75%); p=0.073] than in non-cancerous mucosa (NCM) [6/16 (38%)]. Indeed, an immunohistochemical analysis of early-stage ESCC showed that carcinoma cells expressing FGFR3IIIc stained positively with SCC-112, a tumor marker, and Ki67, a cell proliferation marker, suggesting that the expression of FGFR3IIIc promotes cell proliferation. We used EC-GI-10 cells endogenously expressing FGFR3IIIc as a model of ESCC to provide mechanistic insight into the role of FGFR3IIIc in ESCC. The knockdown of endogenous FGFR3 using siRNA treatment significantly abrogated cell proliferation and the overexpression of FGFR3IIIc in cells with enhanced cell proliferation. EC-GI-10 cells and ESCC from patients with EC showed endogenous expression of FGF2, a specific ligand for FGFR3IIIc, suggesting that the upregulated expression of FGFR3IIIc may create autocrine FGF signaling in ESCC. Taken together, FGFR3IIIc may have the potential to be an early-stage tumor marker and a molecular target for ESCC therapy.

  16. Overexpression of transcriptional coactivator AIB1 promotes hepatocellular carcinoma progression by enhancing cell proliferation and invasiveness.

    PubMed

    Xu, Y; Chen, Q; Li, W; Su, X; Chen, T; Liu, Y; Zhao, Y; Yu, C

    2010-06-10

    Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator for nuclear receptors and other transcription factors. AIB1 has an important role in malignancy of several cancers such as breast and prostate cancers. However, its involvement in human hepatocellular carcinoma (HCC) progression remains unclear. Here, we found that AIB1 protein was overexpressed in 23 of 34 human HCC specimens (68%). Down-regulation of AIB1 reduced HCC cell proliferation, migration, invasion, colony formation ability and tumorigenic potential in nude mice. These phenotypic changes caused by AIB1 knockdown correlated with increased expression of the cell cycle inhibitor p21(Cip1/Waf1) and decreased Akt activation and the expression of proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase MMP-9. In agreement with these findings, clinical AIB1-positive HCC expressed higher levels of PCNA than AIB1-negative HCC. A positive correlation was established between the levels of AIB1 protein and PCNA protein in HCC, suggesting that AIB1 may contribute to HCC cell proliferation. In addition, MMP-9 expression in AIB1-postive HCC was significantly higher than that in AIB1-negative HCC, suggesting that AIB1-postive HCC may be more invasive. Collectively, our results show that overexpression of AIB1 promotes human HCC progression by enhancing cell proliferation and invasiveness. Therefore, AIB1 is a master regulator of human HCC growth and might be a useful molecular target for HCC prognosis and treatment.

  17. Cancer/testis antigen NY-SAR-35 enhances cell proliferation, migration, and invasion.

    PubMed

    Song, Myung-Ha; Kim, Ye-Rin; Lee, Jun-Won; Lee, Chang-Hun; Lee, Sang-Yull

    2016-02-01

    The cancer/testis antigen NY-SAR-35 is aberrantly expressed in various cancer tissues and cancer cell lines but not in normal tissues except for the testis. A previous study demonstrated that the expression of NY-SAR-35 is activated by hypomethylation in cancer cells. However, the functions of this antigen remain unexplored. In the present study, we investigated the role of NY-SAR‑35 in human embryonic kidney (HEK) 293 cells using exogenous expression system of the gene. NY-SAR‑35 was predominantly expressed at the cytoplasm and was mainly observed in spermatogonia and spermatocytes. Expression of NY-SAR-35 in stable HEK293 transfectant clones was 2-fold higher than the control cells promoting cell growth and proliferation. NY-SAR-35 overexpression also enhanced cell migration and invasion ~2-fold and 4-fold more than the control, respectively. In contrast, small interfering RNA-mediated knockdown of NY-SAR-35 suppressed cell proliferation, migration, and invasion in HEK293 stable transfectants. We concluded that NY-SAR-35 as a cancer/testis antigen enhanced cell proliferation and invasion.

  18. GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

    PubMed

    Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

    2016-01-01

    Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS. PMID:26709648

  19. Astrocyte Contributions to Flow/Pressure-Evoked Parenchymal Arteriole Vasoconstriction

    PubMed Central

    Kim, Ki Jung; Iddings, Jennifer A.; Stern, Javier E.; Blanco, Víctor M.; Croom, Deborah; Kirov, Sergei A.

    2015-01-01

    Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca2+ dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca2+ imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca2+. Astrocytic Ca2+ responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca2+. In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca2+) and enhanced when high Ca2+ or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K+ and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone. PMID:26019339

  20. Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction.

    PubMed

    Kim, Ki Jung; Iddings, Jennifer A; Stern, Javier E; Blanco, Víctor M; Croom, Deborah; Kirov, Sergei A; Filosa, Jessica A

    2015-05-27

    Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca(2+) dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca(2+) imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca(2+). Astrocytic Ca(2+) responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca(2+). In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca(2+)) and enhanced when high Ca(2+) or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K(+) and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone. PMID:26019339

  1. Enhancement of primary neuronal cell proliferation using printing-transferred carbon nanotube sheets.

    PubMed

    Kang, Dong-Wan; Sun, Fangfang; Choi, Yoon Ji; Zou, Fengming; Cho, Won-Ho; Choi, Byung-Kwan; Koh, Kwangnak; Lee, Jaebeom; Han, In Ho

    2015-05-01

    Artificial nerve guidance conduits (aNGCs) prepared from polymer scaffolds and carbon nanotubes (CNTs) possess unique chemical and physical properties, and have been widely used in preclinical trials to promote neuronal differentiation and growth. However, there have been only a few reports on the clinical applicability of CNT sheets for proliferation of primary neuronal cells due to safety concerns. The present study assesses the ability and potential applicability of multiwalled CNTs (MWNTs) composited with polydimethylsiloxane (PDMS) sheets to promote and enhance the proliferation of primary neuronal cells. In this study, the aqueous MWNT dispersion was filtered, and the PDMS/MWNT sheets were prepared using a simple printing transfer method. Characterization of PDMS/MWNT sheets demonstrated their unique physical properties such as superior mechanical strength and electroconductivity when compared with PDMS sheets. The effect of the PDMS/MWNT sheets on the neural cell proliferation and cytotoxicity was evaluated using MTT and alamar blue assays. Our results indicate the viability and proliferation of primary neuronal cells and Schwann cells in PDMS/MWNT sheets increased over twice when compared with a noncoated dish that is not usual in the primary neuronal cell growth control (p < 0.05). In addition, PDMS/MWNT sheets enhanced the adhesion and viability of the cells compared with poly-l-lysine coated dishes, which are most commonly used for improving cell adherence. Additionally, the PDMS/MWNT sheets exhibited excellent biocompatibility for culturing neuronal and Schwann cells. Overall, all assessments indicate that PDMS/MWNT sheets are ideal candidates for the development of artificial nerve conduits for clinical use following peripheral nerve injury. PMID:25087551

  2. Elusive roles for reactive astrocytes in neurodegenerative diseases

    PubMed Central

    Ben Haim, Lucile; Carrillo-de Sauvage, Maria-Angeles; Ceyzériat, Kelly; Escartin, Carole

    2015-01-01

    Astrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes (hypertrophy, remodeling of processes) and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND. PMID:26283915

  3. Activation of Sonic Hedgehog Leads to Survival Enhancement of Astrocytes via the GRP78-Dependent Pathway in Mice Infected with Angiostrongylus cantonensis

    PubMed Central

    Chen, Kuang-Yao; Cheng, Chien-Ju; Wang, Lian-Chen

    2015-01-01

    Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytes may protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh) via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infected BABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated with corresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP) and Shh were increased in astrocyte cocultured with living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated in cocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higher in astrocytes pretreated with recombinant Shh. These findings suggest that the expression of Shh may inhibit cell death by activating Bcl-2 through a GRP78-dependent pathway. PMID:25961032

  4. HIV protease inhibitors disrupt astrocytic glutamate transporter function and neurobehavioral performance

    PubMed Central

    Vivithanaporn, Pornpun; Asahchop, Eugene L.; Acharjee, Shaona; Baker, Glen B.; Power, Christopher

    2016-01-01

    Objective: The neurotoxic actions of the HIV protease inhibitors, amprenavir (APV) and lopinavir (LPV) were investigated. Design: With combination antiretroviral therapy (cART), HIV-infected persons exhibit neurocognitive impairments, raising the possibility that cART might exert adverse central nervous system (CNS) effects. We examined the effects of LPV and APV using in-vitro and in-vivo assays of CNS function. Methods: Gene expression, cell viability and amino-acid levels were measured in human astrocytes, following exposure to APV or LPV. Neurobehavioral performance, amino-acid levels and neuropathology were examined in HIV-1 Vpr transgenic mice after treatment with APV or LPV. Results: Excitatory amino-acid transporter-2 (EAAT2) expression was reduced in astrocytes treated with LPV or APV, especially LPV (P < 0.05), which was accompanied by reduced intracellular l-glutamate levels in LPV-treated cells (P < 0.05). Treatment of astrocytes with APV or LPV reduced the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 (P < 0.05) although cell survival was unaffected. Exposure of LPV to astrocytes augmented glutamate-evoked transient rises in [Cai] (P < 0.05). Vpr mice treated with LPV showed lower concentrations of l-glutamate, l-aspartate and l-serine in cortex compared with vehicle-treated mice (P < 0.05). Total errors in T-maze assessment were increased in LPV and APV-treated animals (P < 0.05). EAAT2 expression was reduced in the brains of protease inhibitor-treated animals, which was associated with gliosis (P < 0.05). Conclusion: These results indicated that contemporary protease inhibitors disrupt astrocyte functions at therapeutic concentrations with enhanced sensitivity to glutamate, which can lead to neurobehavioral impairments. ART neurotoxicity should be considered in future therapeutic regimens for HIV/AIDS. PMID:26558720

  5. Endothelin, Astrocytes and Glaucoma

    PubMed Central

    Prasanna, Ganesh; Krishnamoorthy, Raghu; Yorio, Thomas

    2010-01-01

    It has become increasingly clear that astrocytes may play an important role in the genesis of glaucoma. Astrogliosis occurs in response to ocular stress or the presence of noxious stimuli. Agents that appear to stimulate reactive gliosis are becoming increasingly clear. One class of agents that is emerging is the endothelins (ETs; specifically, ET-1). In this review we examine the interactions of ET-1 with astrocytes and provide examples where ET-1 appears to contribute to activation of astrocytes and play a role in the neurodegenerative effects that accompany such reactivation resulting in astrogliosis. These actions are presented in the context of glaucoma although information is also presented with respect to ET-1's role in the central nervous system and brain. While much has been learned with respect to ET-1/astrocyte interactions, there are still a number of questions concerning the potential therapeutic implications of these findings. Hopefully this review will stimulate others to examine this potential. PMID:20849847

  6. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

    SciTech Connect

    Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun . E-mail: dli2@slu.edu

    2006-11-01

    Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), {delta}p85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.

  7. Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.

    PubMed

    Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

    2015-01-01

    Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

  8. Canonical Wnt signaling transiently stimulates proliferation and enhances neurogenesis in neonatal neural progenitor cultures

    SciTech Connect

    Hirsch, Cordula; Campano, Louise M.; Woehrle, Simon; Hecht, Andreas . E-mail: andreas.hecht@mol-med.uni-freiburg.de

    2007-02-01

    Canonical Wnt signaling triggers the formation of heterodimeric transcription factor complexes consisting of {beta}-catenin and T cell factors, and thereby controls the execution of specific genetic programs. During the expansion and neurogenic phases of embryonic neural development canonical Wnt signaling initially controls proliferation of neural progenitor cells, and later neuronal differentiation. Whether Wnt growth factors affect neural progenitor cells postnatally is not known. Therefore, we have analyzed the impact of Wnt signaling on neural progenitors isolated from cerebral cortices of newborn mice. Expression profiling of pathway components revealed that these cells are fully equipped to respond to Wnt signals. However, Wnt pathway activation affected only a subset of neonatal progenitors and elicited a limited increase in proliferation and neuronal differentiation in distinct subsets of cells. Moreover, Wnt pathway activation only transiently stimulated S-phase entry but did not support long-term proliferation of progenitor cultures. The dampened nature of the Wnt response correlates with the predominant expression of inhibitory pathway components and the rapid actuation of negative feedback mechanisms. Interestingly, in differentiating cell cultures activation of canonical Wnt signaling reduced Hes1 and Hes5 expression suggesting that during postnatal neural development, Wnt/{beta}-catenin signaling enhances neurogenesis from progenitor cells by interfering with Notch pathway activity.

  9. Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

    PubMed

    Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

    2016-01-13

    The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds. PMID:26652045

  10. Loss of MiR-664 Expression Enhances Cutaneous Malignant Melanoma Proliferation by Upregulating PLP2

    PubMed Central

    Ding, Zhenhua; Jian, Sun; Peng, Xuebiao; Liu, Yimin; Wang, Jianyu; Zheng, Li; Ou, Chengshan; Wang, Yinghui; Zeng, Weixia; Zhou, Meijuan

    2015-01-01

    Abstract Proteolipid protein 2 (PLP2) has been shown to be upregulated in several cancers, including breast cancer, hepatocellular carcinoma, osteosarcoma, and melanoma. PLP2 specifically binds to phosphatidylinositol 3 kinase to activate the protein kinase B pathway to enhance cell proliferation, adhesion, and invasion in melanoma cells. Therefore, we speculated that PLP2 exhibits oncogenic potential. However, the regulatory mechanisms of PLP2 in cancer cells remain unclear. Herein, we found that microRNA (miR)-664 expression was significantly downregulated in cutaneous malignant melanoma (CMM) cells and tissues compared with normal human melanocytes and benign melanocytic naevi. MiR-664 expression level was significantly correlated with patient survival. Ectopic expression of miR-664 reduced CMM cell proliferation and anchorage-independent growth, whereas the inhibition of miR-664 induced these effects. Furthermore, inhibition of miR-664 in CMM cells resulted in modulation of their entry into the G1/S transitional phase, which was caused by downregulation of the cyclin-dependent kinase inhibitor P21 and upregulation of the cell-cycle regulator cyclin D1. Moreover, we demonstrated that miR-664 downregulated PLP2 expression by directly targeting the PLP2 untranslated region. Taken together, our results suggest that miR-664 may play an important role in suppressing proliferation of CMM cells and present a novel mechanism of miR-mediated direct suppression of PLP2 expression in cancer cells. PMID:26287415

  11. Surface modification of SU-8 for enhanced cell attachment and proliferation within microfluidic chips.

    PubMed

    Hamid, Qudus; Wang, Chengyang; Snyder, Jessica; Sun, Wei

    2015-02-01

    Advances in micro-electro-mechanical systems (MEMS) have led to an increased fabrication of micro-channels. Microfabrication techniques are utilized to develop microfluidic channels for continuous nutrition supply to cells inside a micro-environment. The ability of cells to build tissues and maintain tissue-specific functions depends on the interaction between cells and the extracellular matrix (ECM). SU-8 is a popular photosensitive epoxy-based polymer in MEMS. The patterning of bare SU-8 alone does not provide the appropriate ECM necessary to develop microsystems for biological applications. Manipulating the chemical composition of SU-8 will enhance the biological compatibility, giving the fabricated constructs the appropriate ECM needed to promote a functional tissue array. This article investigates three frequently used surface treatment techniques: (1) plasma treatment, (2) chemical reaction, and (3) deposition treatment to determine which surface treatment is the most beneficial for enhancing the biological properties of SU-8. The investigations presented in this article demonstrated that the plasma, gelatin, and sulfuric acid treatments have a potential to enhance SU-8's surface for biological application. Of course each treatment has their advantages and disadvantages (application dependent). Cell proliferation was studied with the use of the dye Almar Blue, and a micro-plate reader. After 14 days, cell proliferation to plasma treated surfaces was statistically significantly enhanced (p < 0.00001), compared to untreated surfaces. The plasma treated surface is suggested to be the better of the three treatments for biological enhancement followed by gelatin and sulfuric acid treatments, respectively. PMID:24919697

  12. Inhibition of proliferation of normal and transformed neural cells by blood group-related oligosaccharides

    PubMed Central

    1992-01-01

    A synthetic tetrasaccharide structurally related to blood groups and selectin ligands inhibited division of astrocytes, gliomas, and neuroblastomas at micromolar concentrations. The compound was cytostatic for primary astrocytes in culture, but cytotoxic for fast proliferating cell lines. PMID:1512552

  13. In vivo but not in vitro leptin enhances lymphocyte proliferation in Siberian hamsters (Phodopus sungorus).

    PubMed

    Demas, Gregory E

    2010-04-01

    Mounting an immune response requires a relatively substantial investment of energy and marked reductions in energy availability can suppress immune function and presumably increase disease susceptibility. We have previously demonstrated that a moderate reduction in energy stores by partial surgical lipectomy impairs humoral immunity of Siberian hamsters (Phodopus sungorus) and is mediated, in part, by changes in the adipose tissue hormone leptin. The goals of the present study were to assess the role of leptin in cell-mediated immunity and to determine if the potential effects of leptin on immunity are via the direct actions of this hormone on lymphocytes, or indirect, via the sympathetic nervous system (SNS). In Experiment 1, hamsters received osmotic minipumps containing either murine leptin (0.5 microl/h) or vehicle alone for 10 days and splenocyte proliferation in response to the T-cell mitogen Concanavalin A (Con A) was determined. In Experiment 2, Con A-induced splenocyte proliferation was tested in the presence or absence of leptin in vitro. In Experiment 3, exogenous leptin was administered to intact or sympathetically denervated hamsters. Hamsters treated with in vivo leptin displayed increased splenocyte proliferation compared with control hamsters receiving vehicle. In contrast, in vitro leptin had no effect on splenocyte proliferation. Sympathetic denervation attenuated, but did not block, leptin-induced increases in immunity. Taken together, these results are consistent with the idea that leptin can enhance cell-mediated immunity; the SNS appears to contribute, least in part, to leptin-induced increases in immunity. Importantly, these findings confirm previous studies that leptin serves as an important endocrine link between energy balance and immunity.

  14. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    SciTech Connect

    Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  15. In vivo astrocytic Ca2+ signaling in health and brain disorders

    PubMed Central

    Ding, Shinghua

    2013-01-01

    Astrocytes are the predominant glial cell type in the CNS. Although astrocytes are electrically nonexcitable, their excitability is manifested by their Ca2+ signaling, which serves as a mediator of neuron–glia bidirectional interactions via tripartite synapses. Studies from in vivo two-photon imaging indicate that in healthy animals, the properties of spontaneous astrocytic Ca2+ signaling are affected by animal species, age, wakefulness and the location of astrocytes in the brain. Intercellular Ca2+ waves in astrocytes can be evoked by a variety of stimulations. In animal models of some brain disorders, astrocytes can exhibit enhanced Ca2+ excitability featured as regenerative intercellular Ca2+ waves. This review first briefly summarizes the astrocytic Ca2+ signaling pathway and the procedure of in vivo two-photon Ca2+ imaging of astrocytes. It subsequently summarizes in vivo astrocytic Ca2+ signaling in health and brain disorders from experimental studies of animal models, and discusses the possible mechanisms and therapeutic implications underlying the enhanced Ca2+ excitability in astrocytes in brain disorders. Finally, this review summarizes molecular genetic approaches used to selectively manipulate astrocyte function in vivo and their applications to study the role of astrocytes in synaptic plasticity and brain disorders. PMID:24376372

  16. Mast cells and histamine enhance the proliferation of non-small cell lung cancer cells.

    PubMed

    Stoyanov, Evgeniy; Uddin, Mohib; Mankuta, David; Dubinett, Steven M; Levi-Schaffer, Francesca

    2012-01-01

    Non-small cell lung cancer (NSCLC) is the most common form of lung cancer with an extremely low survival rate. It is characterized by a chronic inflammatory process with intense mast cell infiltrate that is associated with reduced survival. The aim of this study was to test the hypothesis that mast cells have an enhancing effect on NSCLC proliferation. To assess the tumor-promoting potential of mast cells, we used the human alveolar basal adenocarcinoma (A549) and the mouse Lewis lung carcinoma (LLC) cell lines, umbilical cord blood-derived mast cells (CBMC) and the mast cell-deficient mouse Sash model. The proliferation rate of A549/LLC cells was markedly increased by mast cells and histamine. Histamine proliferating activity was mediated via H(1), H(2) and H(4) receptors and caused ERK phosphorylation. LLC induced in Sash mice or in wild-type mice treated with the mast cell stabilizer nedocromil sodium displayed an accelerated growth (number of metastic colonies in the lungs, total lung area and lung/total mice weight ratio). In summary, we have shown a significant effect of mast cells and histamine in enhancing NSCLC/LLCX growth in vitro, while in a mouse LLC model in vivo we have found that mast cells are important negative regulators of cancer development. Therefore our results would indicate a pro-tumorogenic effect of the mast cells in vitro on established lung tumor cell lines, and anti-tumorogenic effect in mice at lung cancer induction. In conclusion, mast cell/anti-histamine targeted therapies should carefully consider this dual effect. PMID:21733595

  17. Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation

    PubMed Central

    Yu, Jian; Chen, Liguang; Cui, Bing; Widhopf, George F.; Shen, Zhouxin; Wu, Rongrong; Zhang, Ling; Zhang, Suping; Briggs, Steven P.; Kipps, Thomas J.

    2015-01-01

    Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation. PMID:26690702

  18. Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

    SciTech Connect

    Raesaenen, Kati; Vaheri, Antti

    2010-06-10

    The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

  19. Grb10 deletion enhances muscle cell proliferation, differentiation and GLUT4 plasma membrane translocation.

    PubMed

    Mokbel, Nancy; Hoffman, Nolan J; Girgis, Christian M; Small, Lewin; Turner, Nigel; Daly, Roger J; Cooney, Gregory J; Holt, Lowenna J

    2014-11-01

    Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes.

  20. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

    PubMed Central

    Lakhkar, Nilay J; M Day, Richard; Kim, Hae-Won; Ludka, Katarzyna; Mordan, Nicola J; Salih, Vehid; Knowles, Jonathan C

    2015-01-01

    In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5) that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications. PMID:26668711

  1. Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

    PubMed Central

    Pelto, Jani; Björninen, Miina; Pälli, Aliisa; Talvitie, Elina; Hyttinen, Jari; Mannerström, Bettina; Suuronen Seppanen, Riitta; Kellomäki, Minna; Miettinen, Susanna; Haimi, Suvi

    2013-01-01

    An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering. PMID:23126228

  2. Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-L-lactic acid fibers

    PubMed Central

    Zuidema, Jonathan M.; Hyzinski-García, María C.; Vlasselaer, Kristien Van; Zaccor, Nicholas; Plopper, George E.; Mongin, Alexander A.; Gilbert, Ryan J.

    2014-01-01

    Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake. PMID:24246642

  3. Calcium dynamics in astrocyte processes during neurovascular coupling

    PubMed Central

    Otsu, Yo; Couchman, Kiri; Lyons, Declan G; Collot, Mayeul; Agarwal, Amit; Mallet, Jean-Maurice; Pfrieger, Frank W; Bergles, Dwight E; Charpak, Serge

    2015-01-01

    Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca2+) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca2+ signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca2+ sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca2+ increases in astrocyte processes but not in somata. These Ca2+ increases systematically precede the onset of functional hyperemia by 1–2 s, reestablishing astrocytes as potential regulators of neurovascular coupling. PMID:25531572

  4. Discovery of Novel Small Molecules that Activate Satellite Cell Proliferation and Enhance Repair of Damaged Muscle.

    PubMed

    Billin, Andrew N; Bantscheff, Marcus; Drewes, Gerard; Ghidelli-Disse, Sonja; Holt, Jason A; Kramer, Henning F; McDougal, Alan J; Smalley, Terry L; Wells, Carrow I; Zuercher, William J; Henke, Brad R

    2016-02-19

    Skeletal muscle progenitor stem cells (referred to as satellite cells) represent the primary pool of stem cells in adult skeletal muscle responsible for the generation of new skeletal muscle in response to injury. Satellite cells derived from aged muscle display a significant reduction in regenerative capacity to form functional muscle. This decrease in functional recovery has been attributed to a decrease in proliferative capacity of satellite cells. Hence, agents that enhance the proliferative abilities of satellite cells may hold promise as therapies for a variety of pathological settings, including repair of injured muscle and age- or disease-associated muscle wasting. Through phenotypic screening of isolated murine satellite cells, we identified a series of 2,4-diaminopyrimidines (e.g., 2) that increased satellite cell proliferation. Importantly, compound 2 was effective in accelerating repair of damaged skeletal muscle in an in vivo mouse model of skeletal muscle injury. While these compounds were originally prepared as c-Jun N-terminal kinase 1 (JNK-1) inhibitors, structure-activity analyses indicated JNK-1 inhibition does not correlate with satellite cell activity. Screening against a broad panel of kinases did not result in identification of an obvious molecular target, so we conducted cell-based proteomics experiments in an attempt to identify the molecular target(s) responsible for the potentiation of the satellite cell proliferation. These data provide the foundation for future efforts to design improved small molecules as potential therapeutics for muscle repair and regeneration.

  5. Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors

    PubMed Central

    Hedlund, Eva; Belnoue, Laure; Theofilopoulos, Spyridon; Salto, Carmen; Bye, Chris; Parish, Clare; Deng, Qiaolin; Kadkhodaei, Banafsheh; Ericson, Johan; Arenas, Ernest; Perlmann, Thomas; Simon, András

    2016-01-01

    Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis. PMID:27246266

  6. XPC inhibits NSCLC cell proliferation and migration by enhancing E-Cadherin expression

    PubMed Central

    Cui, Tiantian; Srivastava, Amit Kumar; Han, Chunhua; Yang, Linlin; Zhao, Ran; Zou, Ning; Qu, Meihua; Duan, Wenrui; Zhang, Xiaoli; Wang, Qi-En

    2015-01-01

    Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair. Deletion of XPC is associated with early stages of human lung carcinogenesis, and reduced XPC mRNA levels predict poor patient outcome for non-small cell lung cancer (NSCLC). However, the mechanisms linking loss of XPC expression and poor prognosis in lung cancer are still unclear. Here, we report evidence that XPC silencing drives proliferation and migration of NSCLC cells by down-regulating E-Cadherin. XPC knockdown enhanced proliferation and migration while decreasing E-Cadherin expression in NSCLC cells with an epithelial phenotype. Restoration of E-Cadherin in these cells suppressed XPC knockdown-induced cell growth both in vitro and in vivo. Mechanistic studies showed that the loss of XPC repressed E-Cadherin expression by activating the ERK pathway and upregulating Snail expression. Our findings indicate that XPC silencing-induced reduction of E-Cadherin expression contributes, at least in part, to the poor outcome of NSCLC patients with low XPC expression. PMID:25871391

  7. Utility of Social Modeling for Proliferation Assessment - Enhancing a Facility-Level Model for Proliferation Resistance Assessment of a Nuclear Enegry System

    SciTech Connect

    Coles, Garill A.; Brothers, Alan J.; Gastelum, Zoe N.; Olson, Jarrod; Thompson, Sandra E.

    2009-10-26

    The Utility of Social Modeling for Proliferation Assessment project (PL09-UtilSocial) investigates the use of social and cultural information to improve nuclear proliferation assessments, including nonproliferation assessments, Proliferation Resistance (PR) assessments, safeguards assessments, and other related studies. These assessments often use and create technical information about a host State and its posture towards proliferation, the vulnerability of a nuclear energy system (NES) to an undesired event, and the effectiveness of safeguards. This objective of this project is to find and integrate social and technical information by explicitly considering the role of cultural, social, and behavioral factors relevant to proliferation; and to describe and demonstrate if and how social science modeling has utility in proliferation assessment. This report describes a modeling approach and how it might be used to support a location-specific assessment of the PR assessment of a particular NES. The report demonstrates the use of social modeling to enhance an existing assessment process that relies on primarily technical factors. This effort builds on a literature review and preliminary assessment performed as the first stage of the project and compiled in PNNL-18438. [ T his report describes an effort to answer questions about whether it is possible to incorporate social modeling into a PR assessment in such a way that we can determine the effects of social factors on a primarily technical assessment. This report provides: 1. background information about relevant social factors literature; 2. background information about a particular PR assessment approach relevant to this particular demonstration; 3. a discussion of social modeling undertaken to find and characterize social factors that are relevant to the PR assessment of a nuclear facility in a specific location; 4. description of an enhancement concept that integrates social factors into an existing, technically

  8. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    SciTech Connect

    Tu Qisheng; Valverde, Paloma . E-mail: paloma.valverde@tufts.edu; Chen, Jake

    2006-03-24

    Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

  9. GPR171 expression enhances proliferation and metastasis of lung cancer cells

    PubMed Central

    Dho, So Hee; Lee, Kwang-Pyo; Jeong, Dongjun; Kim, Chang-Jin; Chung, Kyung-Sook; Kim, Ji Young; Park, Bum-Chan; Park, Sung Sup; Kim, Seon-Young; Kwon, Ki-Sun

    2016-01-01

    G protein-coupled receptors (GPCRs) are among the most significant therapeutic targets and some of them promote the growth and metastasis of cancer. Here, we show that an increase in the levels of GPR171 is crucial for lung cancer tumor progression in vitro and in vivo. Immunostaining of clinical samples indicated that GPR171 was overexpressed in 46.8% of lung carcinoma tissues. Depletion of GPR171 with an anti-GPR171 antibody decreased proliferation of lung carcinoma cells and attenuated tumor progression in a mouse xenograft model. Knockdown of GPR171 also inhibited migration and invasion of the lung cancer cell lines. Notably, inhibition of GPR171 synergistically enhanced the tumoricidal activity of an epidermal growth factor receptor (EGFR) inhibitor in lung cancer cells. These results indicate that GPR171 blockade is a promising antineoplastic strategy and provide a preclinical rationale for combined inhibition of GPR171 and EGFR. PMID:26760963

  10. FBI-1 enhances ETS-1 signaling activity and promotes proliferation of human colorectal carcinoma cells.

    PubMed

    Zhu, Min; Li, Mingyang; Zhang, Fan; Feng, Fan; Chen, Weihao; Yang, Yutao; Cui, Jiajun; Zhang, Dong; Linghu, Enqiang

    2014-01-01

    In this study, we investigated a potential regulatory role of FBI-1 in transcription factor activity of ETS-1. The protein interaction was identified between ETS-1 and FBI-1 in lovo cells. The accumulating data showed that FBI-1 promoted the recruitment of ETS-1 to endogenous promoter of its target genes and increase ETS-1 accumulation in the nuclear. Our work also indicated that the FBI-1 enhances ETS-1 transcription factor activity via down-regulating p53-mediated inhibition on ETS-1. Further, FBI-1 plays a role in regulation of colorectal carcinoma cells proliferation. These findings supported that FBI-1 might be a potential molecule target for treating colorectal carcinoma.

  11. FBI-1 Enhances ETS-1 Signaling Activity and Promotes Proliferation of Human Colorectal Carcinoma Cells

    PubMed Central

    Chen, Weihao; Yang, Yutao; Cui, Jiajun; Zhang, Dong; Linghu, Enqiang

    2014-01-01

    In this study, we investigated a potential regulatory role of FBI-1 in transcription factor activity of ETS-1. The protein interaction was identified between ETS-1 and FBI-1 in lovo cells. The accumulating data showed that FBI-1 promoted the recruitment of ETS-1 to endogenous promoter of its target genes and increase ETS-1 accumulation in the nuclear. Our work also indicated that the FBI-1 enhances ETS-1 transcription factor activity via down-regulating p53-mediated inhibition on ETS-1. Further, FBI-1 plays a role in regulation of colorectal carcinoma cells proliferation. These findings supported that FBI-1 might be a potential molecule target for treating colorectal carcinoma. PMID:24857950

  12. Acetylation Enhances the Promoting Role of AIB1 in Breast Cancer Cell Proliferation

    PubMed Central

    You, Dingyun; Zhao, Hongbo; Wang, Yan; Jiao, Yang; Lu, Minnan; Yan, Shan

    2016-01-01

    The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator, which is overexpressed in various types of human cancers, including breast cancer. However, the molecular mechanisms regulating AIB1 function remain largely unknown. In this study, we present evidence demonstrating that AIB1 is acetylated by MOF in human breast cancer cells. Moreover, we also found that the acetylation of AIB1 enhances its function in promoting breast cancer cell proliferation. We further showed that the acetylation of AIB1 is required for its recruitment to E2F1 target genes by E2F1. More importantly, we found that the acetylation levels of AIB1 are greatly elevated in human breast cancer cells compared with that in non-cancerous cells. Collectively, our results shed light on the molecular mechanisms that regulate AIB1 function in breast cancer. PMID:27665502

  13. Nanoparticle-mediated conversion of primary human astrocytes into neurons and oligodendrocytes.

    PubMed

    Li, Xiaowei; Kozielski, Kristen; Cheng, Yu-Hao; Liu, Huanhuan; Zamboni, Camila Gadens; Green, Jordan; Mao, Hai-Quan

    2016-06-21

    Central nervous system (CNS) diseases and injuries are accompanied by reactive gliosis and scarring involving the activation and proliferation of astrocytes to form hypertrophic and dense structures, which present a significant barrier to neural regeneration. Engineering astrocytes to functional neurons or oligodendrocytes may constitute a novel therapeutic strategy for CNS diseases and injuries. Such direct cellular programming has been successfully demonstrated using viral vectors via the transduction of transcriptional factors, such as Sox2, which could program resident astrocytes into neurons in the adult brain and spinal cord, albeit the efficiency was low. Here we report a non-viral nanoparticle-based transfection method to deliver Sox2 or Olig2 into primary human astrocytes and demonstrate the effective conversion of the astrocytes into neurons and oligodendrocyte progenitors following the transgene expression of Sox2 and Olig2, respectively. This approach is highly translatable for engineering astrocytes to repair injured CNS tissues. PMID:27328202

  14. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips.

    PubMed

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M

    2016-10-14

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched  nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter-towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma. PMID:27587351

  15. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips.

    PubMed

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M

    2016-10-14

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched  nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter-towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  16. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips

    NASA Astrophysics Data System (ADS)

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M.

    2016-10-01

    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter—towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  17. Probing astrocytes with carbon nanotubes and assessing their effects on astrocytic structural and functional properties

    NASA Astrophysics Data System (ADS)

    Gottipati, Manoj K.

    Single-walled carbon nanotubes, chemically-functionalized with polyethylene glycol (SWCNT-PEG) have been shown to modulate the morphology and proliferation characteristics of astrocytes in culture, when applied to the cells as colloidal solutes or as films upon which the cells can attach and grow. These changes were associated with a change in the immunoreactivity of the astrocyte-specific protein, glial fibrillary acidic protein (GFAP); the solutes and films caused an increase and a decrease in GFAP levels, respectively. To assess if these morpho-functional changes induced by the SWCNT-PEG modalities are dependent on GFAP or if the changes in GFAP levels are independent events, I used astrocytes isolated from GFAP knockout mice and found that selected changes induced by the SWCNT-PEG modalities are mediated by GFAP, namely the changes in perimeter, shape and cell death for colloidal solutes and the rate of proliferation for films. Since the loss GFAP has been shown to hamper the trafficking of glutamate transporters to the surface of astrocytes, which plays a vital role in the uptake of extracellular glutamate and maintaining homeostasis in the brain and spinal cord, in a subsequent study, I assessed if the SWCNT-PEG solute causes any change in the glutamate uptake characteristics of astrocytes. Using a radioactive glutamate uptake assay and immunolabeling, I found that SWCNT-PEG solute causes an increase in the uptake of glutamate from the extracellular space along with an increase in the immunoreactivity of the glutamate transporter, L-glutamate L-aspartate transporter (GLAST), on their cell surface, a likely consequence of the increase in GFAP levels induced by the SWCNT-PEG solute. These results imply that SWCNT-PEG could potentially be used as a viable candidate in neural prosthesis applications to prevent glutamate excitotoxicity, a pathological process observed in brain and spinal cord injuries, and alleviate the death toll of neurons surrounding the injury

  18. Enhancement of methylnitrosourea skin carcinogenesis by inhibiting cell proliferation with hydroxyurea or skin extracts.

    PubMed

    Iversen, O H

    1982-01-01

    To study the relationship between epidermal DNA synthesis and carcinogenesis, groups of hairless mice were given a single skin application of 2 mg N-methyl-N-nitrosourea (MNU) in acetone. One group received no pretreatment, another group was injected i.p. with 5 mg hydroxyurea (HU) 30 min before MNU, and a further group with 0.5 mg Colcemid 3 h before MNU. Other groups were injected i.p. 14 and 4 h before MNU with either saline, 5 mg crude aqueous skin extract, 2 mg dialysed skin extracts of two types, or 2 mg dialysed extracts of liver or heart muscle, respectively. All substances were dissolved in 0.5 ml distilled water. Cell kinetic studies showed that the three skin extracts inhibited epidermal DNA synthesis and mitosis. HU inhibited DNA synthesis and increased the mitotic rate. The other pretreatments had no effect on epidermal DNA synthesis. There was a significant enhancement of the production of skin tumors in the groups pretreated with epidermal extracts or HU. MNU is a short-acting carcinogen with a half-life in the cell of 30 min. Hence, the results show that when DNA synthesis is inhibited at the time of MNU application, more tumors are produced in the skin. A possible explanation of the enhancement is that a compensatory wave of proliferation a short time after carcinogen binding may fix a DNA injury before repair can take place.

  19. Plumbagin Promotes the Generation of Astrocytes from Rat Spinal Cord Neural Progenitors Via Activation of the Transcription Factor Stat3

    PubMed Central

    Luo, Yongquan; Mughal, Mohamed; Ouyang, Xin; Jiang, Haiyang; Luo, Tae-Gen Son Weiming; Yu, Qian-Sheng; Greig, Nigel H.; Mattson, Mark P.

    2010-01-01

    Plumbagin (5-hydroxy-2-methyl-1,4 naphthoquinone) is a naturally occurring low molecular weight lipophilic phytochemical derived from roots of plants of the Plumbago genus. Plumbagin has been reported to have several clinically relevant biological activities in non-neural cells including antiatherosclerotic, anticoagulant, anticarcinogenic, antitumor and bactericidal effects. In a recent screen of a panel of botanical pesticides we identified plumbagin as having neuroprotective activity. In the present study we determined if plumbagin could modify the developmental fate of rat E14.5 embryonic neural progenitor cells (NPC). Plumbagin exhibited no cytotoxicity when applied to cultured NPC at concentrations below 1 µM. At a concentration of 0.1 µM, plumbagin significantly enhanced the proliferation of NPC as indicated by a 17% increase in the percentage of cells incorporating bromo-deoxyuridine. plumbagin at a concentration of 0.1 pM (but not 0.1 µM), stimulated the production of astrocytes as indicated by increased GFAP expression. Plumbagin selectively induced the proliferation and differentiation of glial progenitor cells without affecting the proliferation or differentiation of neuron-restricted progenitors. Plumbagin (0.1 pM) rapidly activated the transcription factor Stat3 in NPC, and a Stat3 inhibitor peptide prevented both plumbagin-induced astrocyte formation and proliferation. These findings demonstrate the ability of a low molecular weight naturally occurring phytochemical to control the fate of glial progenitor cells by a mechanism involving the Stat3 signaling pathway. PMID:20456019

  20. Constitutive Expression of Human Telomerase Enhances the Proliferation Potential of Human Mesenchymal Stem Cells

    PubMed Central

    Bischoff, David S.; Makhijani, Nalini S.

    2012-01-01

    Abstract Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential. PMID:23515239

  1. Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

    SciTech Connect

    Tang, Yiting; Liu, Lan; Sheng, Ming; Xiong, Kai; Huang, Lei; Gao, Qian; Wei, Jingliang; Wu, Tianwen; Yang, Shulin; Liu, Honglin; Mu, Yulian; Li, Kui

    2015-06-10

    Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1

  2. Simultaneous neuron- and astrocyte-specific fluorescent marking

    SciTech Connect

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; and others

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

  3. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    SciTech Connect

    Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  4. Ethanolamine enhances the proliferation of intestinal epithelial cells via the mTOR signaling pathway and mitochondrial function.

    PubMed

    Yang, Huansheng; Xiong, Xia; Li, Tiejun; Yin, Yulong

    2016-05-01

    Ethanolamine (Etn), which is the base constituent of phosphatidylethanolamine, a major phospholipid in animal cell membranes, is required for the proliferation of many types of mammalian epithelial cells. However, it is not clear whether the proliferation of intestinal epithelial cells requires Etn. The present study was conducted to examine the effects of Etn on the proliferation of intestinal epithelial cells and to elucidate the underlying mechanisms. The addition of Etn at 100 or 200 μM was found to enhance the proliferation of IPEC-1 cells. The expression of cell cycle-related proteins CDK4, RB3, cyclin A, and PCNA was also enhanced by Etn. Moreover, the expression or phosphorylation levels of the mammalian target of rapamycin (mTOR) signaling pathway protein and the expression of proteins related to mitochondrial function were also affected by Etn in IPEC-1 cells. These results indicate that Etn promotes the proliferation of intestinal epithelial cells by exerting effects on mTOR signaling pathway and mitochondrial function.

  5. Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

    PubMed

    Oviedo, Pilar J; Sobrino, Agua; Laguna-Fernandez, Andrés; Novella, Susana; Tarín, Juan J; García-Pérez, Miguel-Angel; Sanchís, Juan; Cano, Antonio; Hermenegildo, Carlos

    2011-03-30

    Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells.

  6. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  7. Adipokines enhance oleic acid-induced proliferation of vascular smooth muscle cells by inducing CD36 expression.

    PubMed

    Schlich, Raphaela; Lamers, Daniela; Eckel, Jürgen; Sell, Henrike

    2015-01-01

    Adipose tissue is not only releasing lipids but also various adipokines that are both dysregulated in the obese state and may contribute to obesity-associated vascular dysfunction and cardiovascular risk. We have previously shown that the combination of adipocyte-conditioned medium (CM) and oleic acid (OA) increases proliferation of human vascular smooth muscle cells (VSMC) in a synergistic way. We identified vascular endothelial growth factor (VEGF) as a component within CM that is responsible for most of the observed effects. In this study, we investigate novel mechanisms that underlie the combined effects of adipokine and oleic acid-induced proliferation of VSMC. Oleic acid leads to significant lipid accumulation in VSMC that is further enhanced by the combined treatment with CM. Accordingly CM stimulates CD36 expression in VSMC while OA is not affecting CD36. Silencing of CD36 was established and prevents lipid accumulation in all tested conditions. CD36 silencing also abrogates CM- and OA-induced proliferation and considerably reduces proliferation induced by the combination of CM and OA. At the same time, VEGF secretion and VEGF-receptor 1 (VEGF-R1) by VSMC was not affected by CD36 silencing. However, VEGF was not able to induce any proliferation in VSMC after CD36 silencing that also blunted VEGF-induced extracellular signal-regulated kinase (ERK) activation. Finally, combined silencing of CD36 together with a blocking antibody against VEGF prevented most of CMOA-induced proliferation. In conclusion, our results demonstrate that CD36 is mediating CM-induced proliferation of VSMC. Induction of CD36 by adipokines enhances the response of VSMC towards VEGF and OA.

  8. The Transcriptional Repressor ZNF503/Zeppo2 Promotes Mammary Epithelial Cell Proliferation and Enhances Cell Invasion.

    PubMed

    Shahi, Payam; Slorach, Euan M; Wang, Chih-Yang; Chou, Jonathan; Lu, Angela; Ruderisch, Aline; Werb, Zena

    2015-02-01

    The NET (nocA, Nlz, elB, TLP-1) subfamily of zinc finger proteins is an important mediator during developmental processes. The evolutionary conserved zinc finger protein ZNF503/Zeppo2 (zinc finger elbow-related proline domain protein 2, Zpo2) plays critical roles during embryogenesis. We found that Zpo2 is expressed in adult tissue and examined its function. We found that ZPO2 is a nuclearly targeted transcriptional repressor that is expressed in mammary epithelial cells. Elevated Zpo2 levels increase mammary epithelial cell proliferation. Zpo2 promotes cellular invasion through down-regulation of E-cadherin and regulates the invasive phenotype in a RAC1-dependent manner. We detect elevated Zpo2 expression during breast cancer progression in a MMTV-PyMT transgenic mouse model. Tumor transplant experiments indicated that overexpression of Zpo2 in MMTV-PyMT mammary tumor cell lines enhances lung metastasis. Our findings suggest that Zpo2 plays a significant role in mammary gland homeostasis and that deregulation of Zpo2 may promote breast cancer development.

  9. Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells

    PubMed Central

    Warth, Benedikt; Del Favero, Giorgia; Wiesenberger, Gerlinde; Puntscher, Hannes; Woelflingseder, Lydia; Fruhmann, Philipp; Sarkanj, Bojan; Krska, Rudolf; Schuhmacher, Rainer; Adam, Gerhard; Marko, Doris

    2016-01-01

    The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority. PMID:27659167

  10. The Neuropeptide VGF Produces Antidepressant-Like Behavioral Effects and Enhances Proliferation in the Hippocampus

    PubMed Central

    Thakker-Varia, Smita; Krol, Jennifer Jernstedt; Nettleton, Jacob; Bilimoria, Parizad M.; Bangasser, Debra A.; Shors, Tracey J.; Black, Ira B.; Alder, Janet

    2012-01-01

    Brain-derived neurotrophic factor (BDNF) is upregulated in the hippocampus by antidepressant treatments, and BDNF produces antidepressant-like effects in behavioral models of depression. In our previous work, we identified genes induced by BDNF and defined their specific roles in hippocampal neuronal development and plasticity. To identify genes downstream of BDNF that may play roles in psychiatric disorders,we examined a subset of BDNF-induced genes also regulated by 5-HT (serotonin), which includes the neuropeptide VGF (nonacronymic). To explore the function of VGF in depression, we first investigated the expression of the neuropeptide in animal models of depression. VGF was downregulated in the hippocampus after both the learned helplessness and forced swim test (FST) paradigms. Conversely, VGF infusion in the hippocampus of mice subjected to FST reduced the time spent immobile for up to 6 d, thus demonstrating a novel role for VGF as an antidepressant-like agent. Recent evidence indicates that chronic treatment of rodents with antidepressants increases neurogenesis in the adult dentate gyrus and that neurogenesis is required for the behavioral effects of antidepressants. Our studies using [3H]thymidine and bromodeoxyuridine as markers of DNA synthesis indicate that chronic VGF treatment enhances proliferation of hippocampal progenitor cells both in vitro and in vivo with survival up to 21 d. By double immunocytochemical analysis of hippocampal neurons, we demonstrate that VGF increases the number of dividing cells that express neuronal markers in vitro. Thus, VGF may act downstream of BDNF and exert its effects as an antidepressant-like agent by enhancing neurogenesis in the hippocampus. PMID:17989282

  11. Local generation of glia is a major astrocyte source in postnatal cortex

    PubMed Central

    Ge, Woo-Ping; Miyawaki, Atsushi; Gage, Fred H.; Jan, Yuh Nung; Jan, Lily Yeh

    2013-01-01

    Glial cells constitute nearly 50% of the cells in the human brain1. Astrocytes, which make up the largest glial population, are crucial to the regulation of synaptic connectivity during postnatal development2. Because defects in astrocyte generation are associated with severe neurological disorders such as brain tumours3, it is important to understand how astrocytes are produced. Astrocytes reportedly arise from two sources4–6: radial glia in the ventricular zone and progenitors in the subventricular zone, with the contribution from each region shifting with time. During the first three weeks of postnatal development, the glial cell population, which contains predominantly astrocytes, expands 6–8-fold in the rodent brain7. Little is known about the mechanisms underlying this expansion. Here we show that a major source of glia in the postnatal cortex in mice is the local proliferation of differentiated astrocytes. Unlike glial progenitors in the subventricular zone, differentiated astrocytes undergo symmetric division, and their progeny integrate functionally into the existing glial network as mature astrocytes that form endfeet with blood vessels, couple electrically to neighbouring astrocytes, and take up glutamate after neuronal activity. PMID:22456708

  12. Astrocytes in Multiple Sclerosis: A Product of their Environment

    PubMed Central

    Nair, Aji; Frederick, Terra J.; Miller, Stephen D.

    2010-01-01

    It has long been thought that astrocytes, like other glial cells, simply provide a support mechanism for neuronal function in the healthy and inflamed central nervous system (CNS). However, recent evidence suggests that astrocytes play an active and dual role in CNS inflammatory diseases such as multiple sclerosis (MS). Astrocytes not only have the ability to enhance immune responses and inhibit myelin repair, but they can also be protective and limit CNS inflammation while supporting oligodendrocyte and axonal regeneration. The particular impact of these cells on the pathogenesis and repair of an inflammatory demyelinating process is dependent upon a number of factors, including the stage of the disease, the type and microenvironment of the lesion, and the interactions with other cell types and factors that influence their activation. In this review, we summarize recent data supporting the idea that astrocytes play a complex role in the regulation of CNS autoimmunity. PMID:18516496

  13. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

    SciTech Connect

    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang; Ko, Kinarm; Koh, Yong-Gon

    2014-08-08

    Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

  14. Pioglitazone enhances small-sized adipocyte proliferation in subcutaneous adipose tissue.

    PubMed

    Kajita, Kazuo; Mori, Ichiro; Hanamoto, Takayuki; Ikeda, Takahide; Fujioka, Kei; Yamauchi, Masahiro; Okada, Hideyuki; Usui, Taro; Takahashi, Noriko; Kitada, Yoshihiko; Taguchi, Kohichiro; Kajita, Toshiko; Uno, Yoshihiro; Morita, Hiroyuki; Ishizuka, Tatsuo

    2012-01-01

    The possibility that mature adipocytes proliferate has not been fully investigated. In this study, we demonstrate that adipocytes can proliferate. 5-bromo-2'-deoxyuridine (BrdU)-labeled adipocyte like cells, most of which were less than 30 μm in diameter, were observed in adipose tissue. Proliferating cell nuclear antigen (PCNA) was simultaneously detected in BrdU-labeled nuclei. Observation of individual mature adipocytes of smeared specimens on glass slides revealed that small sized adipocytes more frequently incorporated BrdU. Cultured mature adipocytes using the ceiling-cultured method showed clustering of proliferating cells in small-sized adipocytes. These small cultured adipocytes, but not large ones, extensively incorporated BrdU. Quantified analysis of BrdU incorporation demonstrated that mature visceral adipocytes, including epididymal, mesenteric and perirenal adipocytes, proliferated more actively than subcutaneous ones. On the other hand, treatment with pioglitazone (Pio), a ligand of peroxisome proliferator-activated receptor γ, containing food for 2w, elevated BrdU incorporation and expression of PCNA in mature adipocytes isolated from subcutaneous, but not visceral adipose tissue. Moreover, Pio induced increased BrdU-labeled small-sized subcutaneous adipocytes, which was associated with an increased number of total small adipocytes in subcutaneous adipose tissue. In conclusion, mature adipocytes have a subgroup representing the potential to replicate, and this proliferation is more active in visceral adipocytes. Treatment with Pio increases proliferation in subcutaneous adipocytes. These results may explain the mechanism of Pio-induced hyperplasia especially in subcutaneous adipocytes.

  15. Enhanced osteoblast proliferation and corrosion resistance of commercially pure titanium through surface nanostructuring by ultrasonic shot peening and stress relieving.

    PubMed

    Jindal, Shitu; Bansal, Rajesh; Singh, Bijay P; Pandey, Rajiv; Narayanan, Shankar; Wani, Mohan R; Singh, Vakil

    2014-07-01

    This investigation was carried out to study the effect of a novel process of surface modification, surface nanostructuring by ultrasonic shot peening, on osteoblast proliferation and corrosion behavior of commercially pure titanium (c p-Ti) in simulated body fluid. A mechanically polished disc of c p-Ti was subjected to ultrasonic shot peening with stainless steel balls to create nanostructure at the surface. A nanostructure (<20 nm) with inhomogeneous distribution was revealed by atomic force and scanning electron microscopy. There was an increase of approximately 10% in cell proliferation, but there was drastic fall in corrosion resistance. Corrosion rate was increased by 327% in the shot peened condition. In order to examine the role of residual stresses associated with the shot peened surface on these aspects, a part of the shot peened specimen was annealed at 400°C for 1 hour. A marked influence of annealing treatment was observed on surface structure, cell proliferation, and corrosion resistance. Surface nanostructure was much more prominent, with increased number density and sharper grain boundaries; cell proliferation was enhanced to approximately 50% and corrosion rate was reduced by 86.2% and 41% as compared with that of the shot peened and the as received conditions, respectively. The highly significant improvement in cell proliferation, resulting from annealing of the shot peened specimen, was attributed to increased volume fraction of stabilized nanostructure, stress recovery, and crystallization of the oxide film. Increase in corrosion resistance from annealing of shot peened material was related to more effective passivation. Thus, the surface of c p-Ti, modified by this novel process, possessed a unique quality of enhancing cell proliferation as well as the corrosion resistance and could be highly effective in reducing treatment time of patients adopting dental and orthopedic implants of titanium and its alloys.

  16. Don't fence me in: Harnessing the beneficial roles of astrocytes for spinal cord repair

    PubMed Central

    White, Robin E.; Jakeman, Lyn B.

    2008-01-01

    Astrocytes comprise a heterogeneous cell population that plays a complex role in repair after spinal cord injury. Reactive astrocytes are major contributors to the glial scar that is a physical and chemical barrier to axonal regeneration. Yet, consistent with a supportive role in development, astrocytes secrete neurotrophic factors and protect neurons and glia spared by the injury. In development and after injury, local cues are modulators of astrocyte phenotype and function. When multipotent cells are transplanted into the injured spinal cord, they differentiate into astrocytes and other glial cells as opposed to neurons, which is commonly viewed as a challenge to be overcome in developing stem cell technology. However, several examples show that astrocytes provide support and guidance for axonal growth and aid in improving functional recovery after spinal cord injury. Notably, transplantation of astrocytes of a developmentally immature phenotype promotes tissue sparing and axonal regeneration. Furthermore, interventions that enhance endogenous astrocyte migration or reinvasion of the injury site result in greater axonal growth. These studies demonstrate that astrocytes are dynamic, diverse cells that have the capacity to promote axon growth after injury. The ability of astrocytes to be supportive of recovery should be exploited in devising regenerative strategies. PMID:18820411

  17. Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury.

    PubMed

    Labombarda, Florencia; González, Susana; Lima, Analia; Roig, Paulina; Guennoun, Rachida; Schumacher, Michael; De Nicola, Alejandro F

    2011-09-01

    Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.

  18. The Role of Astrocytes in Multiple Sclerosis Progression

    PubMed Central

    Correale, Jorge; Farez, Mauricio F.

    2015-01-01

    Multiple sclerosis (MS) is an inflammatory disorder causing central nervous system (CNS) demyelination and axonal injury. Although its etiology remains elusive, several lines of evidence support the concept that autoimmunity plays a major role in disease pathogenesis. The course of MS is highly variable; nevertheless, the majority of patients initially present a relapsing–remitting clinical course. After 10–15 years of disease, this pattern becomes progressive in up to 50% of untreated patients, during which time clinical symptoms slowly cause constant deterioration over a period of many years. In about 15% of MS patients, however, disease progression is relentless from disease onset. Published evidence supports the concept that progressive MS reflects a poorly understood mechanism of insidious axonal degeneration and neuronal loss. Recently, the type of microglial cell and of astrocyte activation and proliferation observed has suggested contribution of resident CNS cells may play a critical role in disease progression. Astrocytes could contribute to this process through several mechanisms: (a) as part of the innate immune system, (b) as a source of cytotoxic factors, (c) inhibiting remyelination and axonal regeneration by forming a glial scar, and (d) contributing to axonal mitochondrial dysfunction. Furthermore, regulatory mechanisms mediated by astrocytes can be affected by aging. Notably, astrocytes might also limit the detrimental effects of pro-inflammatory factors, while providing support and protection for oligodendrocytes and neurons. Because of the dichotomy observed in astrocytic effects, the design of therapeutic strategies targeting astrocytes becomes a challenging endeavor. Better knowledge of molecular and functional properties of astrocytes, therefore, should promote understanding of their specific role in MS pathophysiology, and consequently lead to development of novel and more successful therapeutic approaches. PMID:26347709

  19. 1,3-Dinitrobenzene neurotoxicity - Passage effect in immortalized astrocytes.

    PubMed

    Maurer, Laura L; Latham, Jackelyn D; Landis, Rory W; Song, Dong Hoon; Epstein, Tamir; Philbert, Martin A

    2016-03-01

    Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2-8) and high passage DI-TNC1 astrocytes (HP; #17-28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K(+)-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K(+)-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K(+) in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9-16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined. PMID:26769196

  20. A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters*

    PubMed Central

    Shibasaki, Koji; Ikenaka, Kazuhiro; Tamalu, Fuminobu; Tominaga, Makoto; Ishizaki, Yasuki

    2014-01-01

    Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca2+ transients in astrocytes, and these Ca2+ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4+ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4+ astrocytes. After activation, both TRPV4+ and TRPV4− astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4+ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4+ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands. PMID:24737318

  1. Astrocyte-mediated ischemic tolerance.

    PubMed

    Hirayama, Yuri; Ikeda-Matsuo, Yuri; Notomi, Shoji; Enaida, Hiroshi; Kinouchi, Hiroyuki; Koizumi, Schuichi

    2015-03-01

    Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential. PMID:25740510

  2. In vitro synchronization of mammalian astrocytic cultures by serum deprivation.

    PubMed

    Chou, Richard C; Langan, Thomas J

    2003-07-01

    The study of the regulation of cell division cycle in vitro requires cell cultures growing in the same phase of the cycle. The procedure by which cells are arrested in specific phases of the cell cycle is termed synchronization. Synchronization is particularly important in the study of astrocyte biology, as its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G(0)), and, under carefully defined experimental conditions, move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, including centrifugal elutriation, mitotic shake-off, and chemically induced cell cycle arrest. Yet there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocytic cultures from newborn rat brains by serum deprivation. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum down-shift and arrested in the G(0) phase of the cell cycle. This procedure has recently been extended toward the study of cell cycle control in astroglioma cells and astrocytes from injured adult brains. Since it was also employed in recent precursor cloning studies in developmental biology, this procedure will certainly find increasing use in future research.

  3. Astrocytes and NG2-glia: what's in a name?

    PubMed Central

    Nishiyama, Akiko; Yang, Zhongshu; Butt, Arthur

    2005-01-01

    Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2-glia). These NG2-glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. It is well established that NG2-glia give rise to oligodendrocytes. However, the majority of NG2-glia in the adult CNS proliferate very slowly and are non-motile. Both astrocytes and NG2-glia display a stellate morphology and express ion channels and receptors to neurotransmitters used by neurons. Both types of glia make intimate contacts with neurons in grey and white matter, and their functional differences and similarities are only beginning to be unravelled. Recent observations emphasize the need to examine the relationship between astrocytes and NG2-glia, and address the question of whether they represent overlapping or two distinct glial cell populations. To be of any relevance, this classification must relate to specific functions in the neural network. At present, the balance of evidence is that NG2-glia and astrocytes are functionally segregated populations. PMID:16367796

  4. Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

    PubMed Central

    Silva, Rafael Rodrigues; Mariante, Rafael M.; Silva, Andrea Alice; dos Santos, Ana Luiza Barbosa; Roffê, Ester; Santiago, Helton; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. PMID:25695249

  5. Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

    PubMed

    Silva, Rafael Rodrigues; Mariante, Rafael M; Silva, Andrea Alice; dos Santos, Ana Luiza Barbosa; Roffê, Ester; Santiago, Helton; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. PMID:25695249

  6. Propiconazole Enhances Cell Proliferation by Dysregulation of Ras Farnesylation and theMAPK pathway

    EPA Science Inventory

    Previous studies of mice exposed to the hepatotumorigenic fungicide, propiconazole, revealed an increase in hepatic cell proliferation and over-expression of hepatic genes within the cholesterol biosynthesis pathway. Mevalonate, an intermediate in this pathway, has long been a ta...

  7. Astrocytes: Key Regulators of Neuroinflammation.

    PubMed

    Colombo, Emanuela; Farina, Cinthia

    2016-09-01

    Astrocytes are crucial regulators of innate and adaptive immune responses in the injured central nervous system. Depending on timing and context, astrocyte activity may exacerbate inflammatory reactions and tissue damage, or promote immunosuppression and tissue repair. Recent literature has unveiled key factors and intracellular signaling pathways that govern astrocyte behavior during neuroinflammation. Here we have re-visited in vivo studies on astrocyte signaling in neuroinflammatory models focusing on evidences obtained from the analysis of transgenic mice where distinct genes involved in ligand binding, transcriptional regulation and cell communication have been manipulated in astrocytes. The integration of in vivo observations with in vitro data clarifies precise signaling steps, highlights the crosstalk among pathways and identifies shared effector mechanisms in neuroinflammation.

  8. Plutonium partitioning in uranium and plutonium co-recovery system for fast reactor fuel recycling with enhanced nuclear proliferation resistance

    SciTech Connect

    Nakahara, Masaumi; Koma, Yoshikazu; Nakajima, Yasuo

    2013-07-01

    For enhancement of nuclear proliferation resistance, a 'co-processing' method for U and Pu co-recovery was studied. Two concepts, no U scrubbing and no Pu reduction partitioning, were employed to formulate two types of flow sheets by using a calculation code. Their process performance was demonstrated using radioactive solutions derived from an irradiated fast reactor fuel. These experimental results indicated that U and Pu were co-recovered in the U/Pu product, and the Pu content in the U/Pu product increased approximately 2.3 times regardless of using reductant. The proposed no U scrubbing and no Pu reductant flow sheet is applicable to fast reactor fuel reprocessing and enhances its resistance to nuclear proliferation. (authors)

  9. Effects of arsenite in astrocytes on neuronal signaling transduction.

    PubMed

    Wang, Yan; Zhao, Fenghong; Liao, Yingjun; Jin, Yaping; Sun, Guifan

    2013-01-01

    The main purpose of this study was to test the hypothesis that arsenite induces neurotoxicity via effects on astrocytes. Astrocytes were exposed to 0, 5 or 10 μM arsenite in medium for 24 h, and then astrocyte-conditioned medium (ACM) was collected after incubation with fresh medium for 6 h. Primary neuron cultures were divided into four groups due to ACM, which were neurons without ACM exposure (group I) and neurons exposed to ACM from 0, 5 or 10 μM arsenite treated astrocytes (group II-IV). Protein expression of N-methyl-d-aspartate receptors (NR1, NR2A, NR2B), calmodulin-dependent protein kinase II (CaMKII) and adenylate cyclase (AC) in neurons were measured after incubation with ACM for 4, 8 or 12 h. Morphological changes and synaptic formation were observed after a 72 h-incubation with ACM. Compared to group II, synaptic formation and protein expression of NR2A, NR2B, CaMKII and AC in group III and IV were significantly suppressed. Moreover, synaptic formation and protein expression of CaMKII and AC in group II were significantly enhanced when compared with group I. Taken together, findings from this study suggested that arsenic in astrocytes might impair synaptic formation through disturbing astrocytic effects on neuronal signal transduction.

  10. Two-pore Domain Potassium Channels in Astrocytes

    PubMed Central

    Ryoo, Kanghyun

    2016-01-01

    Two-pore domain potassium (K2P) channels have a distinct structure and channel properties, and are involved in a background K+ current. The 15 members of the K2P channels are identified and classified into six subfamilies on the basis of their sequence similarities. The activity of the channels is dynamically regulated by various physical, chemical, and biological effectors. The channels are expressed in a wide variety of tissues in mammals in an isoform specific manner, and play various roles in many physiological and pathophysiological conditions. To function as channels, the K2P channels form dimers, and some isoforms form heterodimers that provide diversity in channel properties. In the brain, TWIK1, TREK1, TREK2, TRAAK, TASK1, and TASK3 are predominantly expressed in various regions, including the cerebral cortex, dentate gyrus, CA1-CA3, and granular layer of the cerebellum. TWIK1, TREK1, and TASK1 are highly expressed in astrocytes, where they play specific cellular roles. Astrocytes keep leak K+ conductance, called the passive conductance, which mainly involves TWIK1-TREK1 heterodimeric channel. TWIK1 and TREK1 also mediate glutamate release from astrocytes in an exocytosis-independent manner. The expression of TREK1 and TREK2 in astrocytes increases under ischemic conditions, that enhance neuroprotection from ischemia. Accumulated evidence has indicated that astrocytes, together with neurons, are involved in brain function, with the K2P channels playing critical role in these astrocytes. PMID:27790056

  11. Astrocytes, Synapses and Brain Function: A Computational Approach

    NASA Astrophysics Data System (ADS)

    Nadkarni, Suhita

    2006-03-01

    Modulation of synaptic reliability is one of the leading mechanisms involved in long- term potentiation (LTP) and long-term depression (LTD) and therefore has implications in information processing in the brain. A recently discovered mechanism for modulating synaptic reliability critically involves recruitments of astrocytes - star- shaped cells that outnumber the neurons in most parts of the central nervous system. Astrocytes until recently were thought to be subordinate cells merely participating in supporting neuronal functions. New evidence, however, made available by advances in imaging technology has changed the way we envision the role of these cells in synaptic transmission and as modulator of neuronal excitability. We put forward a novel mathematical framework based on the biophysics of the bidirectional neuron-astrocyte interactions that quantitatively accounts for two distinct experimental manifestation of recruitment of astrocytes in synaptic transmission: a) transformation of a low fidelity synapse transforms into a high fidelity synapse and b) enhanced postsynaptic spontaneous currents when astrocytes are activated. Such a framework is not only useful for modeling neuronal dynamics in a realistic environment but also provides a conceptual basis for interpreting experiments. Based on this modeling framework, we explore the role of astrocytes for neuronal network behavior such as synchrony and correlations and compare with experimental data from cultured networks.

  12. Glucose deprivation increases hydrogen peroxide level in immunostimulated rat primary astrocytes.

    PubMed

    Choi, Ji Woong; Shin, Chan Young; Yoo, Byoung Kwon; Choi, Min Sik; Lee, Woo Jong; Han, Byoung Hee; Kim, Won-Ki; Kim, Hyoung-Chun; Ko, Kwang Ho

    2004-03-01

    Activated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H(2)O(2) production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H(2)O(2) in astrocytes. Treatment with lipopolysaccharide plus interferon-gamma for 48 hr evoked astroglial H(2)O(2) production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H(2)O(2) level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H(2)O(2) in glucose-deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H(2)O(2) level and LDH release in glucose-deprived immunostimulated astrocytes. H(2)O(2), either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose-deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H(2)O(2).

  13. Astrocyte fatty acid binding protein-7 is a marker for neurogenic niches in the rat hippocampus.

    PubMed

    Young, John K; Heinbockel, Thomas; Gondré-Lewis, Marjorie C

    2013-12-01

    Recent research has determined that newborn neurons in the dentate gyrus of the hippocampus of the macaque are frequently adjacent to astrocytes immunoreactive for fatty acid binding protein-7 (FABP7). To investigate if a similar relationship between FABP7-positive (FABP7+) astrocytes and proliferating cells exists in the rodent brain, sections of brains from juvenile rats were stained by immunohistochemistry to demonstrate newborn cells (antibody to Ki67 protein) and FABP7+ astrocytes. In rat brains, FABP7+ astrocytes were particularly abundant in the dentate gyrus of the hippocampus and were frequently close to dividing cells immunoreactive for Ki67 protein. FABP7+ astrocytes were also present in the olfactory bulbs, arcuate nucleus of the hypothalamus, and in the dorsal medulla subjacent to the area postrema, sites where more modest numbers of newborn neurons can also be found. These data suggest that regional accumulations of FABP7+ astrocytes may represent reservoirs of cells having the potential for neurogenesis. Because FABP7+ astrocytes are particularly abundant in the hippocampus, and since the gene for FABP7 has been linked to Alzheimer's disease, age-related changes in FABP7+ astrocytes (mitochondrial degeneration) may be relevant to age-associated disorders of the hippocampus.

  14. Stimulation of Mesothelial Cell Proliferation by Exudate Macrophages Enhances Serosal Wound Healing in a Murine Model

    PubMed Central

    Mutsaers, Steven E.; Whitaker, Darrel; Papadimitriou, John M.

    2002-01-01

    Examination of thermally induced serosal lesions in mice displayed collections of inflammatory cells, predominantly macrophages, on and surrounding the wound within 48 hours of injury. Furthermore, by 2 days a large number of uninjured mesothelial cells adjacent to the wound were synthesizing DNA. From these findings, it was hypothesized that macrophages play a major role in serosal repair by stimulating mesothelial cell proliferation. Again, using a murine model of mesothelial regeneration, depletion of circulating monocytes significantly delayed serosal healing whereas addition of peritoneal exudate cells to the wound site 36 hours before injury increased the healing rate. In vivo assessment of mesothelial cell proliferation using tritiated thymidine incorporation and autoradiography demonstrated that peritoneal exudate cells stimulated mesothelial cell proliferation (12.44 ± 1.63% labeling index, compared with controls in which medium only was used 4.48 ± 0.71%). The mesothelial proliferation was predominantly because of macrophage-secreted products with molecular weights of 36 to 53 kd or 67 to 100 kd. These data support the hypothesis that macrophages play an important role in serosal healing by stimulating mesothelial cell proliferation. PMID:11839589

  15. [Novel function of astrocytes revealed by optogenetics].

    PubMed

    Beppu, Kaoru; Matsui, Ko

    2014-12-01

    Astrocytes respond to neuronal activity. However, whether astrocytic activity has any significance in brain function is unknown. Signaling pathway leading from astrocytes to neurons would be required for astrocytes to participate in neuronal functions and, here, we investigated the presence of such pathway. Optogenetics was used to manipulate astrocytic activity. A light-sensitive protein, channelrhodopsin-2 (ChR2), was selectively expressed in astrocytes. Photostimulation of these astrocytes induced glutamate release which modulated neuronal activity and animal behavior. Such glutamate release was triggered by intracellular acidification produced by ChR2 photoactivation. Astrocytic acidification occurs upon brain ischemia, and we found that another optogenetic tool, archaerhodopsin (ArchT), could counter the acidification and suppress astrocytic glutamate release. Controlling of astrocytic pH may become a therapeutic strategy upon ischemia.

  16. Endogenous Cannabinoid Signaling Is Required for Voluntary Exercise-induced Enhancement of Progenitor Cell Proliferation in the Hippocampus

    PubMed Central

    Hill, Matthew N.; Titterness, Andrea K.; Morrish, Anna C.; Carrier, Erica J.; Lee, Tiffany T.-Y.; Gil-Mohapel, Joana; Gorzalka, Boris B.; Hillard, Cecilia J.; Christie, Brian R.

    2009-01-01

    Voluntary exercise and endogenous cannabinoid activity have independently been shown to regulate hippocampal plasticity. The aim of the current study was to determine whether the endocannabinoid system is regulated by voluntary exercise and if these changes contribute to exercise-induced enhancement of cell proliferation. In Experiment 1, eight days of free access to a running wheel increased the agonist binding site density of the cannabinoid CB1 receptor; CB1 receptor-mediated GTPγS binding; and the tissue content of the endocannabinoid anandamide in the hippocampus but not in the prefrontal cortex. In Experiment 2, the CB1 receptor antagonist AM251 (1 mg/kg) was administered daily to animals given free access to a running wheel for 8 days, after which cell proliferation in the hippocampus was examined through immunohistochemical analysis of the cell cycle protein Ki-67. Voluntary exercise increased proliferation of progenitor cells, as evidenced by the increase in the number of Ki-67 positive cells in the granule cell layer of the dentate gyrus in the hippocampus. However, this effect was abrogated by concurrent treatment with AM251, indicating that the increase in endocannabinoid signaling in the hippocampus is required for the exercise-induced increase in cell proliferation. These data demonstrate that the endocannabinoid system in the hippocampus is sensitive to environmental change and suggest that it is a mediator of experience-induced plasticity. PMID:19489006

  17. Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

    PubMed

    Miyahara, Daichi; Oishi, Isao; Makino, Ryuichi; Kurumisawa, Nozomi; Nakaya, Ryuma; Ono, Tamao; Kagami, Hiroshi; Tagami, Takahiro

    2016-04-22

    An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2. PMID:26727404

  18. Platelet-derived growth factors enhance proliferation of human stromal stem cells.

    PubMed

    Lucarelli, Enrico; Beccheroni, Amira; Donati, Davide; Sangiorgi, Luca; Cenacchi, Annarita; Del Vento, Anna M; Meotti, Carolina; Bertoja, Annarosa Zambon; Giardino, Roberto; Fornasari, Pier M; Mercuri, Mario; Picci, Piero

    2003-08-01

    Studies on new procedures for bone reconstruction suggest that autologous cells seeded on a resorbable scaffold can improve the treatment of bone defects. It is important to develop culture conditions for ex vivo expansion of stromal stem cells (SSC) that do not compromise their self-renewing and differentiation capability. Bone marrow SSC and platelet gel (PG) obtained by platelet-rich plasma provide an invaluable source for autologous progenitor cells and growth factors for bone reconstruction. In this study the effect of platelet-rich plasma (PRP) released by PG on SSC proliferation and differentiation was investigated. MTT assay was used to investigate the effect of PRP on proliferation: results showed that PRP induced SSC proliferation. The effect was dose dependent and 10% PRP is sufficient to induce a marked cell proliferation. Untreated cells served as controls. Upon treatment with 10% PRP, cells entered logarithmic growth. Removal of PRP restored the characteristic proliferation rate. Because SSC can gradually lose their capability to differentiate along the chondrogenic and osteogenic lineage during subculture in vitro, we tested whether 10% PRP treatment affected SSC ability to mineralize. SSC were first exposed to 10% PRP for five passages, at passage 6 PRP was washed away and plated cells were treated with dexamethasone (DEX). DEX induced a three-fold increase in the number of alkaline phosphatase positive cells and induced mineralization that is consistent with the differentiation of osteochondroprogenitor cells. In conclusion, 10% PRP promotes SSC proliferation; cells expanded with 10% PRP can mineralize the extracellular matrix once PRP is withdrawn.

  19. [Knock-down of apollon gene by antisense oligodeoxynucleotide inhibits the proliferation of Lovo cells and enhances chemo-sensitivity].

    PubMed

    He, Jin-hua; Zhang, Xiao-ying; Wu, Feng-yun; Liao, Xiao-li; Wang, Wei; Jiang, Jian-wei

    2011-02-01

    In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P < 0.05). After Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon

  20. Theophylline potentiates lipopolysaccharide-induced NO production in cultured astrocytes.

    PubMed

    Ogawa, Mizue; Takano, Katsura; Kawabe, Kenji; Moriyama, Mitsuaki; Ihara, Hideshi; Nakamura, Yoichi

    2014-01-01

    Elucidation of the functions of astrocytes is important for understanding of the pathogenic mechanism of various neurodegenerative diseases. Theophylline is a common drug for bronchial asthma and occasionally develops side-effects, such as acute encephalopathy; although the pathogenic mechanism of the side-effects is unknown. The lipopolysaccharide (LPS)-induced nitricoxide (NO) production is generally used for an index of the activation of astrocyte in vitro. In this study, in order to elucidate the effect of theophylline on the astrocytic functions, we examined the LPS-induced NO production and the expression of iNOS in cultured rat cortex astrocytes.Theophylline alone could not induce the NO production; however, NO production induced by LPS was enhanced by theophylline in a dose-dependent manner; and by isobutylmethylxanthine, a phosphodiesterase inhibitor. The theophylline enhancement of LPS-induced NO production was further increased by dibutyryl cyclic AMP, a membrane-permeable cAMP analog; and by forskolin, an adenylate cyclase activator. When the cells were preincubated with Rp-8-Br-cAMP, an inhibitor of protein kinase A, the theophylline enhancement of LPS-induced NO production was decreased. The extent of iNOS protein expression induced by LPS was also enhanced by theophylline.It is likely that phosphodiesterase inhibition is a major action mechanism for the theophylline enhancement of LPS-induced NO production in astrocytes. Theophylline-induced acute encephalopathy might be due to the hyper-activation of astrocytes via cAMP signaling to produce excess amount of NO.

  1. Extremely low-frequency electromagnetic fields enhance the proliferation and differentiation of neural progenitor cells cultured from ischemic brains.

    PubMed

    Cheng, Yannan; Dai, Yiqin; Zhu, Ximin; Xu, Haochen; Cai, Ping; Xia, Ruohong; Mao, Lizhen; Zhao, Bing-Qiao; Fan, Wenying

    2015-10-21

    In the mammalian brain, neurogenesis persists throughout the embryonic period and adulthood in the subventricular zone of the lateral ventricle and the granular zone (dentate gyrus) of the hippocampus. Newborn neural progenitor cells (NPCs) in the two regions play a critical role in structural and functional plasticity and neural regeneration after brain injury. Previous studies have reported that extremely low-frequency electromagnetic fields (ELF-EMF) could promote osteogenesis, angiogenesis, and cardiac stem cells' differentiation, which indicates that ELF-EMF might be an effective tool for regenerative therapy. The present studies were carried out to examine the effects of ELF-EMF on hippocampal NPCs cultured from embryonic and adult ischemic brains. We found that exposure to ELF-EMF (50 Hz, 0.4 mT) significantly enhanced the proliferation capability both in embryonic NPCs and in ischemic NPCs. Neuronal differentiation was also enhanced after 7 days of cumulative ELF-EMF exposure, whereas glial differentiation was not influenced markedly. The expression of phosphorylated Akt increased during the proliferation process when ischemic NPCs were exposed to ELF-EMF. However, blockage of the Akt pathway abolished the ELF-EMF-induced proliferation of ischemic NPCs. These data show that ELF-EMF promotes neurogenesis of ischemic NPCs and suggest that this effect may occur through the Akt pathway.Video abstract, Supplemental Digital Content 1, http://links.lww.com/WNR/A347.

  2. [Lily polysaccharide 1 enhances the effect of metformin on proliferation and apoptosis of human breast carcinoma cells].

    PubMed

    Hou, Jin; Li, Fen; Li, Xinhua; Mei, Qibing; Mi, Man

    2016-06-01

    Objective To investigate the effect of metformin, alone or in combination with Lily polysaccharide 1 (LP1), on cell viability and apoptosis in MCF-7 human breast cancer cells. Methods LP1 (0.5, 1.0 mg/mL) and metformin (5, 10, 20, 50 mmol/L) were added into MCF-7 cell culture medium, followed by incubating for 72 hours in carbon dioxide incubators at 37DegreesCelsius. MCF-7 cell proliferation was determined using MTT assay; the apoptosis and cell cycle of MCF-7 cells were examined using annexin V-FITC/PI double staining combined with flow cytometry; Western blotting was used to determine the content of Bcl-2, Bax, adenosine monophosphate-activated protein kinase (AMPK) and phosphorated AMPK (p-AMPK) proteins. Results Metformin-induced inhibition of MCF-7 cell proliferation was significantly enhanced when 1 mg/mL LP1 was added in. Compared with the control group and the metformin only group, more cells were arrested to G1 and the apoptosis rate was raised obviously in the metformin and LP1 combination group. LP1 promoted the downregulated expression of Bcl-2 and the upregulated expression of Bax induced by metformin, but it didn't show any impact on the metformin-activated AMPK pathway. Conclusion LP1 enhances the proliferation-inhibitory and apoptosis-promoting effect of metformin on human breast carcinoma cells. The mechanism may be related with Bcl-2 downregulation and Bax upregulation. PMID:27371846

  3. Encapsulation of basic fibroblast growth factor by polyelectrolyte multilayer microcapsules and its controlled release for enhancing cell proliferation.

    PubMed

    She, Zhen; Wang, Chunxia; Li, Jun; Sukhorukov, Gleb B; Antipina, Maria N

    2012-07-01

    Basic fibroblast growth factor (FGF2) is an important protein for cellular activity and highly vulnerable to environmental conditions. FGF2 protected by heparin and bovine serum albumin was loaded into the microcapsules by a coprecipitation-based layer-by-layer encapsulation method. Low cytotoxic and biodegradable polyelectrolytes dextran sulfate and poly-L-arginine were used for capsule shell assembly. The shell thickness-dependent encapsulation efficiency was measured by enzyme-linked immunosorbent assay. A maximum encapsulation efficiency of 42% could be achieved by microcapsules with a shell thickness of 14 layers. The effects of microcapsule concentration and shell thickness on cytotoxicity, FGF2 release kinetics, and L929 cell proliferation were evaluated in vitro. The advantage of using microcapsules as the carrier for FGF2 controlled release for enhancing L929 cell proliferation was analyzed. PMID:22657385

  4. Overexpression of intraislet ghrelin enhances β-cell proliferation after streptozotocin-induced β-cell injury in mice.

    PubMed

    Bando, Mika; Iwakura, Hiroshi; Ariyasu, Hiroyuki; Koyama, Hiroyuki; Hosoda, Kiminori; Adachi, Souichi; Nakao, Kazuwa; Kangawa, Kenji; Akamizu, Takashi

    2013-07-01

    Previously, we reported that exogenous administration of ghrelin ameliorates glucose metabolism in a neonate streptozotocin (STZ)-induced diabetic rat model through enhancement of β-cell proliferation. However, it was not clear whether the observed β-cell proliferation was a direct or indirect effect (e.g., via orexigenic or growth hormone-stimulated pathways) of ghrelin activity. Here, we aimed to investigate whether ghrelin directly impacts β-cell proliferation after STZ-induced injury in mice. Seven-week-old male rat insulin II promoter-ghrelin internal ribosomal sequence ghrelin O-acyltransferase transgenic (RIP-GG Tg) mice, which have elevated pancreatic ghrelin levels, but only minor changes in plasma ghrelin levels when fed a medium-chain triglyceride-rich diet, were treated with STZ. Then, serum insulin, pancreatic insulin mRNA expression, and islet histology were evaluated. We found that the serum insulin levels, but not blood glucose levels, of RIP-GG Tg mice were significantly ameliorated 14 days post-STZ treatment. Pancreatic insulin mRNA expression was significantly elevated in RIP-GG Tg mice, and β-cell numbers in islets were increased. Furthermore, the number of phospho-histone H3⁺ or Ki67⁺ proliferating β-cells was significantly elevated in RIP-GG Tg mice, whereas the apoptotic indexes within the islets, as determined by TUNEL assay, were not changed. These results indicate that ghrelin can directly stimulate β-cell proliferation in vivo after β-cell injury even without its orexigenic or GH-stimulating activities, although it did not have enough impact to normalize the glucose tolerance in adult mice.

  5. Volume transmission signalling via astrocytes

    PubMed Central

    Hirase, Hajime; Iwai, Youichi; Takata, Norio; Shinohara, Yoshiaki; Mishima, Tsuneko

    2014-01-01

    The influence of astrocytes on synaptic function has been increasingly studied, owing to the discovery of both gliotransmission and morphological ensheathment of synapses. While astrocytes exhibit at best modest membrane potential fluctuations, activation of G-protein coupled receptors (GPCRs) leads to a prominent elevation of intracellular calcium which has been reported to correlate with gliotransmission. In this review, the possible role of astrocytic GPCR activation is discussed as a trigger to promote synaptic plasticity, by affecting synaptic receptors through gliotransmitters. Moreover, we suggest that volume transmission of neuromodulators could be a biological mechanism to activate astrocytic GPCRs and thereby to switch synaptic networks to the plastic mode during states of attention in cerebral cortical structures. PMID:25225097

  6. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

    PubMed Central

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, Mª Dolores; Vila, Jose Mª; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L.

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  7. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    PubMed

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, M Dolores; Vila, Jose M; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  8. Decreased functions of astrocytes on carbon nanofiber materials.

    PubMed

    McKenzie, Janice L; Waid, Michael C; Shi, Riyi; Webster, Thomas J

    2004-01-01

    Carbon nanofibers possess excellent conductivity properties, which may be beneficial in the design of more effective neural prostheses; however, limited evidence on their cytocompatibility properties currently exists. The objective of the present in vitro study was to determine cytocompatibility properties of formulations containing carbon nanofibers pertinent to neural implant applications. Substrates were prepared from four different types of carbon fibers, two with nanoscale diameters (nanophase, or less than or equal to 100 nm) and two with conventional diameters (or greater than 100 nm). Within these two categories, both a high and a low surface energy fiber were investigated and tested. Carbon fibers were compacted in a manual hydraulic press via a uniaxial loading cycle. Astrocytes (glial scar tissue-forming cells) were seeded onto the substrates for adhesion, proliferation, and long-term function studies (such as total intracellular protein and alkaline phosphatase activity). Results provided the first evidence that astrocytes preferentially adhered and proliferated on carbon fibers that had the largest diameter and the lowest surface energy. Based on these results, composite substrates were also formed using different weight percentages (0-25 wt%) of the nanophase, high surface energy fibers in a polycarbonate urethane matrix. Results provided the first evidence of decreased adhesion of astrocytes with increasing weight percents of the high surface energy carbon nanofibers in the polymer composite; this further demonstrates that formulations containing carbon fibers in the nanometer regime may limit astrocyte functions leading to decreased glial scar tissue formation. Positive interactions with neurons, and, at the same time, limited astrocyte functions leading to decreased gliotic scar tissue formation are essential for increased neuronal implant efficacy.

  9. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    SciTech Connect

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen; Lu, Yan; Shen, Pingping

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  10. Optimization of Heterogeneous Utilization of Thorium in PRWs to Enhance Proliferation Resistance & Reduce Waste

    SciTech Connect

    Mujid Kazimi

    2003-12-18

    Typical pressurized water reactors, although loaded with uranium fuel, produce 225 to 275 kg of plutonium per gigawatt year of operation. Although the spent fuel is highly radioactive, it nevertheless offers a potential proliferation pathway because the plutonium is relatively easy to separate, amounts to many critical masses, and aside from the alpha (n reaction on the 240 Pu isotope) does not present any significant intrinsic barrier to weapon assembly

  11. Active sulforhodamine 101 uptake into hippocampal astrocytes.

    PubMed

    Schnell, Christian; Hagos, Yohannes; Hülsmann, Swen

    2012-01-01

    Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes.

  12. Astrocyte reactivity to Fas activation is attenuated in TIMP-1 deficient mice, an in vitro study

    PubMed Central

    Ogier, Crystel; Creidy, Rita; Boucraut, José; Soloway, Paul D; Khrestchatisky, Michel; Rivera, Santiago

    2005-01-01

    Background Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional secreted protein with pleiotropic actions, including the inhibition of matrix metalloproteinases (MMPs), cell death/survival and growth promoting activities. After inflammatory challenge, the levels of TIMP-1 are highly and selectively upregulated in astrocytes among glial cells, but little is know about its role in these neural cells. We investigated the influence of TIMP-1 null mutation in the reactivity of cultured astrocytes to pro-inflammatory stimuli with TNF-α and anti-Fas antibody. Results When compared to WT, mutant astrocytes displayed an overall increased constitutive gelatinase expression and were less responsive to Fas-mediated upregulation of MMP-9, of monocyte chemoattractant protein-1 (MCP-1) and of intercellular cell adhesion molecule-1 (ICAM-1), all markers of astrocyte inflammatory response. In contrast, TNF-α treatment induced all these factors similarly regardless of the astrocyte genotype. The incorporation of 3H-thymidin, a marker of cell proliferation, increased in wild-type (WT) astrocytes after treatment with anti-Fas antibody or recombinant TIMP-1 but not in mutant astrocytes. Finally, lymphocyte chemotaxis was differentially regulated by TNF-α in WT and TIMP-1 deficient astrocytes. Conclusion We provide evidence that the alteration of the MMP/TIMP balance in astrocytes influences their reactivity to pro-inflammatory stimuli and that Fas activation modulates the expression of members of the MMP/TIMP axis. We hypothesise that the Fas/FasL transduction pathway and the MMP/TIMP system interact in astrocytes to modulate their inflammatory response to environmental stimuli. PMID:16316466

  13. CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

    SciTech Connect

    Kim, Ki Chan; Hyun Joo, So; Shin, Chan Young

    2011-06-17

    Highlights: {yields} Expression and phosphorylation of CPEB1 is increased by LPS stimulation in rat primary astrocytes. {yields} JNK regulates expression and phosphorylation of CPEB1 in reactive astrocytes. {yields} Down-regulation of CPEB1 using siRNA inhibits oxidative stress and iNOS induction by LPS stimulation. {yields} CPEB1 may play an important role in regulating inflammatory responses in reactive astrocytes induced by LPS. -- Abstract: Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury, yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.

  14. Immature and Mature Megakaryocytes Enhance Osteoblast Proliferation and Inhibit Osteoclast Formation

    PubMed Central

    Ciovacco, Wendy A.; Cheng, Ying-Hua; Horowitz, Mark C.; Kacena, Melissa A.

    2011-01-01

    Recent data suggests that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC proliferation, that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs. PMID:20052670

  15. Uropathogenic E.coli (UPEC) Infection Induces Proliferation through Enhancer of Zeste Homologue 2 (EZH2)

    PubMed Central

    Penna, Frank; Samiei, Alaleh Najdi; Sidler, Martin; Jiang, Jia-Xin; Ibrahim, Fadi; Tolg, Cornelia; Delgado-Olguin, Paul; Rosenblum, Norman; Bägli, Darius J.

    2016-01-01

    Host-pathogen interactions can induce epigenetic changes in the host directly, as well as indirectly through secreted factors. Previously, uropathogenic Escherichia coli (UPEC) was shown to increase DNA methyltransferase activity and expression, which was associated with methylation-dependent alterations in the urothelial expression of CDKN2A. Here, we showed that paracrine factors from infected cells alter expression of another epigenetic writer, EZH2, coordinate with proliferation. Urothelial cells were inoculated with UPEC, UPEC derivatives, or vehicle (mock infection) at low moi, washed, then maintained in media with Gentamycin. Urothelial conditioned media (CM) and extracellular vesicles (EV) were isolated after the inoculations and used to treat naïve urothelial cells. EZH2 increased with UPEC infection, inoculation-induced CM, and inoculation-induced EV vs. parallel stimulation derived from mock-inoculated urothelial cells. We found that infection also increased proliferation at one day post-infection, which was blocked by the EZH2 inhibitor UNC1999. Inhibition of demethylation at H3K27me3 had the opposite effect and augmented proliferation. CONCLUSION: Uropathogen-induced paracrine factors act epigenetically by altering expression of EZH2, which plays a key role in early host cell proliferative responses to infection. PMID:26964089

  16. Lentivirus-mediated silencing of MPHOSPH8 inhibits MTC proliferation and enhances apoptosis

    PubMed Central

    LI, PEIYONG; YANG, WEIPING; SHEN, BAIYONG; LI, HONGWEI; YAN, JIQI

    2016-01-01

    Thyroid carcinoma (TC) is the most common malignancy of the endocrine organs, and its incidence rate has steadily increased over the last decade. For medullary thyroid cancer (MTC), a type of TC, a high mortality rate has been reported. In previous studies, M-phase phosphoprotein 8 (MPHOSPH8) displayed an elevated expression in various human carcinoma cells. Thus, MPHOSPH8 may be a sensitive biomarker that could be used for the diagnosis and follow-up of MTC. In the present study, plasmids of RNA interference targeting the MPHOSPH8 gene were constructed. Once these lentiviruses targeting MPHOSPH8 were transfected into the MTC cell line TT, cell viability and proliferation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was used to assess the cell cycle distribution and apoptosis. The expression levels of MPHOSPH8 were detected by reverse transcription quantitative-polymerase chain reaction and western blot analyses. Depletion of MPHOSPH8 significantly inhibited cell proliferation. Furthermore, knockdown of MPHOSPH8 in TT cells led to G0/G1 phase cell cycle arrest and apoptosis. The results of the present study suggest that MPHOSPH8 promotes cell proliferation and may be a potential target for anticancer therapy of MTC. PMID:27313751

  17. Chemosensitizing effects of carbon-based nanomaterials in cancer cells: enhanced apoptosis and inhibition of proliferation as underlying mechanisms

    NASA Astrophysics Data System (ADS)

    Erdmann, Kati; Ringel, Jessica; Hampel, Silke; Rieger, Christiane; Huebner, Doreen; Wirth, Manfred P.; Fuessel, Susanne

    2014-10-01

    Recent studies have shown that carbon nanomaterials such as carbon nanofibres (CNFs) and multi-walled carbon nanotubes (CNTs) can exert antitumor activities themselves and sensitize cancer cells to conventional chemotherapeutics such as carboplatin and cisplatin. In the present study, the chemosensitizing effect of CNFs and CNTs on cancer cells of urological origin was investigated regarding the underlying mechanisms. Prostate cancer (DU-145, PC-3) and bladder cancer (EJ28) cells were treated with carbon nanomaterials (CNFs, CNTs) and chemotherapeutics (carboplatin, cisplatin) alone as well as in combination for 24 h. Forty-eight (EJ28) or 72 h (DU-145, PC-3) after the end of treatment the effects on cellular proliferation, clonogenic survival, cell death rate and cell cycle distribution were evaluated. Depending on the cell line, simultaneous administration of chemotherapeutics and carbon nanomaterials produced an additional inhibition of cellular proliferation and clonogenic survival of up to 77% and 98%, respectively, compared to the inhibitory effects of the chemotherapeutics alone. These strongly enhanced antiproliferative effects were accompanied by an elevated cell death rate, which was predominantly mediated via apoptosis and not by necrosis. The antitumor effects of combinations with CNTs were less pronounced than those with CNFs. The enhanced effects of the combinatory treatments on cellular function were mostly of additive to partly synergistic nature. Furthermore, cell cycle analysis demonstrated an arrest at the G2/M phase mediated by a monotreatment with chemotherapeutics. Following combinatory treatments, mostly less than or nearly additive increases of cell fractions in the G2/M phase could be observed. In conclusion, the pronounced chemosensitizing effects of CNFs and CNTs were mediated by an enhanced apoptosis and inhibition of proliferation. The combination of carbon-based nanomaterials and conventional chemotherapeutics represents a novel

  18. The reciprocal interactions between astrocytes and prostate cancer cells represent an early event associated with brain metastasis.

    PubMed

    de Oliveira Barros, Eliane Gouvêa; Palumbo, Antonio; Mello, Pedro Lucas Prado; de Mattos, Rômulo Medina; da Silva, Julianna Henriques; Pontes, Bruno; Viana, Nathan Bessa; do Amaral, Rackele Ferreira; Lima, Flavia Regina Souza; da Costa, Nathalia Meireles; Palmero, Celia Yelimar; Miranda-Alves, Leandro; Takiya, Christina Maeda; Nasciutti, Luiz Eurico

    2014-04-01

    Tumor establishment, growth, and survival are supported by interactions with microenvironment components. Here, we investigated whether the interactions between prostate cancer cells and cortical astrocytes are associated to a potential role for astrocytes in tumor establishment. We demonstrate that astrocytes interact in vitro with prostatic cancers cells derived from different metastatic sites. Astrocytes and their secreted extracellular matrix, stimulate DU145 cell (a brain-derived prostate tumor cell line) proliferation while inhibiting cell death and modulating the expression of several genes related to prostate cancer progression, suggesting the activation of EMT process in these cells. In contrast, DU145 cells and their conditioned medium inhibited cell proliferation and induced cell death of astrocytes. On the other hand, the astrocytes were unable to significantly induce an increment of LNCaP cell (a lymph node-derived prostate tumor cell line) proliferative activity. In addition, LNCaP cells were also unable to induce cell death of astrocytes. Thus, we believe that DU145 cells, but not LNCaP cells, present an even more aggressive behavior when interacting with astrocytes. These results provide an important contribution to the elucidation of the cellular mechanisms involved in the brain microenvironment colonization.

  19. The reciprocal interactions between astrocytes and prostate cancer cells represent an early event associated with brain metastasis.

    PubMed

    de Oliveira Barros, Eliane Gouvêa; Palumbo, Antonio; Mello, Pedro Lucas Prado; de Mattos, Rômulo Medina; da Silva, Julianna Henriques; Pontes, Bruno; Viana, Nathan Bessa; do Amaral, Rackele Ferreira; Lima, Flavia Regina Souza; da Costa, Nathalia Meireles; Palmero, Celia Yelimar; Miranda-Alves, Leandro; Takiya, Christina Maeda; Nasciutti, Luiz Eurico

    2014-04-01

    Tumor establishment, growth, and survival are supported by interactions with microenvironment components. Here, we investigated whether the interactions between prostate cancer cells and cortical astrocytes are associated to a potential role for astrocytes in tumor establishment. We demonstrate that astrocytes interact in vitro with prostatic cancers cells derived from different metastatic sites. Astrocytes and their secreted extracellular matrix, stimulate DU145 cell (a brain-derived prostate tumor cell line) proliferation while inhibiting cell death and modulating the expression of several genes related to prostate cancer progression, suggesting the activation of EMT process in these cells. In contrast, DU145 cells and their conditioned medium inhibited cell proliferation and induced cell death of astrocytes. On the other hand, the astrocytes were unable to significantly induce an increment of LNCaP cell (a lymph node-derived prostate tumor cell line) proliferative activity. In addition, LNCaP cells were also unable to induce cell death of astrocytes. Thus, we believe that DU145 cells, but not LNCaP cells, present an even more aggressive behavior when interacting with astrocytes. These results provide an important contribution to the elucidation of the cellular mechanisms involved in the brain microenvironment colonization. PMID:24488147

  20. Enhanced human osteoblast cell adhesion and proliferation on 316 LS stainless steel by means of CO2 laser surface treatment.

    PubMed

    Hao, L; Lawrence, J; Phua, Y F; Chian, K S; Lim, G C; Zheng, H Y

    2005-04-01

    An effective and novel technique for improving the biocompatibility of a biograde 316 LS stainless steel through the application of CO(2) laser treatment to modify the surface properties of the material is described herein. Different surface properties, such as surface roughness, surface oxygen content, and surface energy for CO(2) laser-treated 316 LS stainless steel, untreated, and mechanically roughened samples were analyzed, and their effects on the wettability characteristics of the material were studied. It was found that modification of the wettability characteristics of the 316 LS stainless steel following CO(2) laser treatment was achieved. This improvement was identified as being mainly due to the change in the polar component of the surface energy. One-day cell adhesion tests showed that cells not only adhered and spread better, but also grew faster on the CO(2) laser-treated sample than on either the untreated or mechanically roughened sample. Further, compared with the untreated sample, MTT cell proliferation analysis revealed that the mechanically roughed surface resulted in a slight enhancement, and CO(2) laser treatment brought about a significant increase in cell proliferation. An increase in the wettability of the 316 LS stainless steel was observed to positively correlate with the cell proliferation.

  1. Platelet-derived growth factor enhances proliferation and matrix synthesis of temporomandibular joint disc-derived cells.

    PubMed

    Hanaoka, Koichi; Tanaka, Eiji; Takata, Takashi; Miyauchi, Mutsumi; Aoyama, Junko; Kawai, Nobuhiko; Dalla-Bona, Diego A; Yamano, Eizo; Tanne, Kazuo

    2006-05-01

    Platelet-derived growth factor (PDGF) is an essential signaling molecule for wound healing and tissue repair. This study was aimed at evaluating the effect of PDGF on the proliferation of temporomandibular joint (TMJ) disc-derived cells and extracellular matrix synthesis. The number of cultured cells were counted by COULTER Z1. The assay for collagen synthesis was performed using a sircol soluble collagen assay. Hyaluronic acid (HA) synthesis was analyzed by a high performance liquid chromatography. The expression of collagens, matrix metalloproteinases (MMPs), and the tissue inhibitors of metalloproteinases (TIMPs) were examined using SYBR Green in terms of the RNA levels. PDGF treatment significantly (P < .01) increased the proliferation rate of the disc-derived cells as compared with the controls when the dose was 5 ng/ mL or greater. Treatment with more than 5 ng/mL PDGF resulted in an amount of collagen synthesis significantly (P < .01) higher than the controls. HA synthesis was maximal with 5 ng/mL PDGF treatment. Quantitative real-time polymerase chain reaction analyses showed that treatment with 5 ng/mL of PDGF-BB upregulated the mitochondrial RNA levels of type I and II collagens, MMPs, and TIMPs within 6 hours. It is concluded that PDGF, if its concentration is optimal, enhanced proliferation and matrix synthesis of TMJ disc-derived cells, indicating that PDGF may be effective for use in tissue engineering of the TMJ disc. PMID:16637732

  2. Foxc2 enhances proliferation and inhibits apoptosis through activating Akt/mTORC1 signaling pathway in mouse preadipocytes

    PubMed Central

    Gan, Lu; Liu, Zhenjiang; Jin, Wei; Zhou, Zhongjie; Sun, Chao

    2015-01-01

    Forkhead box C2 (Foxc2) protein is a transcription factor in regulation of development, metabolism, and immunology. However, the regulatory mechanisms of Foxc2 on proliferation and apoptosis of preadipocytes are unclear. In this study, we found that high-fat-diet-induced obesity elevated the expression of Foxc2 and cyclin E after 6 weeks. Additionally, Foxc2 suppressed preadipocyte differentiation, increased cell counts and augmented G1-S transition of preadipocytes, along with the elevation of cyclin E expression and the reduction levels of p27 and p53. Furthermore, Foxc2 knockdown reduced early apoptotic cells with accompanying reduction of mitochondrial membrane potential and increased fragmentation of genomic DNA. We show that Foxc2 reduces the expression of Bax, caspase-9, and caspase-3 in both serum-starved and palmitic acid-induced cell apoptotic models, which confirms the anti-apoptotic role of Foxc2. Moreover, the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)C1 signaling pathway and the ERK/mTORC1 signaling pathway were activated along with preadipocyte proliferation in response to Foxc2 overexpression, whereas apoptosis marker genes were downregulated during this process. Those effects were blocked by the interference of Foxc2 or signal pathways specific inhibitors. These data collectively reveal that Foxc2 enhances proliferation of preadipocytes and inhibits apoptosis of preadipocytes by activating the Akt/mTORC1 and ERK/mTORC1 signaling pathways. PMID:26113535

  3. Serotonin 1A Receptors on Astrocytes as a Potential Target for the Treatment of Parkinson’s Disease

    PubMed Central

    Miyazaki, Ikuko; Asanuma, Masato

    2016-01-01

    Astrocytes are the most abundant neuron-supporting glial cells in the central nervous system. The neuroprotective role of astrocytes has been demonstrated in various neurological disorders such as amyotrophic lateral sclerosis, spinal cord injury, stroke and Parkinson’s disease (PD). Astrocyte dysfunction or loss-of-astrocytes increases the susceptibility of neurons to cell death, while astrocyte transplantation in animal studies has therapeutic advantage. We reported recently that stimulation of serotonin 1A (5-HT1A) receptors on astrocytes promoted astrocyte proliferation and upregulated antioxidative molecules to act as a neuroprotectant in parkinsonian mice. PD is a progressive neurodegenerative disease with motor symptoms such as tremor, bradykinesia, rigidity and postural instability, that are based on selective loss of nigrostriatal dopaminergic neurons, and with non-motor symptoms such as orthostatic hypotension and constipation based on peripheral neurodegeneration. Although dopaminergic therapy for managing the motor disability associated with PD is being assessed at present, the main challenge remains the development of neuroprotective or disease-modifying treatments. Therefore, it is desirable to find treatments that can reduce the progression of dopaminergic cell death. In this article, we summarize first the neuroprotective properties of astrocytes targeting certain molecules related to PD. Next, we review neuroprotective effects induced by stimulation of 5-HT1A receptors on astrocytes. The review discusses new promising therapeutic strategies based on neuroprotection against oxidative stress and prevention of dopaminergic neurodegeneration. PMID:26795196

  4. Simultaneous neuron- and astrocyte-specific fluorescent marking.

    PubMed

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille; Hashimoto, Ryota; Baba, Akemichi; Hashimoto, Hitoshi

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.

  5. Galunisertib inhibits glioma vasculogenic mimicry formation induced by astrocytes.

    PubMed

    Zhang, Chao; Chen, Wenliang; Zhang, Xin; Huang, Bin; Chen, Aanjing; He, Ying; Wang, Jian; Li, Xingang

    2016-03-15

    Gliomas are among the most lethal primary brain tumors found in humans. In high-grade gliomas, vasculogenic mimicry is often detected and has been correlated with prognosis, thus suggesting its potential as a therapeutic target. Vasculogenic mimicry mainly forms vascular-like channels independent of endothelial cells; however, little is known about the relationship between astrocytes and vasculogenic mimicry. In our study, we demonstrated that the presence of astrocytes promoted vasculogenic mimicry. With suspension microarray technology and in vitro tube formation assays, we identified that astrocytes relied on TGF-β1 to enhance vasculogenic mimicry. We also found that vasculogenic mimicry was inhibited by galunisertib, a promising TGF-β1 inhibitor currently being studied in an ongoing trial in glioma patients. The inhibition was partially attributed to a decrease in autophagy after galunisertib treatment. Moreover, we observed a decrease in VE-cadherin and smooth muscle actin-α expression, as well as down-regulation of Akt and Flk phosphorylation in galunisertib-treated glioma cells. By comparing tumor weight and volume in a xenograft model, we acquired promising results to support our theory. This study expands our understanding of the role of astrocytes in gliomas and demonstrates that galunisertib inhibits glioma vasculogenic mimicry induced by astrocytes.

  6. OPTIMIZATION OF HETEROGENEOUS UTILIZATION OF THORIUM IN PWRS TO ENHANCE PROLIFERATION RESISTANCE AND REDUCE WASTE.

    SciTech Connect

    TODOSOW,M.; KAZIMI,M.

    2004-08-01

    Issues affecting the implementation, public perception and acceptance of nuclear power include: proliferation, radioactive waste, safety, and economics. The thorium cycle directly addresses the proliferation and waste issues, but optimization studies of core design and fuel management are needed to ensure that it fits within acceptable safety and economic margins. Typical pressurized water reactors, although loaded with uranium fuel, produce 225 to 275 kg of plutonium per gigawatt-year of operation. Although the spent fuel is highly radioactive, it nevertheless offers a potential proliferation pathway because the plutonium is relatively easy to separate, amounts to many critical masses, and does not present any significant intrinsic barrier to weapon assembly. Uranium 233, on the other hand, produced by the irradiation of thorium, although it too can be used in weapons, may be ''denatured'' by the addition of natural, depleted or low enriched uranium. Furthermore, it appears that the chemical behavior of thoria or thoria-urania fuel makes it a more stable medium for the geological disposal of the spent fuel. It is therefore particularly well suited for a once-through fuel cycle. The use of thorium as a fertile material in nuclear fuel has been of interest since the dawn of nuclear power technology due to its abundance and to potential neutronic advantages. Early projects include homogeneous mixtures of thorium and uranium oxides in the BORAX-IV, Indian Point I, and Elk River reactors, as well as heterogeneous mixtures in the Shippingport seed-blanket reactor. However these projects were developed under considerably different circumstances than those which prevail at present. The earlier applications preceded the current proscription, for non-proliferation purposes, of the use of uranium enriched to more than 20 w/o in {sup 235}U, and has in practice generally prohibited the use of uranium highly enriched in {sup 235}U. They were designed when the expected burnup of

  7. Mutant Disrupted-In-Schizophrenia 1 in astrocytes: focus on glutamate metabolism

    PubMed Central

    Abazyan, Sofya; Yang, Eun Ju; Abazyan, Bagrat; Xia, Meng; Yang, Chunxia; Rojas, Camilo; Slusher, Barbara; Sattler, Rita; Pletnikov, Mikhail

    2014-01-01

    Disrupted-In-Schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase (SR) to regulate production of D-serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, re-uptake or secretion of GLU remains unexplored. Thus, we studied the effects of dominant-negative mutant DISC1 on various aspects of GLU metabolism using primary astrocyte cultures and the hippocampal tissue from transgenic mice with astrocyte-restricted expression of mutant DISC1. While mutant DISC1 had no significant effects on astrocyte proliferation, GLU re-uptake, Glutaminase or Glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine-serine-cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus as well as elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of NMDA receptors in the hippocampus at postnatal day 21. Our findings indicate that decreased D-serine production by astrocytic mutant DISC1 may lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse. PMID:25131692

  8. Astrocyte reactivity after brain injury—: The role of galectins 1 and 3

    PubMed Central

    Sirko, Swetlana; Irmler, Martin; Gascón, Sergio; Bek, Sarah; Schneider, Sarah; Dimou, Leda; Obermann, Jara; De Souza Paiva, Daisylea; Poirier, Francoise; Beckers, Johannes; Hauck, Stefanie M.; Barde, Yves‐Alain

    2015-01-01

    Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins—Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere‐forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes. GLIA 2015;63:2340–2361 PMID:26250529

  9. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  10. MicroRNA-328 directly targets p21-activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing docetaxel sensitivity

    PubMed Central

    LIU, CHUNHUI; ZHANG, LEI; HUANG, YEQING; LU, KAI; TAO, TAO; CHEN, SHUQIU; ZHANG, XIAOWEN; GUAN, HAN; CHEN, MING; XU, BIN

    2015-01-01

    Prostate cancer (Pca) has one of the highest mortality rates for malignant cancers worldwide. Previous research has demonstrated that numerous genes are aberrantly expressed during Pca onset and development. p21-activated protein kinase 6 (PAK6) is known to be overexpressed in primary and metastatic Pca, however the mechanism of this aberrant expression remains unknown. In the present study, immunohistochemistry demonstrated that PAK6 is overexpressed in castration-resistant Pca (CRPC). Furthermore, PAK6 overexpression was regulated by microRNA (miR)-328. Luciferase reporter assay and western blot analysis indicated that PAK6 was directly targeted by miR-328. Forced expression of miR-328 enhanced docetaxel sensitivity, inhibited cell proliferation and promoted cell apoptosis without affecting the cell cycle. This indicates that miR-328 performs important functions in CRPC progression via PAK6 regulation. This mechanism may be used to enhance the effect of docetaxel. PMID:26459798

  11. Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I

    SciTech Connect

    Rosendahl, Ann H.; Gundewar, Chinmay; Said Hilmersson, Katarzyna; Ni, Lan; Saleem, Moin A.; Andersson, Roland

    2015-01-15

    Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer. - Highlights: • Generation of human conditionally immortalized human pancreatic stellate cell lines. • Temperature-sensitive SV40LT allows switch to primary PSC phenotype characteristics. • Proteome profiling revealed distinct expression patterns between TPSC and NPSC. • Enhanced IGF-I-stimulated proliferation and motility by TPSC compared to NPSC.

  12. Enhanced osteoblast-like cell adhesion and proliferation using sulfonate-bearing polymeric scaffolds.

    PubMed

    Chaterji, Somali; Gemeinhart, Richard A

    2007-12-15

    Orthopedic malfunction, degeneration, or damage remains a serious healthcare issue despite advances in medical technology. Proactive extracellular matrix (ECM)-mimetic scaffolds are being researched to orchestrate the activation of diverse osteogenic signaling cascades, facilitating osteointegration. We hypothesized that sulfonated functionalities incorporated into synthetic hydrogels would simulate anionic, sulfate-bearing proteoglycans, abundant in the ECM. Using this rationale, we successfully developed differentially sulfonated hydrogels, polymerizing a range of sulfopropyl acrylate potassium-acrylamide (SPAK-AM) mole ratios as monomer feeds under room temperature conditions. For anchorage-dependent cells, such as osteoblasts, adhesion is a critical prerequisite for subsequent osteointegration and cell specialization. The introduction of the sulfonated monomer, SPAK, resulted in favorable uptake of serum proteins with proportional increase in adhesion and proliferation rates of model cell lines, which included NIH/3T3 fibroblasts, MG-63 osteoblasts, and MC3T3-E1 subclone 4 preosteoblasts. In fact, higher proportions of sulfonate content (pSPAK75, pSPAK100) exhibited comparable or even higher degrees of adhesion and proliferation, relative to commercial grade tissue culture polystyrene in vitro. These results indicate promising potentials of sulfonated ECM-mimetic hydrogels as potential osteogenic tissue engineering scaffolds. PMID:17584889

  13. Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media▿

    PubMed Central

    Furukawa, Masayuki; Ebmeyer, Jörg; Pak, Kwang; Austin, Darrell A.; Melhus, Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2007-01-01

    Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation. PMID:17325051

  14. Enhancement of B-cell translocation gene-1 expression by prostaglandin E2 in macrophages and the relationship to proliferation.

    PubMed Central

    Suk, K; Sipes, D G; Erickson, K L

    1997-01-01

    Although prostaglandin (PG) E2 is known to suppress various macrophage functions, the molecular mechanisms by which that occurs are largely unknown. To understand better those mechanisms, differential screening of a cDNA library from PGE2-treated macrophages was performed. Subsequently, the DNA sequence of a differentially expressed cDNA clone was determined and the cDNA was identified as B-cell translocation gene-1 (BTG1), a recently cloned antiproliferative gene. A two-to threefold increase in macrophage BTG1 expression was observed after PGE2 treatment. PGE1 and platelet-activating factor, but not leukotrienes B4, and C4, or lipopolysaccharide, also enhanced BTG1 expression. Furthermore, this effect ws mimicked by dibutyryl cAMP which indicated the involvement of elevated cAMP in the PGE2-mediated enhancement of BTG1. Moreover, there was an inverse correlation between BTG1 mRNA expression and macrophage proliferation; however, BTG1 alteration was not associated with macrophage tumoricidal activation. Thus, BTG1 may play a role in PGE2-mediated inhibition of macrophage proliferation and not activation. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:9203975

  15. Fatty Acid Synthase as a Factor Required for Exercise-Induced Cognitive Enhancement and Dentate Gyrus Cellular Proliferation

    PubMed Central

    Chorna, Nataliya E.; Santos-Soto, Iván J.; Carballeira, Nestor M.; Morales, Joan L.; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P.; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis. PMID:24223732

  16. Fatty acid synthase as a factor required for exercise-induced cognitive enhancement and dentate gyrus cellular proliferation.

    PubMed

    Chorna, Nataliya E; Santos-Soto, Iván J; Carballeira, Nestor M; Morales, Joan L; de la Nuez, Janneliz; Cátala-Valentin, Alma; Chornyy, Anatoliy P; Vázquez-Montes, Adrinel; De Ortiz, Sandra Peña

    2013-01-01

    Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN), the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v.) microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis.

  17. Differential roles of astrocyte and microglia in supporting oligodendrocyte development and myelination in vitro.

    PubMed

    Pang, Yi; Fan, Lir-Wan; Tien, Lu-Tai; Dai, Xuemei; Zheng, Baoying; Cai, Zhengwei; Lin, Rick C S; Bhatt, Abhay

    2013-09-01

    Oligodendrocyte (OL) development relies on many extracellular cues, most of which are secreted cytokines from neighboring neural cells. Although it is generally accepted that both astrocytes and microglia are beneficial for OL development, there is a lack of understanding regarding whether astrocytes and microglia play similar or distinct roles. The current study examined the effects of astrocytes and microglia on OL developmental phenotypes including cell survival, proliferation, differentiation, and myelination in vitro. Our data reveal that, although both astrocytes- and microglia-conditioned medium (ACDM and MCDM, respectively) protect OL progenitor cells (OPCs) against growth factor withdrawal-induced apoptosis, ACDM is significantly more effective than MCDM in supporting long-term OL survival. In contrast, MCDM preferentially promotes OL differentiation and myelination. These differential effects of ACDM and MCDM on OL development are highlighted by distinct pattern of cytokine/growth factors in the conditioned medium, which correlates with differentially activated intracellular signaling pathways in OPCs upon exposure to the conditioned medium.

  18. Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington's disease

    PubMed Central

    Valenza, M; Marullo, M; Di Paolo, E; Cesana, E; Zuccato, C; Biella, G; Cattaneo, E

    2015-01-01

    In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington's disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyte–neuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD. PMID:25301063

  19. Surfactant tuning of hydrophilicity of porous degradable copolymer scaffolds promotes cellular proliferation and enhances bone formation.

    PubMed

    Yassin, Mohammed A; Leknes, Knut N; Sun, Yang; Lie, Stein A; Finne-Wistrand, Anna; Mustafa, Kamal

    2016-08-01

    Poly(l-lactide-co-ɛ-caprolactone) (poly(LLA-co-CL)) has been blended with Tween 80 to tune the material properties and optimize cell-material interactions. Accordingly, the aims of this study were fourfold: to evaluate the effect of low concentrations of Tween 80 on the surface microstructure of 3D poly(LLA-co-CL) porous scaffolds: to determine the effect of different concentrations of Tween 80 on proliferation of bone marrow stromal cells (BMSCs) in vitro under dynamic cell culture at 7 and 21 days; to assess the influence of Tween 80 on the degradation rate of poly(LLA-co-CL) at 7 and 21 days; and in a subcutaneous rat model, to evaluate the effect on bone formation of porous scaffolds modified with 3% Tween 80 at 2 and 8 weeks. Blending 3% (w/w) Tween 80 with poly(LLA-co-CL) improves the surface wettability (p < 0.001). Poly(LLA-co-CL)/3% Tween 80 shows significantly increased cellular proliferation at days 7 and 21 (p < 0.001). Moreover, the presence of Tween 80 facilitates the degradation of poly(LLA-co-CL). Two weeks post-implantation, the poly(LLA-co-CL)/3% Tween 80 scaffolds exhibit significant mRNA expression of Runx2 (p = 0.004). After 8 weeks, poly(LLA-co-CL)/3% Tween 80 scaffolds show significantly increased de novo bone formation, demonstrated by μ-CT (p = 0.0133) and confirmed histologically. It can be concluded that blending 3% (w/w) Tween 80 with poly (LLA-co-CL) improves the hydrophilicity and osteogenic potential of the scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2049-2059, 2016.

  20. Human breast cancer biopsies induce eosinophil recruitment and enhance adjacent cancer cell proliferation.

    PubMed

    Szalayova, Gabriela; Ogrodnik, Aleksandra; Spencer, Brianna; Wade, Jacqueline; Bunn, Janice; Ambaye, Abiy; James, Ted; Rincon, Mercedes

    2016-06-01

    Chronic inflammation is known to facilitate cancer progression and metastasis. Less is known about the effect of acute inflammation within the tumor microenvironment, resulting from standard invasive procedures. Recent studies in mouse models have shown that the acute inflammatory response triggered by a biopsy in mammary cancer increases the frequency of distal metastases. Although tumor biopsies are part of the standard clinical practice in breast cancer diagnosis, no studies have reported their effect on inflammatory response. The objective of this study is to (1) determine whether core needle biopsies in breast cancer patients trigger an inflammatory response, (2) characterize the type of inflammatory response present, and (3) evaluate the potential effect of any acute inflammatory response on residual tumor cells. The biopsy wound site was identified in the primary tumor resection tissue samples from breast cancer patients. The inflammatory response in areas adjacent (i.e., immediately around previous biopsy site) and distant to the wound biopsy was investigated by histology and immunohistochemistry analysis. Proliferation of tumor cells was also assayed. We demonstrate that diagnostic core needle biopsies trigger a selective recruitment of inflammatory cells at the site of the biopsy, and they persist for extended periods of time. While macrophages were part of the inflammatory response, an unexpected accumulation of eosinophils at the edge of the biopsy wound was also identified. Importantly, we show that biopsy causes an increase in the proliferation rate of tumor cells located in the area adjacent to the biopsy wound. Diagnostic core needle biopsies in breast cancer patients do induce a unique acute inflammatory response within the tumor microenvironment and have an effect on the surrounding tumor cells. Therefore, biopsy-induced inflammation could have an impact on residual tumor cell progression and/or metastasis in human breast cancer. These findings

  1. Astrocyte-Dependent Vulnerability to Excitotoxicity in Spermine Oxidase-Overexpressing Mouse.

    PubMed

    Cervetto, Chiara; Vergani, Laura; Passalacqua, Mario; Ragazzoni, Milena; Venturini, Arianna; Cecconi, Francesco; Berretta, Nicola; Mercuri, Nicola; D'Amelio, Marcello; Maura, Guido; Mariottini, Paolo; Voci, Adriana; Marcoli, Manuela; Cervelli, Manuela

    2016-03-01

    Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

  2. Glutamate Mediated Astrocytic Filtering of Neuronal Activity

    PubMed Central

    Herzog, Nitzan; De Pittà, Maurizio; Jacob, Eshel Ben; Berry, Hugues; Hanein, Yael

    2014-01-01

    Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity. PMID:25521344

  3. The BMP signaling pathway leads to enhanced proliferation in serous ovarian cancer-A potential therapeutic target.

    PubMed

    Peng, Jin; Yoshioka, Yumiko; Mandai, Masaki; Matsumura, Noriomi; Baba, Tsukasa; Yamaguchi, Ken; Hamanishi, Junzo; Kharma, Budiman; Murakami, Ryusuke; Abiko, Kaoru; Murphy, Susan K; Konishi, Ikuo

    2016-04-01

    Members of the transforming growth factor-β (TGF-β) superfamily transduce signals via SMAD proteins. SMAD2 and SMAD3 mediate TGF-β signaling, whereas SMAD1, SMAD5, and SMAD8/9 transduce bone morphogenetic protein (BMP) signals. We would like to identify the function of BMP/SMAD5 signaling in serous ovarian cancer. The protein levels of total SMAD5 and phosphorylated SMAD5 (pSMAD5) were examined by immunohistochemical analysis using clinical serous ovarian cancer samples. Following treatment with either recombinant BMP2 (rBMP2) or Dorsomorphin (DM), western blotting was performed to observe pSMAD5 protein in the cytoplasm and the nucleus, separately. Cell proliferation was detected in SMAD5 knockdown serous ovarian cancer cell lines cultured with DM or rBMP2. The impact of DM or rBMP2 on tumor growth was observed in a mouse model of serous ovarian cancer. An inverse correlation was observed between pSMAD5 levels in the nucleus and the prognosis of patients with serous ovarian cancer. The treatment of SK-OV-3 with rBMP2 stimulated pSMAD5 translocation from the cytoplasm to the nucleus, and the addition of DM inhibited this effect. The proliferation of ovarian cancer cell lines was enhanced by BMP2 and suppressed by DM via SMAD5 in vitro. In vitro and in vivo experiments clearly demonstrated BMP2-stimulated proliferation of serous ovarian cancer and inhibition of this effect by DM. Our data suggests that BMP/SMAD5 signaling plays an important role and, therefore, becomes a potential therapeutic target in serous ovarian cancer. © 2015 Wiley Periodicals, Inc.

  4. Functional Oxygen Sensitivity of Astrocytes.

    PubMed

    Angelova, Plamena R; Kasymov, Vitaliy; Christie, Isabel; Sheikhbahaei, Shahriar; Turovsky, Egor; Marina, Nephtali; Korsak, Alla; Zwicker, Jennifer; Teschemacher, Anja G; Ackland, Gareth L; Funk, Gregory D; Kasparov, Sergey; Abramov, Andrey Y; Gourine, Alexander V

    2015-07-22

    In terrestrial mammals, the oxygen storage capacity of the CNS is limited, and neuronal function is rapidly impaired if oxygen supply is interrupted even for a short period of time. However, oxygen tension monitored by the peripheral (arterial) chemoreceptors is not sensitive to regional CNS differences in partial pressure of oxygen (PO2 ) that reflect variable levels of neuronal activity or local tissue hypoxia, pointing to the necessity of a functional brain oxygen sensor. This experimental animal (rats and mice) study shows that astrocytes, the most numerous brain glial cells, are sensitive to physiological changes in PO2 . Astrocytes respond to decreases in PO2 a few millimeters of mercury below normal brain oxygenation with elevations in intracellular calcium ([Ca(2+)]i). The hypoxia sensor of astrocytes resides in the mitochondria in which oxygen is consumed. Physiological decrease in PO2 inhibits astroglial mitochondrial respiration, leading to mitochondrial depolarization, production of free radicals, lipid peroxidation, activation of phospholipase C, IP3 receptors, and release of Ca(2+) from the intracellular stores. Hypoxia-induced [Ca(2+)]i increases in astrocytes trigger fusion of vesicular compartments containing ATP. Blockade of astrocytic signaling by overexpression of ATP-degrading enzymes or targeted astrocyte-specific expression of tetanus toxin light chain (to interfere with vesicular release mechanisms) within the brainstem respiratory rhythm-generating circuits reveals the fundamental physiological role of astroglial oxygen sensitivity; in low-oxygen conditions (environmental hypoxia), this mechanism increases breathing activity even in the absence of peripheral chemoreceptor oxygen sensing. These results demonstrate that astrocytes are functionally specialized CNS oxygen sensors tuned for rapid detection of physiological changes in brain oxygenation. Significance statement: Most, if not all, animal cells possess mechanisms that allow them to

  5. Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation.

    PubMed

    Shi, Changcan; Yuan, Wenjie; Khan, Musammir; Li, Qian; Feng, Yakai; Yao, Fanglian; Zhang, Wencheng

    2015-05-01

    Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

  6. Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α

    SciTech Connect

    Gao, Hui; Xie, Jing; Peng, Jianjun; Han, Yantao; Jiang, Qixiao; Han, Mei; Wang, Chunbo

    2015-03-15

    Gallbladder cancer (GBC) is an aggressive malignancy of the bile duct, which is associated with a low (5-year) survival and poor prognosis. The transcription factor HIF-1α is implicated in the angiogenesis, cell survival, epithelial mesenchymal transition (EMT) and invasiveness of GBC. In this study, we have investigated the role of HIF-1α in the pathobilogy of GBC and effect of hispidulin on the molecular events controlled by this transcription factor. We observed that hispidulin caused induction of apoptosis, blockade of growth and cell cycle progression in GBC cells. Our results have demonstrated for the first time that hispidulin-exerted anti-tumor effect involved the suppression of HIF-1α signaling. Hispidulin was found to repress the expression of HIF-1α protein dose-dependently without affecting the HIF-1α mRNA expression. In addition, the inhibition of HIF-1α protein synthesis was revealed to be mediated through the activation of AMPK signaling. Hispidulin also sensitized the tumor cells to Gemcitabine and 5-Fluoroucil by down-regulating HIF-1α/P-gp signaling. Given the low cost and exceedingly safe profile, hispidulin appears to be a promising and novel chemosensitizer for GBC treatment. - Highlights: • Hispidulin inhibits proliferation of gallbladder cancer cells by targeting HIF-1α. • Hispidulin regulates HIF-1α via activating AMPK signaling. • Hispidulin sensitized the GBC cells to chemotherapeutics by down-regulating P-gp.

  7. TAp73 enhances the pentose phosphate pathway and supports cell proliferation.

    PubMed

    Du, Wenjing; Jiang, Peng; Mancuso, Anthony; Stonestrom, Aaron; Brewer, Michael D; Minn, Andy J; Mak, Tak W; Wu, Mian; Yang, Xiaolu

    2013-08-01

    TAp73 is a structural homologue of the pre-eminent tumour suppressor p53. However, unlike p53, TAp73 is rarely mutated, and instead is frequently overexpressed in human tumours. It remains unclear whether TAp73 affords an advantage to tumour cells and if so, what the underlying mechanism is. Here we show that TAp73 supports the proliferation of human and mouse tumour cells. TAp73 activates the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). By stimulating G6PD, TAp73 increases PPP flux and directs glucose to the production of NADPH and ribose, for the synthesis of macromolecules and detoxification of reactive oxygen species (ROS). The growth defect of TAp73-deficient cells can be rescued by either enforced G6PD expression or the presence of nucleosides plus an ROS scavenger. These findings establish a critical role for TAp73 in regulating metabolism, and connect TAp73 and the PPP to oncogenic cell growth.

  8. Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study

    PubMed Central

    Luo, Li-ke; Wei, Qing-jun; Liu, Lei; Zheng, Li; Zhao, Jin-min

    2015-01-01

    As the main active constituent of Andrographis paniculata that was applied in treatment of many diseases including inflammation in ancient China, andrographolide (ANDRO) was found to facilitate reduction of edema and analgesia in arthritis. This suggested that ANDRO may be promising anti-inflammatory agent to relieve destruction and degeneration of cartilage after inflammation. In this study, the effect of ANDRO on rabbit articular chondrocytes in vitro was investigated. Results showed that not more than 8 μM ANDRO did no harm to chondrocytes (P < 0.05). DNA content and glycosaminoglycan (GAG) /DNA were, respectively, improved in ANDRO groups comparing to the control (P < 0.05). ANDRO could promote expression of aggrecan, collagen II, and Sox9 genes while downregulating expression of collagen I gene (P < 0.05). Furthermore, hypertrophy that may result in chondrocyte ossification could not be detected in all groups (P > 0.05). The viability assay, hematoxylin-eosin, safranin O, and immunohistochemical staining also showed better performances in ANDRO groups. As to the doses, 3 μM ANDRO showed the best performance. The results indicate that ANDRO can accelerate proliferation of rabbit articular chondrocytes in vitro and meanwhile maintain the phenotype, which may provide valuable references for further exploration on arthritis. PMID:25802548

  9. Surface tailored organobentonite enhances bacterial proliferation and phenanthrene biodegradation under cadmium co-contamination.

    PubMed

    Mandal, Asit; Biswas, Bhabananda; Sarkar, Binoy; Patra, Ashok K; Naidu, Ravi

    2016-04-15

    Co-contamination of soil and water with polycyclic aromatic hydrocarbon (PAH) and heavy metals makes biodegradation of the former extremely challenging. Modified clay-modulated microbial degradation provides a novel insight in addressing this issue. This study was conducted to evaluate the growth and phenanthrene degradation performance of Mycobacterium gilvum VF1 in the presence of a palmitic acid (PA)-grafted Arquad® 2HT-75-based organobentonite in cadmium (Cd)-phenanthrene co-contaminated water. The PA-grafted organobentonite (ABP) adsorbed a slightly greater quantity of Cd than bentonite at up to 30mgL(-1) metal concentration, but its highly negative surface charge imparted by carboxylic groups indicated the potential of being a significantly superior adsorbent of Cd at higher metal concentrations. In systems co-contained with Cd (5 and 10mgL(-1)), the Arquad® 2HT-75-modified bentonite (AB) and PA-grafted organobentonite (ABP) resulted in a significantly higher (72-78%) degradation of phenanthrene than bentonite (62%) by the bacterium. The growth and proliferation of bacteria were supported by ABP which not only eliminated Cd toxicity through adsorption but also created a congenial microenvironment for bacterial survival. The macromolecules produced during ABP-bacteria interaction could form a stable clay-bacterial cluster by overcoming the electrostatic repulsion among individual components. Findings of this study provide new insights for designing clay modulated PAH bioremediation technologies in mixed-contaminated water and soil.

  10. Magnetofection Mediated Transient NANOG Overexpression Enhances Proliferation and Myogenic Differentiation of Human Hair Follicle Derived Mesenchymal Stem Cells.

    PubMed

    Son, Seoyoung; Liang, Mao-Shih; Lei, Pedro; Xue, Xiaozheng; Furlani, Edward P; Andreadis, Stelios T

    2015-07-15

    We used magnetofection (MF) to achieve high transfection efficiency into human mesenchymal stem cells (MSCs). A custom-made magnet array, matching well-to-well to a 24-well plate, was generated and characterized. Theoretical predictions of magnetic force distribution within each well demonstrated that there was no magnetic field interference among magnets in adjacent wells. An optimized protocol for efficient gene delivery to human hair follicle derived MSCs (hHF-MSCs) was established using an egfp-encoding plasmid, reaching approximately ∼50% transfection efficiency without significant cytotoxicity. Then we applied the optimized MF protocol to express the pluripotency-associated transcription factor NANOG, which was previously shown to reverse the effects of organismal aging on MSC proliferation and myogenic differentiation capacity. Indeed, MF-mediated NANOG delivery increased proliferation and enhanced the differentiation of hHF-MSCs into smooth muscle cells (SMCs). Collectively, our results show that MF can achieve high levels of gene delivery to MSCs and, therefore, may be employed to moderate or reverse the effects of cellular senescence or reprogram cells to the pluripotent state without permanent genetic modification.

  11. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials. PMID:25637448

  12. Porous titania surfaces on titanium with hierarchical macro- and mesoporosities for enhancing cell adhesion, proliferation and mineralization.

    PubMed

    Han, Guang; Müller, Werner E G; Wang, Xiaohong; Lilja, Louise; Shen, Zhijian

    2015-02-01

    Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5 μm. An additional mesoporous titania top layer following the contour of the macropores, of 100-200 nm thickness and with a pore diameter of 10nm, was formed by using the evaporation-induced self-assembly (EISA) method with titanium (IV) tetraethoxide as the precursor. A coherent laminar titania surface layer was thus obtained, creating a hierarchical macro- and mesoporous surface that was characterized by high-resolution electron microscopy. The interfacial bonding between the surface layers and the titanium matrix was characterized by the scratch test that confirmed a stable and strong bonding of titania surface layers on titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy. The results proved that the porous titania surface with hierarchical macro- and mesoporosities was hydrophilic that significantly promoted cell attachment and spreading. A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, compared with the titania surface with solo scale topography.

  13. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.

  14. PROX1 promotes hepatocellular carcinoma proliferation and sorafenib resistance by enhancing β-catenin expression and nuclear translocation.

    PubMed

    Liu, Y; Ye, X; Zhang, J-B; Ouyang, H; Shen, Z; Wu, Y; Wang, W; Wu, J; Tao, S; Yang, X; Qiao, K; Zhang, J; Liu, J; Fu, Q; Xie, Y

    2015-10-29

    Aberrant activation of the Wnt/β-catenin pathway is frequent in hepatocellular carcinoma (HCC) and contributes to HCC initiation and progression. This abnormal activation may result from somatic mutations in the genes of the Wnt/β-catenin pathway and/or dysregulation of the Wnt/β-catenin pathway. The mechanism for the latter remains poorly understood. Prospero-related homeobox 1 (PROX1) is a downstream target of the Wnt/β-catenin pathway in human colorectal cancer and elevated PROX1 expression promotes malignant progression. However, the Wnt/β-catenin pathway does not regulate PROX1 expression in the liver and HCC cells. Here we report that PROX1 promotes HCC cell proliferation in vitro and tumor growth in HCC xenograft mice. PROX1 and β-catenin levels are positively correlated in tumor tissues as well as in cultured HCC cells. PROX1 can upregulate β-catenin transcription by stimulating the β-catenin promoter and enhance the nuclear translocation of β-catenin in HCC cells, which leads to the activation of the Wnt/β-catenin pathway. Moreover, we show that increase in PROX1 expression renders HCC cells more resistant to sorafenib treatment, which is the standard therapy for advanced HCC. Overall, we have pinpointed PROX1 as a critical factor activating the Wnt/β-catenin pathway in HCC, which promotes HCC proliferation and sorafenib resistance.

  15. Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1

    SciTech Connect

    Moon, Jai-Hee; Yoon, Byung Sun; Kim, Bona; Park, Gyuman; Jung, Hye-Youn; Maeng, Isaac; Jun, Eun Kyoung; Yoo, Seung Jun; Kim, Aeree; Oh, Sejong; Whang, Kwang Youn; Kim, Hyunggee; Kim, Dong-Wook; Kim, Ki Dong; You, Seungkwon

    2008-06-27

    Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

  16. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    SciTech Connect

    Wang, Suna Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-04-15

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions.

  17. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  18. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory.

    PubMed

    Orr, Anna G; Hsiao, Edward C; Wang, Max M; Ho, Kaitlyn; Kim, Daniel H; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B; Masliah, Eliezer; Conklin, Bruce R; Mucke, Lennart

    2015-03-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer's disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  19. 10-Shogaol, an Antioxidant from Zingiber officinale for Skin Cell Proliferation and Migration Enhancer

    PubMed Central

    Chen, Chung-Yi; Cheng, Kuo-Chen; Chang, Andy Y; Lin, Ying-Ting; Hseu, You-Cheng; Wang, Hui-Min

    2012-01-01

    In this work, one of Zingiber officinale components, 10-shogaol, was tested with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, metal chelating ability, and reducing power to show antioxidant activity. 10-Shogaol promoted human normal epidermal keratinocytes and dermal fibroblasts cell growths. 10-Shogaol enhanced growth factor production in transforming growth factor-β (TGF-β), platelet derived growth factor-αβ (PDGF-αβ) and vascular endothelial growth factors (VEGF) of both cells. In the in vitro wound healing assay for 12 or 24 h, with 10-shogaol, the fibroblasts and keratinocytes migrated more rapidly than the vehicle control group. Thus, this study substantiates the target compound, 10-shogaol, as an antioxidant for human skin cell growth and a migration enhancer with potential to be a novel wound repair agent. PMID:22408422

  20. 10-Shogaol, an antioxidant from Zingiber officinale for skin cell proliferation and migration enhancer.

    PubMed

    Chen, Chung-Yi; Cheng, Kuo-Chen; Chang, Andy Y; Lin, Ying-Ting; Hseu, You-Cheng; Wang, Hui-Min

    2012-01-01

    In this work, one of Zingiber officinale components, 10-shogaol, was tested with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, metal chelating ability, and reducing power to show antioxidant activity. 10-Shogaol promoted human normal epidermal keratinocytes and dermal fibroblasts cell growths. 10-Shogaol enhanced growth factor production in transforming growth factor-β (TGF-β), platelet derived growth factor-αβ (PDGF-αβ) and vascular endothelial growth factors (VEGF) of both cells. In the in vitro wound healing assay for 12 or 24 h, with 10-shogaol, the fibroblasts and keratinocytes migrated more rapidly than the vehicle control group. Thus, this study substantiates the target compound, 10-shogaol, as an antioxidant for human skin cell growth and a migration enhancer with potential to be a novel wound repair agent.

  1. JC virus promoter/enhancers contain TATA box-associated Spi-B-binding sites that support early viral gene expression in primary astrocytes.

    PubMed

    Marshall, Leslie J; Moore, Lisa D; Mirsky, Matthew M; Major, Eugene O

    2012-03-01

    JC virus (JCV) is the aetiological agent of the demyelinating disease progressive multifocal leukoencephalopathy, an AIDS defining illness and serious complication of mAb therapies. Initial infection probably occurs in childhood. In the working model of dissemination, virus persists in the kidney and lymphoid tissues until immune suppression/modulation causes reactivation and trafficking to the brain where JCV replicates in oligodendrocytes. JCV infection is regulated through binding of host factors such as Spi-B to, and sequence variation in the non-coding control region (NCCR). Although NCCR sequences differ between sites of persistence and pathogenesis, evidence suggests that the virus that initiates infection in the brain disseminates via B-cells derived from latently infected haematopoietic precursors in the bone marrow. Spi-B binds adjacent to TATA boxes in the promoter/enhancer of the PML-associated JCV Mad-1 and Mad-4 viruses but not the non-pathogenic, kidney-associated archetype. The Spi-B-binding site of Mad-1/Mad-4 differs from that of archetype by a single nucleotide, AAAAGGGAAGGGA to AAAAGGGAAGGTA. Point mutation of the Mad-1 Spi-B site reduced early viral protein large T-antigen expression by up to fourfold. Strikingly, the reverse mutation in the archetype NCCR increased large T-antigen expression by 10-fold. Interestingly, Spi-B protein binds the NCCR sequence flanking the viral promoter/enhancer, but these sites are not essential for early viral gene expression. The effect of mutating Spi-B-binding sites within the JCV promoter/enhancer on early viral gene expression strongly suggests a role for Spi-B binding to the viral promoter/enhancer in the activation of early viral gene expression.

  2. SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma.

    PubMed

    Zhu, Ying; Zhu, Meng-Xiao; Zhang, Xiao-Dan; Xu, Xiu-E; Wu, Zhi-Yong; Liao, Lian-Di; Li, Li-Yan; Xie, Yang-Min; Wu, Jian-Yi; Zou, Hai-Ying; Xie, Jian-Jun; Li, En-Min; Xu, Li-Yan

    2016-06-01

    Epigenetic alterations, including DNA methylation and histone modifications, are involved in the regulation of cancer initiation and progression. SET and MYND domain-containing protein 3 (SMYD3), a methyltransferase, plays an important role in transcriptional regulation during human cancer progression. However, SMYD3 expression and its function in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, SMYD3 expression was studied by immunohistochemistry in a tumor tissue microarray from 131 cases of ESCC patients. Statistical analysis showed that overall survival of patients with high SMYD3 expressing in primary tumors was significantly lower than that of patients with low SMYD3-expressing tumors (P = .008, log-rank test). Increased expression of SMYD3 was found to be associated with lymph node metastasis in ESCC (P = .036) and was an independent prognostic factor for poor overall survival (P = .025). RNAi-mediated knockdown of SMYD3 suppressed ESCC cell proliferation, migration, and invasion in vitro and inhibited local tumor invasion in vivo. SMYD3 regulated transcription of EZR and LOXL2 by directly binding to the sequences of the promoter regions of these target genes, as demonstrated by a chromatin immunoprecipitation assay. Immunohistochemical staining of ESCC tissues also confirmed that protein levels of EZR and LOXL2 positively correlated with SMYD3 expression, and the Spearman correlation coefficients (rs) were 0.78 (n = 81; P < .01) and 0.637 (n = 103; P < .01), respectively. These results indicate that SMYD3 enhances tumorigenicity in ESCC through enhancing transcription of genes involved in proliferation, migration, and invasion. PMID:26980013

  3. SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma.

    PubMed

    Zhu, Ying; Zhu, Meng-Xiao; Zhang, Xiao-Dan; Xu, Xiu-E; Wu, Zhi-Yong; Liao, Lian-Di; Li, Li-Yan; Xie, Yang-Min; Wu, Jian-Yi; Zou, Hai-Ying; Xie, Jian-Jun; Li, En-Min; Xu, Li-Yan

    2016-06-01

    Epigenetic alterations, including DNA methylation and histone modifications, are involved in the regulation of cancer initiation and progression. SET and MYND domain-containing protein 3 (SMYD3), a methyltransferase, plays an important role in transcriptional regulation during human cancer progression. However, SMYD3 expression and its function in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, SMYD3 expression was studied by immunohistochemistry in a tumor tissue microarray from 131 cases of ESCC patients. Statistical analysis showed that overall survival of patients with high SMYD3 expressing in primary tumors was significantly lower than that of patients with low SMYD3-expressing tumors (P = .008, log-rank test). Increased expression of SMYD3 was found to be associated with lymph node metastasis in ESCC (P = .036) and was an independent prognostic factor for poor overall survival (P = .025). RNAi-mediated knockdown of SMYD3 suppressed ESCC cell proliferation, migration, and invasion in vitro and inhibited local tumor invasion in vivo. SMYD3 regulated transcription of EZR and LOXL2 by directly binding to the sequences of the promoter regions of these target genes, as demonstrated by a chromatin immunoprecipitation assay. Immunohistochemical staining of ESCC tissues also confirmed that protein levels of EZR and LOXL2 positively correlated with SMYD3 expression, and the Spearman correlation coefficients (rs) were 0.78 (n = 81; P < .01) and 0.637 (n = 103; P < .01), respectively. These results indicate that SMYD3 enhances tumorigenicity in ESCC through enhancing transcription of genes involved in proliferation, migration, and invasion.

  4. Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

    PubMed Central

    Muñoz, Manuel F.; Puebla, Mariela; Figueroa, Xavier F.

    2015-01-01

    Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process

  5. Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation.

    PubMed

    Rashid, Rafi; Lim, Natalie Sheng Jie; Chee, Stella Min Ling; Png, Si Ning; Wohland, Thorsten; Raghunath, Michael

    2014-12-01

    Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. Since standard cell culture media lack this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix (ECM) deposition, is impaired in vitro. To bring back physiological crowdedness to in vitro systems, we have previously introduced carbohydrate-based macromolecules to culture media and have achieved marked improvements with mixed MMC in terms of ECM deposition and differentiation of mesenchymal stem cells (MSCs). We show here that although this system is successful, it is limited, due to viscosity, to only 33% of the fractional volume occupancy (FVO) of full serum, which we calculated to have an FVO of approximately 54% v/v. We show here that full-serum FVO can be achieved using polyvinylpyrrolidone (PVP) 360 kDa. Under these conditions, ECM deposition in human fibroblasts and MSCs is on par, if not stronger than, with original MMC protocols using carbohydrates, but with a viscosity that is not significantly changed. In addition, we have found that the proliferation rate for bone marrow-derived MSCs and fibroblasts increases slightly in the presence of PVP360, similar to that observed with carbohydrate-based crowders. A palette of MMC compounds is now emerging that enables us to tune the crowdedness of culture media seamlessly from interstitial fluid (9% FVO), in which the majority of tissue cells might be based, to serum environments mimicking intravascular conditions. Despite identical FVO's, individual crowder size effects play a role and different cell types appear to have preferences in terms of FVO and the crowder that this is achieved with. However, in the quest of crowders that we have predicted to have a smoother regulatory approval path, PVP is a highly interesting compound, as it has been widely used in the medical and food industries and shows a novel promising use in cell culture and

  6. MicroRNA-132 enhances transition from inflammation to proliferation during wound healing.

    PubMed

    Li, Dongqing; Wang, Aoxue; Liu, Xi; Meisgen, Florian; Grünler, Jacob; Botusan, Ileana R; Narayanan, Sampath; Erikci, Erdem; Li, Xi; Blomqvist, Lennart; Du, Lei; Pivarcsi, Andor; Sonkoly, Enikö; Chowdhury, Kamal; Catrina, Sergiu-Bogdan; Ståhle, Mona; Landén, Ning Xu

    2015-08-01

    Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

  7. Mild heat stress enhances differentiation and proliferation of Japanese quail myoblasts and enhances slow muscle fiber characteristics.

    PubMed

    Choi, Y M; Chen, P R; Shin, S; Zhang, J; Hwang, S; Lee, K

    2016-08-01

    The objective of this study was to investigate the effect of mild heat stress on muscle fiber hyperplastic and hypertrophic growth in quail primary myogenic cells to better understand the mechanisms leading to increased skeletal muscle development in avian embryos incubated at a higher temperature. Compared to control cultures maintained at 37°C, incubation at 39°C enhanced myotube length (P < 0.01) and diameter (P < 0.001) at 3 days after differentiation (D3). This enlargement of the myotubes incubated at 39°C can be explained by differences in the fusion index (56.7 vs. 46.2%, P < 0.05) and nuclei number per myotube (18.1 vs. 10.8, P < 0.001) compared to the control cells at D3. Additionally, a higher density of myotubes at D3 in cultures exposed to a higher temperature were related to higher levels of Pax-7 (P < 0.05) compared to the control cells incubated continuously at 37°C. These results indicated a higher proliferative capacity in cells exposed to mild heat stress compared to the control cells. On the other hand, mild heat stress enhanced protein levels of slow myosin heavy chain isoform (P < 0.01) and cytochrome c oxidase subunit IV (P < 0.01) compared to the control cells at D3. These discrepancies in protein expression indicated maintenance of slow muscle fiber type characteristics in myotubes incubated at 39°C. Our results suggest that mild heat stress plays a significant role in myogenic mechanisms related to muscle mass and development.

  8. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    SciTech Connect

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki; Kawada, Teruo

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  9. Proliferation enhancement of budding yeast and mammalian cells with periodic oxygen radical treatment

    NASA Astrophysics Data System (ADS)

    Mori, Yosuke; Kobayashi, Jun; Murata, Tomiyasu; Hahizume, Hiroshi; Hori, Masaru; Ito, Masafumi

    2015-09-01

    Recently, nonequilibrium atmospheric-pressure plasmas have been intensively studied for biological applications. However, the each effect of species in plasmas to biological tissue has not been clarified yet because various factors exist in the plasmas. Accordingly, we have studied effects of atomic oxygen dose on cell growth such as budding yeast and mouse NIH3T3 fibroblasts of mammalian cells. Both of cells were suspended with PBS, and treated using oxygen radical source. In order to prevent the radicals from reacting with the ambient air, the treatment region was surrounded by a plastic cover and purged with Ar. The proliferative effect of 15 % was observed at the O3Pj dose of around 1 . 0 ×1017 cm-3 in NIH3T3 cells as well as in yeast cells. Moreover, periodic oxygen treatment enhanced the effect in budding yeast cells. The best interval of periodic oxygen radical treatment was around 2 hours, which is almost the same period as that of their cell cycle. With the optimum interval time, we have investigated the effect of the number of the treatments. As the number of treatments increases, the growth rate of budding yeast cells was gradually enhanced and saturated at thrice treatments. This work was partly supported by JSPS KAKENHI Grant Numbers 26286072 and project for promoting Research Center in Meijo University.

  10. Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

    PubMed Central

    Schneider, L; Fumagalli, M; d'Adda di Fagagna, F

    2012-01-01

    The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes – the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for γH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR. PMID:21979466

  11. HSPB1 Enhances SIRT2-Mediated G6PD Activation and Promotes Glioma Cell Proliferation

    PubMed Central

    Cao, Fei; Chen, Mantao; Zheng, Xiujue; Zhan, Renya

    2016-01-01

    Heat shock proteins belong to a conserved protein family and are involved in multiple cellular processes. Heat shock protein 27 (Hsp27), also known as heat HSPB1, participates in cellular responses to not only heat shock, but also oxidative or chemical stresses. However, the contribution of HSPB1 to anti-oxidative response remains unclear. Here, we show that HSPB1 activates G6PD in response to oxidative stress or DNA damage. HSPB1 enhances the binding between G6PD and SIRT2, leading to deacetylation and activation of G6PD. Besides, HSPB1 activates G6PD to sustain cellular NADPH and pentose production in glioma cells. High expression of HSPB1 correlates with poor survivalrate of glioma patients. Together, our study uncovers the molecular mechanism by which HSPB1 activates G6PD to protect cells from oxidative and DNA damage stress. PMID:27711253

  12. Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B.

    PubMed

    Cho, Hyun-Soo; Shimazu, Tadahiro; Toyokawa, Gouji; Daigo, Yataro; Maehara, Yoshihiko; Hayami, Shinya; Ito, Akihiro; Masuda, Ken; Ikawa, Noriko; Field, Helen I; Tsuchiya, Eiju; Ohnuma, Shin-ichi; Ponder, Bruce A J; Yoshida, Minoru; Nakamura, Yusuke; Hamamoto, Ryuji

    2012-01-01

    Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis. PMID:22990868

  13. Endocytosis-mediated HIV-1 entry and its significance in the elusive behavior of the virus in astrocytes.

    PubMed

    Chauhan, Ashok; Mehla, Rajeev; Vijayakumar, Theophilus Sunder; Handy, Indhira

    2014-05-01

    Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rabs 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab 5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system.

  14. Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin

    PubMed Central

    Ohshima, Mihoko; Miyake, Mio; Takeda, Masanori; Kamijima, Michihiro

    2011-01-01

    Background. Staphylococcal enterotoxin B (SEB) may be associated with the exacerbation of atopic dermatitis. We investigated whether SEB causes proliferation of sensory C-fibers and subsequent enhancement of plasma leakage induced by sensorineural stimulation in rat skin. Methods. SEB was applied intracutaneously to the abdomen of preweaning and adult rats. Evans blue dye leakage into the skin induced by topical 10% formalin was measured as an index of neurogenic skin vascular permeability. Local expression of substance P, tachykinin NK1 receptors, and nerve growth factor was assessed immunohistochemically. In addition, we assessed the effects of topical tacrolimus on these skin responses induced by SEB. Results. Increased neurogenic skin plasma leakage was seen 7 days after SEB treatment in 2 different age groups. Innervation of substance P-immunoreactive nerves and expression of tachykinin NK1 receptors and nerve growth factor were also promoted by SEB, peaking at 7 days, 7 days, and 56 h after SEB treatment, respectively. Tacrolimus markedly inhibited these skin changes. Conclusions. SEB increased the innervation of sensory C-fibers and tachykinin NK1 receptors in rat skin, probably because of upregulated production of neurotrophins, including nerve growth factor, leading to enhancement of neurogenic skin inflammation. T cell activation induced by SEB may initiate these changes. PMID:21252260

  15. Capsaicin enhances anti-proliferation efficacy of pirarubicin via activating TRPV1 and inhibiting PCNA nuclear translocation in 5637 cells.

    PubMed

    Zheng, Long; Chen, Jiaqi; Ma, Zhenkun; Liu, Wei; Yang, Fei; Yang, Zhao; Wang, Ke; Wang, Xinyang; He, Dalin; Li, Lei; Zeng, Jin

    2016-01-01

    The recurrence of bladder cancer after surgery with or without chemotherapy remains a major challenge in bladder cancer treatment. Previous studies have shown that transient receptor potential vanilloid 1 (TRPV1) acts as a tumor suppressor through inducing apoptosis in bladder cancer cells. However, whether activation of TRPV1 has any synergistic effects with pirarubicin (THP), one of main drugs used in urinary bladder instillation chemotherapy to improve chemotherapeutic efficacy has remained elusive. The present study verified that TRPV1 was differentially expressed in bladder cancer cell lines. Furthermore, activation of TRPV1 by capsaicin was shown to induce growth inhibition of 5637 cells in which TRPV1 was highly expressed, while the growth of T24 cells, which express TRPV1 at low levels, was not affected. In addition, the present study demonstrated that activation of TRPV1 enhanced the anti‑proliferative effects of pirarubicin using an MTT assay and cell cycle analysis. Finally, immunofluorescent microscopy revealed that activation of TRPV1 prevented the translocation of proliferating cell nuclear antigen to the nucleus. This phenomenon was reversed by pre‑treatment with capsazepine, a specific TRPV1 antagonist. In conclusion, the present study confirmed the anti‑tumor activity of TRPV1 against bladder cancer. Activation of TRPV1 may be applied as a novel strategy to treat bladder cancer or enhance the therapeutic efficacy of traditional chemotherapeutic drugs.

  16. Alpha-tubulin enhanced renal tubular cell proliferation and tissue repair but reduced cell death and cell-crystal adhesion

    PubMed Central

    Manissorn, Juthatip; Khamchun, Supaporn; Vinaiphat, Arada; Thongboonkerd, Visith

    2016-01-01

    Adhesion of calcium oxalate (CaOx) crystals on renal tubular epithelial cells is a critical event for kidney stone disease that triggers many cascades of cellular response. Our previous expression proteomics study identified several altered proteins in MDCK renal tubular cells induced by CaOx crystals. However, functional significance of those changes had not been investigated. The present study thus aimed to define functional roles of such proteome data. Global protein network analysis using STRING software revealed α-tubulin, which was decreased, as one of central nodes of protein-protein interactions. Overexpression of α-tubulin (pcDNA6.2-TUBA1A) was then performed and its efficacy was confirmed. pcDNA6.2-TUBA1A could maintain levels of α-tubulin and its direct interacting partner, vimentin, after crystal exposure. Also, pcDNA6.2-TUBA1A successfully reduced cell death to almost the basal level and increased cell proliferation after crystal exposure. Additionally, tissue repair capacity was improved in pcDNA6.2-TUBA1A cells. Moreover, cell-crystal adhesion was reduced by pcDNA6.2-TUBA1A. Finally, levels of potential crystal receptors (HSP90, HSP70, and α-enolase) on apical membrane were dramatically reduced to basal levels by pcDNA6.2-TUBA1A. These findings implicate that α-tubulin has protective roles in kidney stone disease by preventing cell death and cell-crystal adhesion, but on the other hand, enhancing cell proliferation and tissue repair function. PMID:27363348

  17. Elevated expression of KIF18A enhances cell proliferation and predicts poor survival in human clear cell renal carcinoma

    PubMed Central

    CHEN, QI; CAO, BIN; NAN, NING; WANG, YU; ZHAI, XU; LI, YOUFANG; CHONG, TIE

    2016-01-01

    The function of kinesin family member 18A (KIF18A) in human renal cell carcinoma (RCC) is unclear. The purpose of the current study was to determine the expression and prognostic significance of KIF18A in RCC. Specimens from 273 RCC patients undergoing nephrectomies were studied. Expression of KIF18A mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR) or quantitative PCR, and the expression of KIF18A protein was examined by immunohistochemistry and western blotting. The expression of KIF18A in clear-cell RCC cell lines was decreased using small interfering RNA targeting KIF18A, and increased by transfection with KIF18A cDNA. The proliferative ability of RCC cells in vitro and in vivo was detected by WST-1 assay and an animal xenograft model with BALB/c nude mice, respectively. The association between KIF18A expression and overall survival was calculated using Kaplan-Meier analysis. The results showed that KIF18A expression was significantly increased in RCC tissues compared with normal kidney tissues. The level of KIF18A expression was significantly associated with tumor stage, histological grade, metastasis and tumor size. Moreover, KIF18A increased the proliferation of RCC cells in vitro and in vivo. KIF18A expression was upregulated in RCC and enhanced the proliferation of RCC cells. Therefore, it appears that KIF18A plays a key role in the carcinogenesis and progression of RCC, and is a novel candidate prognostic marker for RCC patients. Furthermore, silencing KIF18A expression may serve as a new therapeutic strategy against RCC. PMID:27347065

  18. Extracellular miR-1246 promotes lung cancer cell proliferation and enhances radioresistance by directly targeting DR5.

    PubMed

    Yuan, Dexiao; Xu, Jinping; Wang, Juan; Pan, Yan; Fu, Jiamei; Bai, Yang; Zhang, Jianghong; Shao, Chunlin

    2016-05-31

    MiRNAs in the circulation have been demonstrated to be a type of signaling molecule involved in intercellular communication but little is known about their role in regulating radiosensitivity. This study aims to investigate the effects of extracellular miRNAs induced by ionizing radiation (IR) on cell proliferation and radiosensitivity. The miRNAs in the conditioned medium (CM) from irradiated and non-irradiated A549 lung cancer cells were compared using a microarray assay and the profiles of 21 miRNAs up and down-regulated by radiation were confirmed by qRT-PCR. One of these miRNAs, miR-1246, was especially abundant outside the cells and had a much higher level compared with that inside of cells. The expressions of miR-1246 in both A549 and H446 cells increased along with irradiation dose and the time post-irradiation. By labeling exosomes and miR-1246 with different fluorescence dyes, it was found that the extracellular miR-1246 could shuttle from its donor cells to other recipient cells by a non-exosome associated pathway. Moreover, the treatments of cells with miR-1246 mimic or its antisense inhibitor showed that the extracellular miR-1246 could enhance the proliferation and radioresistance of lung cancer cells. A luciferase reporter-gene transfer experiment demonstrated that the death receptor 5 (DR5) was the direct target of miR-1246, and the kinetics of DR5 expression was opposite to that of miR-1246 in the irradiated cells. Our results show that the oncogene-like extracellular miR-1246 could act as a signaling messenger between irradiated and non-irradiated cells, more importantly, it contributes to cell radioresistance by directly suppressing the DR5 gene. PMID:27129166

  19. ZNF217 is associated with poor prognosis and enhances proliferation and metastasis in ovarian cancer.

    PubMed

    Li, Jing; Song, Lanlin; Qiu, Yuwen; Yin, Ailan; Zhong, Mei

    2014-01-01

    ZNF217 is an alternatively spliced Kruppel-like transcription factor that has recently been implicated to play a role in human carcinogenesis. Here, we used immunohistochemistry (IHC) to show that ZNF217 protein is overexpressed in nearly 60% of ovarian tumor samples. The disease-free survival time was shorter in patients with positive ZNF217 expression than in ZNF217-negative patients (P=0.042). Fluorescence in situ hybridization (FISH) analysis showed ZNF217 genomic amplification in the poorly differentiated tumors, suggesting that ZNF217 is associated with the progression of ovarian cancer. Invasion was enhanced in HO-8910 cells stably transfected with constructs carrying full-length ZNF217 relative to cells transfected with the empty vector. To confirm our findings in vivo, we performed a tumorigenicity assay in nude mice inoculated with the HO-8910 overexpressing ZNF217 cells. As expected, tumors grown in the ZNF217 group were more invasive and prone to metastasis than those formed control groups. Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes.

  20. Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells

    SciTech Connect

    Grayson, Warren L.; Zhao, Feng; Bunnell, Bruce; Ma, Teng . E-mail: teng@eng.fsu.edu

    2007-07-06

    Changes in oxygen concentrations affect many of the innate characteristics of stem and progenitor cells. Human mesenchymal stem cells (hMSCs) were maintained under hypoxic atmospheres (2% O{sub 2}) for up to seven in vitro passages. This resulted in approximately 30-fold higher hMSC expansion over 6 weeks without loss of multi-lineage differentiation capabilities. Under hypoxia, hMSCs maintained their growth-rates even after reaching confluence, resulting in the formation of multiple cell layers. Hypoxic hMSCs also displayed differences in the cell and nuclear morphologies as well as enhanced ECM formation and organization. These changes in cellular characteristics were accompanied by higher mRNA levels of Oct-4 and HIF-2{alpha}, as well as increased expression levels of connexin-43, a protein used in gap junction formation. The results from this study demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation.

  1. ZNF217 is associated with poor prognosis and enhances proliferation and metastasis in ovarian cancer.

    PubMed

    Li, Jing; Song, Lanlin; Qiu, Yuwen; Yin, Ailan; Zhong, Mei

    2014-01-01

    ZNF217 is an alternatively spliced Kruppel-like transcription factor that has recently been implicated to play a role in human carcinogenesis. Here, we used immunohistochemistry (IHC) to show that ZNF217 protein is overexpressed in nearly 60% of ovarian tumor samples. The disease-free survival time was shorter in patients with positive ZNF217 expression than in ZNF217-negative patients (P=0.042). Fluorescence in situ hybridization (FISH) analysis showed ZNF217 genomic amplification in the poorly differentiated tumors, suggesting that ZNF217 is associated with the progression of ovarian cancer. Invasion was enhanced in HO-8910 cells stably transfected with constructs carrying full-length ZNF217 relative to cells transfected with the empty vector. To confirm our findings in vivo, we performed a tumorigenicity assay in nude mice inoculated with the HO-8910 overexpressing ZNF217 cells. As expected, tumors grown in the ZNF217 group were more invasive and prone to metastasis than those formed control groups. Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes. PMID:25031722

  2. Enhancement of cells proliferation and control of bioactivity of strontium doped glass

    NASA Astrophysics Data System (ADS)

    Oudadesse, H.; Dietrich, E.; Bui, X. V.; Le Gal, Y.; Pellen, P.; Cathelineau, G.

    2011-08-01

    Bioactivity and chemical reactivity of bioactive glass offer the ability to bond for soft and hard biological tissues. In this work, synthesis was carried out by using melting and rapid quenching. Strontium was introduced as trace element at different contents in the glass matrix, according to its concentration in the bone matrix. This chemical element presents a high interest in the bone metabolism activity. Investigations were conducted on the surface of biomaterials by using in vitro assay after immersion in SBF. Several physico-chemical methods such as SEM, FTIR, NMR, ICP-OES and MTT test were employed to highlight the effects of the Sr. The in vitro experiments showed that after soaking in SBF, the behaviour of pure glass is different compared to glass doped with Sr. NMR analyses showed in the 29Si MAS-NMR that glass matrix undergoes some changes after in vitro assays particularly the emergence of new components attributed to Q 3(OH). The presence of Sr slowed down the bioactivity of glass after immersion in SBF. The non toxic character of compounds was confirmed. Introduction of Sr at 0.1 wt % induce an enhancement of cells at about 14.3%.

  3. MHC class I protects motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis (ALS)

    PubMed Central

    Braun, Lyndsey; Meyer, Kathrin; Frakes, Ashley E.; Ferraiuolo, Laura; Likhite, Shibi; Bevan, Adam K.; Foust, Kevin D.; McConnell, Michael J.; Walker, Christopher M.; Kaspar, Brian K.

    2016-01-01

    Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic towards motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of motor neurons to cell death remains unclear. Here, we report that astrocytes derived from mice bearing ALS mutations and from individuals with ALS reduce expression of major histocompatibility complex class I (MHCI) on MNs. Reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MN against astrocyte toxicity. A single MHCI molecule, HLA-F, protects MNs from ALS astrocyte-mediated toxicity, while knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, an inhibitory receptor that recognizes MHCI, on astrocytes results in enhanced MN death. These data indicate that in ALS upon loss of MHCI expression MNs become vulnerable to astrocyte-mediated toxicity. PMID:26928464

  4. Enhanced alveolar monocytic phagocyte (macrophage) proliferation in tobacco and marijuana smokers

    SciTech Connect

    Barbers, R.G.; Evans, M.J.; Gong, H. Jr.; Tashkin, D.P. )

    1991-05-01

    We tested the hypothesis that enhanced cell division accounted for the augmented numbers of monocytic phagocytes with characteristics attributed to alveolar macrophages (AM) found in the lungs of habitual tobacco (T) and marijuana (M) smokers. The monocytic phagocytes, that is, alveolar macrophages, were obtained by bronchoalveolar lavage (BAL) from 12 nonsmoking subjects; 10 subjects who smoked T only (TS); 13 subjects who smoked M only (MS); and 6 smokers of both T and M (MTS). The replication of these cells was determined by measuring the incorporation of ({sup 3}H)thymidine into the DNA of dividing cells and visually counting 2,000 cells on autoradiographically prepared cytocentrifuge cell preparations. This study demonstrated that the number of ({sup 3}H)thymidine-labeled monocytic phagocytes with characteristics of alveolar macrophages from either TS or MS have a higher proliferative index compared to cells (macrophages) from nonsmokers, p less than 0.05 by one-way ANOVA. The total number of BAL macrophages that are in mitosis in TS (17.90 +/- 4.50 labeled AM x 10(3)/ml) or MTS (10.50 +/- 4.20 labeled AM x 10(3)/ml) are 18- and 10-fold greater, respectively, than the number obtained from nonsmokers (1.01 +/- 0.18 labeled AM x 10(3)/ml). Interestingly, the number of ({sup 3}H)thymidine-labeled macrophages from MS (2.90 +/- 0.66 labeled AM x 10(3)/ml) are also greater than the number obtained from nonsmokers, although this is not statistically significant. The stimulus augmenting alveolar macrophage replication is as yet unknown but may likely be found in the T or M smoke.

  5. Astrocytes increase ATP exocytosis mediated calcium signaling in response to microgroove structures.

    PubMed

    Singh, Ajay V; Raymond, Michael; Pace, Fabiano; Certo, Anthony; Zuidema, Jonathan M; McKay, Christopher A; Gilbert, Ryan J; Lu, X Lucas; Wan, Leo Q

    2015-01-01

    Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 μm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell-substrate interactions, and may benefit the optimization of scaffold design for CNS healing. PMID:25597401

  6. Astrocyte calcium signaling: the third wave.

    PubMed

    Bazargani, Narges; Attwell, David

    2016-02-01

    The discovery that transient elevations of calcium concentration occur in astrocytes, and release 'gliotransmitters' which act on neurons and vascular smooth muscle, led to the idea that astrocytes are powerful regulators of neuronal spiking, synaptic plasticity and brain blood flow. These findings were challenged by a second wave of reports that astrocyte calcium transients did not mediate functions attributed to gliotransmitters and were too slow to generate blood flow increases. Remarkably, the tide has now turned again: the most important calcium transients occur in fine astrocyte processes not resolved in earlier studies, and new mechanisms have been discovered by which astrocyte [Ca(2+)]i is raised and exerts its effects. Here we review how this third wave of discoveries has changed our understanding of astrocyte calcium signaling and its consequences for neuronal function.

  7. Astrocytes Control Synapse Formation, Function, and Elimination

    PubMed Central

    Chung, Won-Suk; Allen, Nicola J.; Eroglu, Cagla

    2015-01-01

    Astrocytes, through their close associations with synapses, can monitor and alter synaptic function, thus actively controlling synaptic transmission in the adult brain. Besides their important role at adult synapses, in the last three decades a number of critical findings have highlighted the importance of astrocytes in the establishment of synaptic connectivity in the developing brain. In this article, we will review the key findings on astrocytic control of synapse formation, function, and elimination. First, we will summarize our current structural and functional understanding of astrocytes at the synapse. Then, we will discuss the cellular and molecular mechanisms through which developing and mature astrocytes instruct the formation, maturation, and refinement of synapses. Our aim is to provide an overview of astrocytes as important players in the establishment of a functional nervous system. PMID:25663667

  8. Astrocytes in the tempest of multiple sclerosis.

    PubMed

    Miljković, Djordje; Timotijević, Gordana; Mostarica Stojković, Marija

    2011-12-01

    Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes. PMID:21443873

  9. Involvement of astrocytes in neurovascular communication.

    PubMed

    Nuriya, M; Hirase, H

    2016-01-01

    The vascular interface of the brain is distinct from that of the peripheral tissue in that astrocytes, the most numerous glial cell type in the gray matter, cover the vasculature with their endfeet. This morphological feature of the gliovascular junction has prompted neuroscientists to suggest possible functional roles of astrocytes including astrocytic modulation of the vasculature. Additionally, astrocytes develop an intricate morphology that intimately apposes neuronal synapses, making them an ideal cellular mediator of neurovascular coupling. In this article, we first introduce the classical anatomical and physiological findings that led to the proposal of various gliovascular interaction models. Next, we touch on the technological advances in the past few decades that enabled investigations and evaluations of neuro-glio-vascular interactions in situ. We then review recent experimental findings on the roles of astrocytes in neurovascular coupling from the viewpoints of intra- and intercellular signalings in astrocytes. PMID:27130410

  10. Methamphetamine Inhibits the Glucose Uptake by Human Neurons and Astrocytes: Stabilization by Acetyl-L-Carnitine

    PubMed Central

    Szlachetka, Adam M.; Haorah, James

    2011-01-01

    Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers. PMID:21556365

  11. High mobility group nucleosome-binding family proteins promote astrocyte differentiation of neural precursor cells.

    PubMed

    Nagao, Motoshi; Lanjakornsiripan, Darin; Itoh, Yasuhiro; Kishi, Yusuke; Ogata, Toru; Gotoh, Yukiko

    2014-11-01

    Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Although previous studies have shown that the STAT signaling pathway or its regulators promote the generation of astrocytes from multipotent neural precursor cells (NPCs) in the developing mammalian brain, the molecular mechanisms that regulate the astrocytic fate decision have still remained largely unclear. Here, we show that the high mobility group nucleosome-binding (HMGN) family proteins, HMGN1, 2, and 3, promote astrocyte differentiation of NPCs during brain development. HMGN proteins were expressed in NPCs, Sox9(+) glial progenitors, and GFAP(+) astrocytes in perinatal and adult brains. Forced expression of either HMGN1, 2, or 3 in NPCs in cultures or in the late embryonic neocortex increased the generation of astrocytes at the expense of neurons. Conversely, knockdown of either HMGN1, 2, or 3 in NPCs suppressed astrocyte differentiation and promoted neuronal differentiation. Importantly, overexpression of HMGN proteins did not induce the phosphorylation of STAT3 or activate STAT reporter genes. In addition, HMGN family proteins did not enhance DNA demethylation and acetylation of histone H3 around the STAT-binding site of the gfap promoter. Moreover, knockdown of HMGN family proteins significantly reduced astrocyte differentiation induced by gliogenic signal ciliary neurotrophic factor, which activates the JAK-STAT pathway. Therefore, we propose that HMGN family proteins are novel chromatin regulatory factors that control astrocyte fate decision/differentiation in parallel with or downstream of the JAK-STAT pathway through modulation of the responsiveness to gliogenic signals. PMID:25069414

  12. Astrocytic glycogenolysis: mechanisms and functions.

    PubMed

    Hertz, Leif; Xu, Junnan; Song, Dan; Du, Ting; Li, Baoman; Yan, Enzhi; Peng, Liang

    2015-02-01

    Until the demonstration little more than 20 years ago that glycogenolysis occurs during normal whisker stimulation glycogenolysis was regarded as a relatively uninteresting emergency procedure. Since then, a series of important astrocytic functions has been shown to be critically dependent on glycogenolytic activity to support the signaling mechanisms necessary for these functions to operate. This applies to glutamate formation and uptake and to release of ATP as a transmitter, stimulated by other transmitters or elevated K(+) concentrations and affecting not only other astrocytes but also most other brain cells. It is also relevant for astrocytic K(+) uptake both during the period when the extracellular K(+) concentration is still elevated after neuronal excitation, and capable of stimulating glycogenolytic activity, and during the subsequent undershoot after intense neuronal activity, when glycogenolysis may be stimulated by noradrenaline. Both elevated K(+) concentrations and several transmitters, including the β-adrenergic agonist isoproterenol and vasopressin increase free cytosolic Ca(2+) concentration in astrocytes, which stimulates phosphorylase kinase so that it activates the transformation of the inactive glycogen phosphorylase a to the active phosphorylase b. Contrary to common belief cyclic AMP plays at most a facilitatory role, and only when free cytosolic Ca(2+) concentration is also increased. Cyclic AMP is not increased during activation of glycogenolysis by either elevated K(+) concentrations or the stimulation of the serotonergic 5-HT(2B) receptor. Not all agents that stimulate glycogenolysis do so by directly activating phophorylase kinase--some do so by activating processes requiring glycogenolysis, e.g. for synthesis of glutamate. PMID:24744118

  13. Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

    PubMed

    Thomas, Stacey L; Schultz, Chad R; Mouzon, Ezekiell; Golembieski, William A; El Naili, Reima; Radakrishnan, Archanna; Lemke, Nancy; Poisson, Laila M; Gutiérrez, Jorge A; Cottingham, Sandra; Rempel, Sandra A

    2015-07-01

    Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

  14. Targeting astrocytes in bipolar disorder.

    PubMed

    Peng, Liang; Li, Baoman; Verkhratsky, Alexei

    2016-06-01

    Astrocytes are homeostatic cells of the central nervous system, which are critical for development and maintenance of synaptic transmission and hence of synaptically connected neuronal ensembles. Astrocytic densities are reduced in bipolar disorder, and therefore deficient astroglial function may contribute to overall disbalance in neurotransmission and to pathological evolution. Classical anti-bipolar drugs (lithium salts, valproic acid and carbamazepine) affect expression of astroglial genes and modify astroglial signalling and homeostatic cascades. Many effects of both antidepressant and anti-bipolar drugs are exerted through regulation of glutamate homeostasis and glutamatergic transmission, through K(+) buffering, through regulation of calcium-dependent phospholipase A2 (that controls metabolism of arachidonic acid) or through Ca(2+) homeostatic and signalling pathways. Sometimes anti-depressant and anti-bipolar drugs exert opposite effects, and some effects on gene expression in drug treated animals are opposite in neurones vs. astrocytes. Changes in the intracellular pH induced by anti-bipolar drugs affect uptake of myo-inositol and thereby signalling via inositoltrisphosphate (InsP3), this being in accord with one of the main theories of mechanism of action for these drugs. PMID:27015045

  15. Astrocytes: Everything but the glue

    PubMed Central

    Gonzalez-Perez, Oscar; Lopez-Virgen, Veronica; Quiñones-Hinojosa, Alfredo

    2015-01-01

    The current knowledge in neuroscience indicates that neural tissue has two major cell populations: neurons and glia (term derived from the Greek word for glue). Neuronal population is characterized by the capacity to produce action potentials, whereas glial cells are typically identified as the subordinate cell population of neurons. To date, this point of view has changed dramatically and growing evidence indicates that glial cells play a crucial role in normal mental functions and the pathogenesis of neurological diseases. Classically, glial cells include four major populations clearly discernible in the adult brain: astrocytes, oligodendrocytes, microglia cells and NG2 glia. Astrocytes, also referred as to astroglia, are by far the most abundant cell lineage in the adult brain. These cells are in close contact with several tissue components of the brain parenchyma including neurons, vasculature, extracellular matrix and other glial populations. Hence, the number and strategic position of astrocytes provide them with exceptional capacity for modulating multiple functions in the neural tissue. PMID:25938129

  16. Disentangling calcium-driven astrocyte physiology.

    PubMed

    Rusakov, Dmitri A

    2015-04-01

    Astrocytes seem to rely on relatively sluggish and spatially blurred Ca(2+) waves to communicate with fast and point-precise neural circuits. This apparent discrepancy could, however, reflect our current inability to understand the microscopic mechanisms involved. Difficulties in detecting and interpreting astrocyte Ca(2+) signals may have led to some prominent controversies in the field. Here, we argue that a deeper understanding of astrocyte physiology requires a qualitative leap in our experimental and analytical strategies.

  17. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    PubMed

    Korenić, Andrej; Boltze, Johannes; Deten, Alexander; Peters, Myriam; Andjus, Pavle; Radenović, Lidija

    2014-01-01

    Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m)) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m), visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m) during reperfusion, whereas GD caused a robust Δψ(m) negativation. In case no Δψ(m) negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m) hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψ(m) during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury. PMID:24587410

  18. Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation.

    PubMed

    Muhit, Md Abdul; Umehara, Kaoru; Noguchi, Hiroshi

    2016-09-01

    Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L-P (1-5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity. PMID:27425446

  19. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  20. Astrocytic Actions on Extrasynaptic Neuronal Currents

    PubMed Central

    Pál, Balázs

    2015-01-01

    In the last few decades, knowledge about astrocytic functions has significantly increased. It was demonstrated that astrocytes are not passive elements of the central nervous system (CNS), but active partners of neurons. There is a growing body of knowledge about the calcium excitability of astrocytes, the actions of different gliotransmitters and their release mechanisms, as well as the participation of astrocytes in the regulation of synaptic functions and their contribution to synaptic plasticity. However, astrocytic functions are even more complex than being a partner of the “tripartite synapse,” as they can influence extrasynaptic neuronal currents either by releasing substances or regulating ambient neurotransmitter levels. Several types of currents or changes of membrane potential with different kinetics and via different mechanisms can be elicited by astrocytic activity. Astrocyte-dependent phasic or tonic, inward or outward currents were described in several brain areas. Such currents, together with the synaptic actions of astrocytes, can contribute to neuromodulatory mechanisms, neurosensory and -secretory processes, cortical oscillatory activity, memory, and learning or overall neuronal excitability. This mini-review is an attempt to give a brief summary of astrocyte-dependent extrasynaptic neuronal currents and their possible functional significance. PMID:26696832

  1. Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes.

    PubMed

    Mares, V; Malík, R; Lisá, V; Sedo, A

    2005-05-20

    Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2'7'dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity. PMID:15893589

  2. Characterization of astrocyte reactivity and gene expression on biomaterials for neural electrodes.

    PubMed

    Ereifej, Evon S; Khan, Saida; Newaz, Golam; Zhang, Jinsheng; Auner, Gregory W; VandeVord, Pamela J

    2011-10-01

    Neural electrode devices hold great promise to help people with the restoration of lost functions. However, research is lacking in the biomaterial design of a stable, long-term device. Glial scarring is initiated when a device is inserted into brain tissue and an inflammatory response ensues. Astrocytes become hypertrophic, hyperplastic, and upregulate glial-fibrillary acidic protein. This study was designed to investigate the astrocyte proliferation, viability, morphology, and gene expression to assess the reactive state of the cells on different material surfaces. Although platinum and silicon have been extensively characterized both in vivo and in vitro for their biocompatibility with neuronal cells, this study used the novel usage of PMMA and SU-8 in neural electrodes by comparative analysis of materials' biocompatibility. This study has shown evidence of noncytotoxicity of SU-8. We have also confirmed the biocompatibility of PMMA with astrocytes. Moreover, we have established sound guidelines of which neural implant materials should meet to be depicted biocompatible. PMID:21812095

  3. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  4. Overexpression of PGC‑1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein.

    PubMed

    Shin, Sung-Won; Yun, Seong-Hoon; Park, Eun-Seon; Jeong, Jin-Sook; Kwak, Jong-Young; Park, Joo-In

    2015-03-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC‑1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC‑1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC‑1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC‑1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC‑1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC‑1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC‑1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC‑1α (PGC‑1α-HEK293 cells) compared to those expressing an empty vector. In PGC‑1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC‑1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC‑1α may promote cell proliferation and tumorigenesis through upregulation of ACBP

  5. Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

    PubMed

    Weihs, Anna M; Fuchs, Christiane; Teuschl, Andreas H; Hartinger, Joachim; Slezak, Paul; Mittermayr, Rainer; Redl, Heinz; Junger, Wolfgang G; Sitte, Harald H; Rünzler, Dominik

    2014-09-26

    Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.

  6. Astrocytes and Developmental White Matter Disorders

    ERIC Educational Resources Information Center

    Sen, Ellora; Levison, Steven W.

    2006-01-01

    There is an increasing awareness that the astrocytes in the immature periventricular white matter are vulnerable to ischemia and respond to inflammation. Here we provide a synopsis of the articles that have evaluated the causes and consequences of developmental brain injuries to white matter astrocytes as well as the consequences of several…

  7. Body protective compound-157 enhances alkali-burn wound healing in vivo and promotes proliferation, migration, and angiogenesis in vitro.

    PubMed

    Huang, Tonglie; Zhang, Kuo; Sun, Lijuan; Xue, Xiaochang; Zhang, Cun; Shu, Zhen; Mu, Nan; Gu, Jintao; Zhang, Wangqian; Wang, Yukun; Zhang, Yingqi; Zhang, Wei

    2015-01-01

    Chemical burns take up a high proportion of burns admissions and can penetrate deep into tissues. Various reagents have been applied in the treatment of skin chemical burns; however, no optimal reagent for skin chemical burns currently exists. The present study investigated the effect of topical body protective compound (BPC)-157 treatment on skin wound healing, using an alkali burn rat model. Topical treatment with BPC-157 was shown to accelerate wound closure following an alkali burn. Histological examination of skin sections with hematoxylin-eosin and Masson staining showed better granulation tissue formation, reepithelialization, dermal remodeling, and a higher extent of collagen deposition when compared to the model control group on the 18th day postwounding. BPC-157 could promote vascular endothelial growth factor expression in wounded skin tissues. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell cycle analysis demonstrated that BPC-157 enhanced the proliferation of human umbilical vein endothelial cells (HUVECs). Transwell assay and wound healing assay showed that BPC-157 significantly promoted migration of HUVECs. We also observed that BPC-157 upregulated the expression of VEGF-a and accelerated vascular tube formation in vitro. Moreover, further studies suggested that BPC-157 regulated the phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as its downstream targets, including c-Fos, c-Jun, and Egr-1, which are key molecules involved in cell growth, migration, and angiogenesis. Altogether, our results indicated that BPC-157 treatment may accelerate wound healing in a model of alkali burn-induced skin injury. The therapeutic mechanism may be associated with accelerated granulation tissue formation, reepithelialization, dermal remodeling, and collagen deposition through ERK1/2 signaling pathway. PMID:25995620

  8. Body protective compound-157 enhances alkali-burn wound healing in vivo and promotes proliferation, migration, and angiogenesis in vitro

    PubMed Central

    Huang, Tonglie; Zhang, Kuo; Sun, Lijuan; Xue, Xiaochang; Zhang, Cun; Shu, Zhen; Mu, Nan; Gu, Jintao; Zhang, Wangqian; Wang, Yukun; Zhang, Yingqi; Zhang, Wei

    2015-01-01

    Chemical burns take up a high proportion of burns admissions and can penetrate deep into tissues. Various reagents have been applied in the treatment of skin chemical burns; however, no optimal reagent for skin chemical burns currently exists. The present study investigated the effect of topical body protective compound (BPC)-157 treatment on skin wound healing, using an alkali burn rat model. Topical treatment with BPC-157 was shown to accelerate wound closure following an alkali burn. Histological examination of skin sections with hematoxylin–eosin and Masson staining showed better granulation tissue formation, reepithelialization, dermal remodeling, and a higher extent of collagen deposition when compared to the model control group on the 18th day postwounding. BPC-157 could promote vascular endothelial growth factor expression in wounded skin tissues. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell cycle analysis demonstrated that BPC-157 enhanced the proliferation of human umbilical vein endothelial cells (HUVECs). Transwell assay and wound healing assay showed that BPC-157 significantly promoted migration of HUVECs. We also observed that BPC-157 upregulated the expression of VEGF-a and accelerated vascular tube formation in vitro. Moreover, further studies suggested that BPC-157 regulated the phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as its downstream targets, including c-Fos, c-Jun, and Egr-1, which are key molecules involved in cell growth, migration, and angiogenesis. Altogether, our results indicated that BPC-157 treatment may accelerate wound healing in a model of alkali burn-induced skin injury. The therapeutic mechanism may be associated with accelerated granulation tissue formation, reepithelialization, dermal remodeling, and collagen deposition through ERK1/2 signaling pathway. PMID:25995620

  9. Inflammatory mediators alter the astrocyte transcriptome and calcium signaling elicited by multiple G-protein-coupled receptors.

    PubMed

    Hamby, Mary E; Coppola, Giovanni; Ao, Yan; Geschwind, Daniel H; Khakh, Baljit S; Sofroniew, Michael V

    2012-10-17

    Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2ry1, Gnao1, Gng7), but some up (for example, P2ry14, P2ry6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.

  10. Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells

    PubMed Central

    Chu, Tony L. H.; Guan, Qiunong; Nguan, Christopher Y. C.; Du, Caigan

    2015-01-01

    Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe’s equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity. PMID:26671563

  11. Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells.

    PubMed

    Chu, Tony L H; Guan, Qiunong; Nguan, Christopher Y C; Du, Caigan

    2015-01-01

    Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe's equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity. PMID:26671563

  12. Nitric Oxide in Astrocyte-Neuron Signaling

    SciTech Connect

    Nianzhen Li

    2002-06-27

    Astrocytes, a subtype of glial cell, have recently been shown to exhibit Ca{sup 2+} elevations in response to neurotransmitters. A Ca{sup 2+} elevation can propagate to adjacent astrocytes as a Ca{sup 2+} wave, which allows an astrocyte to communicate with its neighbors. Additionally, glutamate can be released from astrocytes via a Ca{sup 2+}-dependent mechanism, thus modulating neuronal activity and synaptic transmission. In this dissertation, the author investigated the roles of another endogenous signal, nitric oxide (NO), in astrocyte-neuron signaling. First the author tested if NO is generated during astrocytic Ca{sup 2+} signaling by imaging NO in purified murine cortical astrocyte cultures. Physiological concentrations of a natural messenger, ATP, caused a Ca{sup 2+}-dependent NO production. To test the roles of NO in astrocytic Ca{sup 2+} signaling, the author applied NO to astrocyte cultures via addition of a NO donor, S-nitrosol-N-acetylpenicillamine (SNAP). NO induced an influx of external Ca{sup 2+}, possibly through store-operated Ca{sup 2+} channels. The NO-induced Ca{sup 2+} signaling is cGMP-independent since 8-Br-cGMP, an agonistic analog of cGMP, did not induce a detectable Ca{sup 2+} change. The consequence of this NO-induced Ca{sup 2+} influx was assessed by simultaneously monitoring of cytosolic and internal store Ca{sup 2+} using fluorescent Ca{sup 2+} indicators x-rhod-1 and mag-fluo-4. Blockage of NO signaling with the NO scavenger PTIO significantly reduced the refilling percentage of internal stores following ATP-induced Ca{sup 2+} release, suggesting that NO modulates internal store refilling. Furthermore, locally photo-release of NO to a single astrocyte led to a Ca{sup 2+} elevation in the stimulated astrocyte and a subsequent Ca{sup 2+} wave to neighbors. Finally, the author tested the role of NO inglutamate-mediated astrocyte-neuron signaling by recording the astrocyte-evoked glutamate-dependent neuronal slow inward current (SIC

  13. Store-operated Ca2+ channels blockers inhibit lipopolysaccharide induced astrocyte activation.

    PubMed

    Li, Jian-Hua; Zhao, Shen-Ting; Wu, Cui-Ying; Cao, Xiong; Peng, Miao-Ru; Li, Shu-Ji; Liu, Xiao-Ai; Gao, Tian-Ming

    2013-10-01

    The destruction of calcium homeostasis is an important factor leading to neurological diseases. Store-operated Ca(2+) (SOC) channels are essential for Ca(2+) homeostasis in many cell types. However, whether SOC channels are involved in astrocyte activation induced by lipopolysaccharide (LPS) still remains unknown. In this study, we used LPS as an exogenous stimulation to investigate the role of SOC channels in astrocyte activation. Using calcium imaging technology, we first found that SOC channels blockers, 1-[h-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) and 2-aminoethyldiphenyl borate (2-APB), inhibited LPS induced [Ca(2+)]i increase, which prompted us to speculate that SOC channels may be involved in LPS induced astrocyte activation. Further experiments confirmed our speculation shown as SOC channels blockers inhibited LPS induced astrocyte activation characterized as cell proliferation by MTS and BrdU assay, raise in glial fibrillary acidic protein expression by immunofluorescence and Western Blot and secretion of interleukin 6 (IL-6) and interleukin 1β (IL-1β) by ELISA. So, our studies showed that SOC channels are involved in LPS-induced astrocyte activation.

  14. Fimbria-fornix (FF)-transected hippocampal extracts induce the activation of astrocytes in vitro.

    PubMed

    Zou, Linqing; Li, Haoming; Jin, Guohua; Tian, Meiling; Qin, Jianbing; Zhao, Heyan

    2014-03-01

    Hippocampus is one of the neurogenesis areas in adult mammals, but the function of astrocytes in this area is still less known. In our previous study, the fimbria-fornix (FF)-transected hippocampal extracts promoted the proliferation and neuronal differentiation of radial glial cells in vitro. To explore the effects of hippocampal extracts on gliogenesis, the hippocampal astrocytes were treated by normal or ff-transected hippocampal extracts in vitro. The cells were immunostained by brain lipid-binding protein (BLBP), nestin, and SOX2 to assess their state of activation. The effects of astrocyte-conditioned medium on the neuronal differentiation of hippocampal neural stem cells (NSCs) were also investigated. After treatment of FF-transected hippocampal extracts, the number of BLBP, nestin, and Sox-positive cells were obviously more than the cells which treated by normal hippocampal extracts, these cells maintained a state of activation and the activated astrocyte-conditioned medium also promoted the differentiation of NSCs into more neurons. These findings suggest that the astrocytes can be activated by FF-transected hippocampal extracts and these activated cells also can promote the neuronal differentiation of hippocampal NSCs in vitro.

  15. N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

    PubMed

    Mao, Gaowei; Goswami, Monali; Kalen, Amanda L; Goswami, Prabhat C; Sarsour, Ehab H

    2016-01-01

    The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.

  16. Postnatal Treadmill Exercise Alleviates Prenatal Stress-Induced Anxiety in Offspring Rats by Enhancing Cell Proliferation Through 5-Hydroxytryptamine 1A Receptor Activation

    PubMed Central

    2016-01-01

    Purpose: Stress during pregnancy is a risk factor for the development of anxiety-related disorders in offspring later in life. The effects of treadmill exercise on anxiety-like behaviors and hippocampal cell proliferation were investigated using rats exposed to prenatal stress. Methods: Exposure of pregnant rats to a hunting dog in an enclosed room was used to induce stress. Anxiety-like behaviors of offspring were evaluated using the elevated plus maze test. Immunohistochemistry for the detection of 5-bromo-2ʹ- deoxyuridine and doublecortin (DCX) in the hippocampal dentate gyrus and 5-hydroxytryptamine 1A receptors (5-HT1A) in the dorsal raphe was conducted. Brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) levels in the hippocampus were evaluated by western blot analysis. Results: Offspring of maternal rats exposed to stress during pregnancy showed anxiety-like behaviors. Offspring also showed reduced expression of BDNF, TrkB, and DCX in the dentate gyrus, decreased cell proliferation in the hippocampus, and reduced 5-HT1A expression in the dorsal raphe. Postnatal treadmill exercise by offspring, but not maternal exercise during pregnancy, enhanced cell proliferation and expression of these proteins. Conclusions: Postnatal treadmill exercise ameliorated anxiety-like behaviors in offspring of stressed pregnant rats, and the alleviating effect of exercise on these behaviors is hypothesized to result from enhancement of cell proliferation through 5-HT1A activation in offspring rats. PMID:27230461

  17. Large-scale recording of astrocyte activity

    PubMed Central

    Nimmerjahn, Axel; Bergles, Dwight E.

    2015-01-01

    Astrocytes are highly ramified glial cells found throughout the central nervous system (CNS). They express a variety of neurotransmitter receptors that can induce widespread chemical excitation, placing these cells in an optimal position to exert global effects on brain physiology. However, the activity patterns of only a small fraction of astrocytes have been examined and techniques to manipulate their behavior are limited. As a result, little is known about how astrocytes modulate CNS function on synaptic, microcircuit, or systems levels. Here, we review current and emerging approaches for visualizing and manipulating astrocyte activity in vivo. Deciphering how astrocyte network activity is controlled in different physiological and pathological contexts is critical for defining their roles in the healthy and diseased CNS. PMID:25665733

  18. Probing the Complexities of Astrocyte Calcium Signaling.

    PubMed

    Shigetomi, Eiji; Patel, Sandip; Khakh, Baljit S

    2016-04-01

    Astrocytes are abundant glial cells that tile the entire central nervous system and mediate well-established functions for neurons, blood vessels, and other glia. These ubiquitous cells display intracellular Ca(2+) signals, which have been intensely studied for 25 years. Recently, the use of improved methods has unearthed the panoply of astrocyte Ca(2+) signals and a variable landscape of basal Ca(2+) levels. In vivo studies have started to reveal the settings under which astrocytes display behaviorally relevant Ca(2+) signaling. Studies in mice have emphasized how astrocyte Ca(2+) signaling is altered in distinct neurodegenerative diseases. Progress in the past few years, fueled by methodological advances, has thus reignited interest in astrocyte Ca(2+) signaling for brain function and dysfunction. PMID:26896246

  19. Enhanced Functional Recovery in MRL/MpJ Mice after Spinal Cord Dorsal Hemisection

    PubMed Central

    Thuret, Sandrine; Thallmair, Michaela; Horky, Laura L.; Gage, Fred H.

    2012-01-01

    Adult MRL/MpJ mice have been shown to possess unique regeneration capabilities. They are able to heal an ear-punched hole or an injured heart with normal tissue architecture and without scar formation. Here we present functional and histological evidence for enhanced recovery following spinal cord injury (SCI) in MRL/MpJ mice. A control group (C57BL/6 mice) and MRL/MpJ mice underwent a dorsal hemisection at T9 (thoracic vertebra 9). Our data show that MRL/MpJ mice recovered motor function significantly faster and more completely. We observed enhanced regeneration of the corticospinal tract (CST). Furthermore, we observed a reduced astrocytic response and fewer micro-cavities at the injury site, which appear to create a more growth-permissive environment for the injured axons. Our data suggest that the reduced astrocytic response is in part due to a lower lesion-induced increase of cell proliferation post-SCI, and a reduced astrocytic differentiation of the proliferating cells. Interestingly, we also found an increased number of proliferating microglia, which could be involved in the MRL/MpJ spinal cord repair mechanisms. Finally, to evaluate the molecular basis of faster spinal cord repair, we examined the difference in gene expression changes in MRL/MpJ and C57BL/6 mice after SCI. Our microarray data support our histological findings and reveal a transcriptional profile associated with a more efficient spinal cord repair in MRL/MpJ mice. PMID:22348029

  20. Immunophilin Deficiency Augments Ca2+-Dependent Glutamate Release from Mouse Cortical Astrocytes

    PubMed Central

    Reyes, Reno C.; Perry, Giselle; Lesort, Mathieu; Parpura, Vladimir

    2010-01-01

    SUMMARY Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca2+ dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca2+ (Ca2+cyt) dynamics and consequential Ca2+-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif −/− displayed reduced mechanically-induced Ca2+cyt increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca2+ buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca2+cyt dynamics, but also to an enhanced Ca2+-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse. PMID:21163525

  1. Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.

    PubMed

    Dieb, W; Hafidi, A

    2013-09-01

    Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine.

  2. The Role of Astrocytes in Mediating Exogenous Cell-Based Restorative Therapy for Stroke

    PubMed Central

    Li, Yi; Liu, Zhongwu; Xin, Hongqi; Chopp, Michael

    2014-01-01

    Astrocytes have not been a major therapeutic target for the treatment of stroke, with most research emphasis on the neuron. Given the essential role that astrocytes play in maintaining physiological function of the central nervous system and the very rapid and sensitive reaction astrocytes have in response to cerebral injury or ischemic insult, we propose to replace the neurocentric view for treatment with a more nuanced astrocytic centered approach. In addition, after decades of effort in attempting to develop neuroprotective therapies, which target reduction of the ischemic lesion, there are no effective clinical treatments for stroke, aside from thrombolysis with tissue plasminogen activator, which is used in a small minority of patients. A more promising therapeutic approach, which may affect nearly all stroke patients, may be in promoting endogenous restorative mechanisms, which enhance neurological recovery. A focus of efforts in stimulating recovery post stroke is the use of exogenously administered cells. The present review focuses on the role of the astrocyte in mediating the brain network, brain plasticity, and neurological recovery post stroke. As a model to describe the interaction of a restorative cell-based therapy with astrocytes, which drives recovery from stroke, we specifically highlight the subacute treatment of stroke with multipotent mesenchymal stromal cell therapy. PMID:24272702

  3. Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

    PubMed Central

    Uwechue, Nneka M; Marx, Mari-Carmen; Chevy, Quentin; Billups, Brian

    2012-01-01

    Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins l-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal dl-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes. PMID:22411007

  4. Histone methylation patterns in astrocytes are influenced by age following ischemia.

    PubMed

    Chisholm, Nioka C; Henderson, Michael L; Selvamani, Amutha; Park, Min Jung; Dindot, Scott; Miranda, Rajesh C; Sohrabji, Farida

    2015-01-01

    In animal models, middle-aged females sustain greater ischemia-induced infarction as compared to adult females. This age difference in infarct severity is associated with reduced functional capacity of astrocytes, a critical neural support cell. The impaired response of astrocytes following stroke in middle-aged females may be related to epigenetic alterations, including histone acetylation or methylation. The present study measured the activity of enzymes that regulate histone acetylation and methylation in cerebral cortical astrocytes of adult (6 month) and middle-aged (11+ month) female rats 48 h following middle cerebral artery occlusion. H3K4 histone methyltransferase activity was decreased in astrocytes from middle-aged females. The next experiment therefore examined H3K4me3 (transcriptional enhancer) and H3K9me3 (transcriptional repressor) in astrocytes from adult and middle-aged females using ChIP-seq analysis. Adult females had more enriched H3K4me3 peaks (304 vs. 26) at transcriptional start sites and fewer H3K9me3 enriched peaks than middle-aged females (4 vs. 22), indicating a pattern of less active chromatin in astrocytes in the older group following ischemia. DAVID clustering analysis of H3K4me3 enriched genes found several functional categories, including cell motility, regulation of apoptosis and the vascular endothelial growth factor (VEGF) pathway. H3K4me3 was enriched at the miR-17-20 cluster and VEGFa, and analysis of a separate set of astrocytes confirmed that VEGF protein expression and miR-20 mRNA expression were significantly greater following ischemia in adult females compared to middle-aged females. These data indicate that astrocytes display less active chromatin with aging and provide new insight into possible mechanisms for differences in stroke severity observed during aging. PMID:25565250

  5. CYP2S1 depletion enhances colorectal cell proliferation is associated with PGE2-mediated activation of β-catenin signaling

    SciTech Connect

    Yang, Chao; Li, Changyuan; Li, Minle; Tong, Xuemei; Hu, Xiaowen; Yang, Xuhan; Yan, Xiaomei; He, Lin; Wan, Chunling

    2015-02-15

    Colorectal epithelial cancer is one of the most common cancers in the world and its 5-year survival rate is still relatively low. Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in the oxidative metabolism of a wide range of xenobiotics, including (pro-)carcinogens and endogenous compounds. Although CYP2S1, a member of CYP family, strongly expressed in many extrahepatic tissues, the role of CYP2S1 in cancer remains unclear. To investigate whether CYP2S1 involves in colorectal carcinogenesis, cell proliferation was analyzed in HCT116 cells depleted of CYP2S1 using small hairpin interfering RNA. Our data show that CYP2S1 knockdown promotes cell proliferation through increasing the level of endogenous prostaglandin E2(PGE2). PGE2, in turn, reduces phosphorylation of β-catenin and activates β-catenin signaling, which contributes to the cell proliferation. Furthermore, CYP2S1 knockdown increase tumor growth in xenograft mouse model. In brief, these results demonstrate that CYP2S1 regulates colorectal cancer growth through associated with PGE2-mediated activation of β-catenin signaling. - Highlights: • Knockdown of CYP2S1 expression improve HCT116 cell proliferation in vitro and in vivo. • Elevate PGE2 production in CYP2S1 knockdown cell is associated with its proliferation. • Elevate PGE2 level in CYP2S1 knockdown cells enhance β-catenin accumulation. • β-catenin activate TCF/LEF and target gene expression thus promote cell proliferation.

  6. Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen.

    PubMed

    Wang, Jianling; Wang, Gangduo; Ma, Huaxian; Khan, M Firoze

    2011-01-15

    Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in

  7. Low-level Ga-Al-As laser irradiation enhances osteoblast proliferation through activation of Hedgehog signaling pathway

    NASA Astrophysics Data System (ADS)

    Li, Qiushi; Qu, Zhou; Chen, Yingxin; Liu, Shujie; Zhou, Yanmin

    2014-12-01

    Low-level laser irradiation has been reported to promote bone formation, but the molecular mechanism is still unclear. Hedgehog signaling pathway has been reported to play an important role in promoting bone formation. The aim of the present study was to examine whether low-level Ga-Al-As laser (808 nm) irradiation could have an effect on Hedgehog signaling pathway during osteoblast proliferation in vitro. Mouse osteoblastic cell line MC3T3-E1 was cultured in vitro. The cultures after laser irradiation (3.75J/cm2) were treated with recombinant N-terminals Sonic Hedgehog (N-Shh)or Hedgehog inhibitor cyclopamine (cy). The experiment was divided into 4 group, group 1:laser irradiation, group 2: laser irradiation and N-Shh, group 3: laser irradiation and cy, group 4:control with no laser irradiation. On day 1,2 and 3,cell proliferation was determined by cell counting, Cell Counting Kit-8.On 12 h and 24 h, cell cycle was detected by flow cytometry. Proliferation activity of laser irradiation and N-Shh group was remarkably increased compared with those of laser irradiation group. Proliferation activity of laser irradiation and cy group was remarkably decreased compared with those of laser irradiation group, however proliferation activity of laser irradiation and cy group was remarkably increased compared with those of control group. These results suggest that low-level Ga-Al-As laser irradiation activate Hedgehog signaling pathway during osteoblast proliferation in vitro. Hedgehog signaling pathway is one of the signaling pathways by which low-level Ga-Al-As laser irradiation regulates osteoblast proliferation.

  8. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    SciTech Connect

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline; Sun, Jianmin; Jögi, Annika; Neumann, Drorit; Rönnstrand, Lars; Påhlman, Sven

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  9. Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen

    SciTech Connect

    Wang Jianling; Wang Gangduo; Ma Huaxian; Khan, M. Firoze

    2011-01-15

    Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in

  10. The pathophysiological role of astrocytic endothelin-1.

    PubMed

    Hostenbach, Stéphanie; D'haeseleer, Miguel; Kooijman, Ron; De Keyser, Jacques

    2016-09-01

    In the normal central nervous system, endothelin-1 (ET-1) is found in some types of neurons, epithelial cells of the choroid plexus, and endothelial cells of microvessels, but it is usually not detectable in glial cells. However, in different pathological conditions, astrocytes adapting a reactive phenotype express high levels of ET-1 and its receptors, mainly the ETB receptor. ET-1 released by reactive astrocytes appears mainly to have neurodeleterious effects by mechanisms that include constriction of cerebral arterioles leading to impairment of the cerebral microcirculation, increase of blood brain barrier permeability, inflammation, excitotoxicity, impairment of fast axonal transport, and astrogliosis. A few studies in rodents found that ET-1 increased the astrocytic expression of brain-derived neurotrophic factor, glial cell-line derived neurotrophic factor and neurotropin-3, and the production of endocannabinoids. However, whether this occurs in physiological or pathological conditions is unclear. This review summarizes current knowledge about the role of the astrocytic ET-1 system in acute and chronic neurological conditions, including multiple sclerosis, ischemic stroke and hypoxic/ischemic brain injury, traumatic brain injury, subarachnoid hemorrhage, Alzheimer's disease, Binswanger's disease and post-stroke dementia, amyotrophic lateral sclerosis, and CNS infections. Counteracting the harmful effects of astrocytic ET-1 may represent a promising therapeutic target for mitigating secondary brain damage in a variety of neurological diseases. We also briefly address the role of astrocytic ET-1 in astrocytic tumors and pain. PMID:27132521

  11. Astrocytes regulate cortical state switching in vivo.

    PubMed

    Poskanzer, Kira E; Yuste, Rafael

    2016-05-10

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation-dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  12. ASTROCYTES: EMERGING STARS IN LEUKODYSTROPHY PATHOGENESIS

    PubMed Central

    Lanciotti, Angela; Brignone, Maria Stefania; Bertini, Enrico; Petrucci, Tamara C.; Aloisi, Francesca; Ambrosini, Elena

    2013-01-01

    Astrocytes are the predominant glial cell population in the central nervous system (CNS). Once considered only passive scaffolding elements, astrocytes are now recognised as cells playing essential roles in CNS development and function. They control extracellular water and ion homeostasis, provide substrates for energy metabolism, and regulate neurogenesis, myelination and synaptic transmission. Due to these multiple activities astrocytes have been implicated in almost all brain pathologies, contributing to various aspects of disease initiation, progression and resolution. Evidence is emerging that astrocyte dysfunction can be the direct cause of neurodegeneration, as shown in Alexander’s disease where myelin degeneration is caused by mutations in the gene encoding the astrocyte-specific cytoskeleton protein glial fibrillary acidic protein. Recent studies point to a primary role for astrocytes in the pathogenesis of other genetic leukodystrophies such as megalencephalic leukoencephalopathy with subcortical cysts and vanishing white matter disease. The aim of this review is to summarize current knowledge of the pathophysiological role of astrocytes focusing on their contribution to the development of the above mentioned leukodystrophies and on new perspectives for the treatment of neurological disorders. PMID:24340223

  13. Astrocytes regulate cortical state switching in vivo

    PubMed Central

    Poskanzer, Kira E.; Yuste, Rafael

    2016-01-01

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation–dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  14. Artificial astrocytes improve neural network performance.

    PubMed

    Porto-Pazos, Ana B; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  15. Artificial Astrocytes Improve Neural Network Performance

    PubMed Central

    Porto-Pazos, Ana B.; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  16. Opiates selectively increase intracellular calcium in developing type-1 astrocytes: role of calcium in morphine-induced morphologic differentiation.

    PubMed

    Stiene-Martin, A; Mattson, M P; Hauser, K F

    1993-12-17

    Endogenous opioids and opiate drugs inhibit nervous system maturation, in part, by affecting the growth of astrocytes. Opiates inhibit astrocyte proliferation and cause premature differentiation. The emerging importance of Ca2+ in astrocyte function prompted us to explore whether opiates might affect astrocyte development by altering Ca2+ homeostasis. Astrocyte-enriched cultures were derived from newborn ICR mouse cerebra. Quantitative fluorescent measurements of intracellular free Ca2+ ([Ca2+]i) using Fura-2 as well as fluo-3 and computer-aided image analysis showed that 1 microM morphine significantly increased [Ca2+]i in flat, polyhedral, glial fibrillary acidic protein (GFAP) immunoreactive astrocytes at 2 and 6 min, and at 72 h. Co-administration of 3 microM naloxone blocked morphine-dependent increases in [Ca2+]i. Treatment with 1 microM concentrations of the kappa-opioid receptor agonist, U69,593, but not equimolar amounts of mu ([D-Ala2,MePhe4,Gly(ol)5]enkephalin)- or delta ([D-Pen2,D-Pen5]enkephalin)-opioid receptor agonists, significantly increased [Ca2+]i in astrocytes. To assess the role of Ca2+ in morphine-induced astrocyte differentiation, untreated and 1 microM morphine-treated astrocyte cultures were incubated for 5 days in < 0.01, 0.3, 1.0, or 3.0 mM extracellular Ca2+ ([Ca2+]o), or incubated with 1.0 mM [Ca2+]o in the presence of 1 microM of the Ca2+ ionophore, A23187. The areas of single astrocytes were measured and there was a positive correlation between astrocyte area and [Ca2+]o. Morphine had an additive effect on area and form factor measures when [Ca2+]o was 1.0 mM. High [Ca2+]o (3.0 mM) alone mimicked the action of morphine. Morphine alone had no effect on astrocyte area in the presence of 3.0 mM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    SciTech Connect

    Boku, Shuken; Nakagawa, Shin; Takamura, Naoki; Kato, Akiko; Takebayashi, Minoru; Hisaoka-Nakashima, Kazue; Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  18. The lymphocyte secretome from young adults enhances skeletal muscle proliferation and migration, but effects are attenuated in the secretome of older adults

    PubMed Central

    Al-Dabbagh, Sarah; McPhee, Jamie S; Murgatroyd, Christopher; Butler-Browne, Gillian; Stewart, Claire E; Al-Shanti, Nasser

    2015-01-01

    Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells which can be reversed by exposure to young blood. To investigate the role of immune cells in muscle regeneration, we isolated lymphocytes from whole blood of young and older healthy volunteers and cultured them with, or without, anti-CD3/CD28 activators to induce release of cytokines, interleukins, and growth factors into the media. The secreted proteins were collected to prepare a conditioned media, which was subsequently used to culture C2C12 myoblasts. The conditioned media from the activated young lymphocytes increased the rate of proliferation of myoblasts by around threefold (P < 0.005) and caused an approximate fourfold (P < 0.005) increase in migration compared with nonactivated lymphocyte control media. These responses were characterized by minimal myotube formation (2%), low fusion index (5%), low myosin heavy chain content, and substantial migration. In contrast, myoblasts treated with conditioned media from activated old lymphocytes exhibited a high degree of differentiation, and multi-nucleated myotube formation that was comparable to control conditions, thus showing no effect on proliferation or migration of myoblasts. These results indicate that secreted proteins from lymphocytes of young people enhance the muscle cell proliferation and migration, whereas secreted proteins from lymphocytes of older people may contribute to the attenuated skeletal muscle satellite cell proliferation and migration. PMID:26603449

  19. The lymphocyte secretome from young adults enhances skeletal muscle proliferation and migration, but effects are attenuated in the secretome of older adults.

    PubMed

    Al-Dabbagh, Sarah; McPhee, Jamie S; Murgatroyd, Christopher; Butler-Browne, Gillian; Stewart, Claire E; Al-Shanti, Nasser

    2015-11-01

    Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells which can be reversed by exposure to young blood. To investigate the role of immune cells in muscle regeneration, we isolated lymphocytes from whole blood of young and older healthy volunteers and cultured them with, or without, anti-CD3/CD28 activators to induce release of cytokines, interleukins, and growth factors into the media. The secreted proteins were collected to prepare a conditioned media, which was subsequently used to culture C2C12 myoblasts. The conditioned media from the activated young lymphocytes increased the rate of proliferation of myoblasts by around threefold (P < 0.005) and caused an approximate fourfold (P < 0.005) increase in migration compared with nonactivated lymphocyte control media. These responses were characterized by minimal myotube formation (2%), low fusion index (5%), low myosin heavy chain content, and substantial migration. In contrast, myoblasts treated with conditioned media from activated old lymphocytes exhibited a high degree of differentiation, and multi-nucleated myotube formation that was comparable to control conditions, thus showing no effect on proliferation or migration of myoblasts. These results indicate that secreted proteins from lymphocytes of young people enhance the muscle cell proliferation and migration, whereas secreted proteins from lymphocytes of older people may contribute to the attenuated skeletal muscle satellite cell proliferation and migration. PMID:26603449

  20. Cholesterol depletion by methyl-β-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures.

    PubMed

    Portilho, Débora M; Soares, Carolina P; Morrot, Alexandre; Thiago, Leandro S; Butler-Browne, Gillian; Savino, Wilson; Costa, Manoel L; Mermelstein, Cláudia

    2012-11-01

    Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway.

  1. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

    PubMed Central

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K.; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O.; Wagner, Mary B.; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  2. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells.

    PubMed

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O; Wagner, Mary B; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  3. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

    PubMed Central

    Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

    2015-01-01

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma. PMID:25971279

  4. RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.

    PubMed

    Wang, Zhixing; Coleman, Daniel J; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K

    2011-01-01

    We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

  5. Multiple myeloma cell-derived microvesicles are enriched in CD147 expression and enhance tumor cell proliferation

    PubMed Central

    Arendt, Bonnie K.; Walters, Denise K.; Wu, Xiaosheng; Tschumper, Renee C.; Jelinek, Diane F.

    2014-01-01

    Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells within the bone marrow. There is a growing literature that tumor cells release biologically active microvesicles (MVs) that modify both local and distant microenvironments. In this study, our goals were to determine if MM cells release MVs, and if so, begin to characterize their biologic activity. Herein we present clear evidence that not only do both patient MM cells and human MM cell lines (HMCLs) release MVs, but that these MVs stimulate MM cell growth. Of interest, MM-derived MVs were enriched with the biologically active form of CD147, a transmembrane molecule previously shown by us to be crucial for MM cell proliferation. Using MVs isolated from HMCLs stably transfected with a CD147-GFP fusion construct (CD147GFP), we observed binding and internalization of MV-derived CD147 with HMCLs. Cells with greater CD147GFP internalization proliferated at a higher rate than did cells with less CD147GFP association. Lastly, MVs obtained from CD147 downregulated HMCLs were attenuated in their ability to stimulate HMCL proliferation. In summary, this study demonstrates the significance of MV shedding and MV-mediated intercellular communication on malignant plasma cell proliferation, and identifies the role of MV-enriched CD147 in this process. PMID:25015330

  6. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    PubMed Central

    Wang, Yuan-Yi; Zhu, Qing-San; Wang, Yi-Wei; Yin, Ruo-Feng

    2015-01-01

    Background: Thymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation. Methods: TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage. Results: NP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group. Conclusions: TB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases. PMID:26021512

  7. Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

    PubMed

    Song, SungWon; Miranda, Carlos J; Braun, Lyndsey; Meyer, Kathrin; Frakes, Ashley E; Ferraiuolo, Laura; Likhite, Shibi; Bevan, Adam K; Foust, Kevin D; McConnell, Michael J; Walker, Christopher M; Kaspar, Brian K

    2016-04-01

    Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity. PMID:26928464

  8. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression.

  9. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression. PMID:24909096

  10. Brief alcohol exposure alters transcription in astrocytes via the heat shock pathway

    PubMed Central

    Pignataro, Leonardo; Varodayan, Florence P; Tannenholz, Lindsay E; Protiva, Petr; Harrison, Neil L

    2013-01-01

    Astrocytes are critical for maintaining homeostasis in the central nervous system (CNS), and also participate in the genomic response of the brain to drugs of abuse, including alcohol. In this study, we investigated ethanol regulation of gene expression in astrocytes. A microarray screen revealed that a brief exposure of cortical astrocytes to ethanol increased the expression of a large number of genes. Among the alcohol-responsive genes (ARGs) are glial-specific immune response genes, as well as genes involved in the regulation of transcription, cell proliferation, and differentiation, and genes of the cytoskeleton and extracellular matrix. Genes involved in metabolism were also upregulated by alcohol exposure, including genes associated with oxidoreductase activity, insulin-like growth factor signaling, acetyl-CoA, and lipid metabolism. Previous microarray studies performed on ethanol-treated hepatocyte cultures and mouse liver tissue revealed the induction of almost identical classes of genes to those identified in our microarray experiments, suggesting that alcohol induces similar signaling mechanisms in the brain and liver. We found that acute ethanol exposure activated heat shock factor 1 (HSF1) in astrocytes, as demonstrated by the translocation of this transcription factor to the nucleus and the induction of a family of known HSF1-dependent genes, the heat shock proteins (Hsps). Transfection of a constitutively transcriptionally active Hsf1 construct into astrocytes induced many of the ARGs identified in our microarray study supporting the hypothesis that HSF1 transcriptional activity, as part of the heat shock cascade, may mediate the ethanol induction of these genes. These data indicate that acute ethanol exposure alters gene expression in astrocytes, in part via the activation of HSF1 and the heat shock cascade. PMID:23533150

  11. Astrocytes: The missing link in neurological disease?

    PubMed Central

    Lin, Chia-Ching John; Deneen, Benjamin

    2013-01-01

    The central nervous system (CNS) is comprised of numerous cell types that work in concert to facilitate proper function and homeostasis. Disruption of these carefully orchestrated networks results in neuronal dysfunction, manifesting itself in a variety of neurological disorders. While neuronal dysregulation is causative of symptoms manifest in the clinic, the etiology of these disorders is often more complex than simply a loss of neurons or intrinsic dysregulation of their function. In the adult brain, astrocytes comprise the most abundant cell type and play key roles in CNS physiology, therefore it stands to reason that dysregulation of normal astrocyte function contributes to the etiology and progression of varied neurological disorders. We review here some neurological disorders associated with an astrocyte factor and discuss how the related astrocyte dysfunction contributes to the etiology and/or progression of these disorders. PMID:24365571

  12. Psoralidin inhibits proliferation and enhances apoptosis of human esophageal carcinoma cells via NF-κB and PI3K/Akt signaling pathways

    PubMed Central

    Jin, Zhiliang; Yan, Wei; Jin, Hui; Ge, Changzheng; Xu, Yanhua

    2016-01-01

    Esophageal cancer is the most common gastrointestinal cancer. Psoralidin exhibits antioxidant, anti-apoptotic, anti-inflammatory and antitumor effects, which result in the inhibition of cancer formation. The present study aimed to investigate the effect of psoralidin on esophageal carcinoma proliferation and growth, and to elucidate its underlying mechanism of action. The effect of psoralidin on cell proliferation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and 4′,6-diamidino-2-phenylindole staining assay, the present study demonstrated that psoralidin significantly enhanced apoptosis of human esophageal carcinoma Eca9706 cells. In addition, caspase-3 activity was analyzed with a caspase-3 colorimetric assay kit, while nuclear factor (NF)-κB activity and protein phosphatidylinositol 3-kinase (PI3K)/Akt expression were measured with an NF-κB enzyme-linked immunosorbent assay kit and western blot analysis, respectively. Eca9706 cells were treated with a PI3K agonist in order to investigate the mechanism of action of psoralidin. It was observed that psoralidin was able to decrease the proliferation and promote the cellular apoptosis of Eca9706 cells in a dose-dependent manner. Furthermore, psoralidin was also able to inhibit the caspase-3 activity of Eca9706 cells in a dose-dependent manner. In addition, psoralidin inhibited NF-κB activity and reduced PI3K and Akt protein expression in Eca9706 cells. Notably, the PI3K agonist was able to reverse the effect of psoralidin on Eca9706 cells. The results of the present study demonstrated that psoralidin was able to inhibit proliferation and enhance apoptosis of human esophageal carcinoma cells via the NF-κB and PI3K/Akt signaling pathways. PMID:27446379

  13. Astrocytes as Gatekeepers of GABAB Receptor Function

    PubMed Central

    Beenhakker, Mark P.; Huguenard, John R.

    2010-01-01

    The long-lasting actions of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) result from the activation of metabotropic GABAB receptors. Enhanced GABAB-mediated inhibitory postsynaptic currents (IPSCs) are critical for the generation of generalized thalamocortical seizures. Here, we demonstrate that GABAB-mediated inhibitory postsynaptic currents (IPSCs) recorded in the thalamus are largely defined by GABA diffusion and activation of distal extrasynaptic receptors potentially up to tens of μm from synapses. We also show that this diffusion is differentially regulated by two astrocytic GABA transporters, GAT1 and GAT3, which are localized near and far from synapses, respectively. A biologically constrained model of GABA diffusion and uptake shows how the two GATs differentially modulate amplitude and duration of GABAB IPSCs. Specifically, the perisynaptic expression of GAT1 enables it to regulate GABA levels near synapses and selectively modulate peak IPSC amplitude, which is largely dependent on perisynaptic receptor occupancy. GAT3 expression, on the other hand, is broader and includes distal extrasynaptic regions. As such, GAT3 acts as a gatekeeper to prevent diffusion of GABA away from synapses towards extrasynaptic regions that contain a potentially enormous pool of GABAB receptors. Targeting this gatekeeper function may provide new pharmacotherapeutic opportunities to prevent the excessive GABAB receptor activation that appears necessary for thalamic seizure generation. PMID:21068331

  14. Modulation of neuritogenesis by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; Costa, Lucio G

    2008-11-14

    Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

  15. Ammonia-induced miRNA expression changes in cultured rat astrocytes.

    PubMed

    Oenarto, Jessica; Karababa, Ayse; Castoldi, Mirco; Bidmon, Hans J; Görg, Boris; Häussinger, Dieter

    2016-01-01

    Hepatic encephalopathy is a neuropsychiatric syndrome evolving from cerebral osmotic disturbances and oxidative/nitrosative stress. Ammonia, the main toxin of hepatic encephalopathy, triggers astrocyte senescence in an oxidative stress-dependent way. As miRNAs are critically involved in cell cycle regulation and their expression may be regulated by oxidative stress, we analysed, whether astrocyte senescence is a consequence of ammonia-induced miRNA expression changes. Using a combined miRNA and gene microarray approach, 43 miRNA species which were downregulated and 142 genes which were upregulated by NH4Cl (5 mmol/l, 48 h) in cultured rat astrocytes were found. Ammonia-induced miRNA and gene expression changes were validated by qPCR and 43 potential miRNA target genes, including HO-1, were identified by matching upregulated mRNA species with predicted targets of miRNA species downregulated by ammonia. Inhibition of HO-1 targeting miRNAs which were downregulated by NH4Cl strongly upregulated HO-1 mRNA and protein levels and inhibited astrocyte proliferation in a HO-1-dependent way. Preventing ammonia-induced upregulation of HO-1 by taurine (5 mmol/l) as well as blocking HO-1 activity by tin-protoporphyrine IX fully prevented ammonia-induced proliferation inhibition and senescence. The data suggest that ammonia induces astrocyte senescence through NADPH oxidase-dependent downregulation of HO-1 targeting miRNAs and concomitant upregulation of HO-1 at both mRNA and protein level. PMID:26755400

  16. Reduction of myeloid suppressor cell derived nitric oxide provides a mechanistic basis of lead enhancement of alloreactive CD4{sup +} T cell proliferation

    SciTech Connect

    Farrer, David G.; Hueber, Sara; Laiosa, Michael D.; Eckles, Kevin G.; McCabe, Michael J.

    2008-06-01

    The persistent environmental toxicant and immunomodulator, lead (Pb), has been proposed to directly target CD4{sup +} T cells. However, our studies suggest that CD4{sup +} T cells are an important functional, yet indirect target. In order to identify the direct target of Pb in the immune system and the potential mechanism of Pb-induced immunotoxicity, myeloid suppressor cells (MSCs) were evaluated for their ability to modulate CD4{sup +} T cell proliferation after Pb exposure. Myeloid suppressor cells regulate the adaptive immune response, in part, by inhibiting the proliferation of CD4{sup +} T cells. It is thought that the mechanism of MSC-dependent regulation involves the release of the bioactive gas, nitric oxide (NO), blocking cell signaling cascades downstream of the IL-2 receptor and thus preventing T cells from entering cell-cycle. In mixed lymphocyte culture (MLC), increasing numbers of MSCs suppressed T cell proliferation in a dose-dependent manner, and this suppression is strikingly abrogated with 5 {mu}M lead (Pb) treatment. The Pb-sensitive MSC population is CD11b{sup +}, GR1{sup +}and CD11c{sup -} and thus phenotypically consistent with MSCs described in other literature. Inhibition of NO-synthase (NOS), the enzyme responsible for the production of NO, enhanced alloreactive T cell proliferation in MLC. Moreover, Pb attenuated NO production in MLC, and exogenous replacement of NO restored suppression in the presence of Pb. Significantly, MSC from iNOS-/- mice were unable to suppress T cell proliferation. An MSC-derived cell line (MSC-1) also suppressed T cell proliferation in MLC, and Pb disrupted this suppression by attenuating NO production. Additionally, Pb disrupted NO production in MSC-1 cells in response to treatment with interferon-{gamma} (IFN-{gamma}) and LPS or in response to concanavalin A-stimulated splenocytes. However, neither the abundance of protein nor levels of mRNA for the inducible isoform of NOS (iNOS) were altered with Pb

  17. Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells.

    PubMed

    Nakanishi, Masaya; Niidome, Tetsuhiro; Matsuda, Satoru; Akaike, Akinori; Kihara, Takeshi; Sugimoto, Hachiro

    2007-02-01

    Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. It has been recognized that astrocytes promote neurogenic differentiation of NSPCs, suggesting the importance of cell-cell interactions between glial cells and NSPCs. Recent studies have demonstrated that microglia, one type of glial cells, play an important role in neurogenesis. However, little is known about how activated microglia control the proliferation and differentiation of NSPCs. In this study, we investigated the possibility that microglia-derived soluble factors regulate the behaviour of NSPCs. To this end, NSPCs and microglial cultures were obtained from rat embryonic day 16 subventricular zone (SVZ) and rat postnatal 1 day cortex, respectively, and the conditioned medium from microglia was prepared. Microglial-conditioned medium had no significant effect on the proliferation of NSPCs. In contrast, it increased the percentage of cells positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP) during differentiation. The induction of astrocytic differentiation by microglial-conditioned medium was reduced by the inhibition of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, microglia-derived interleukin (IL)-6 and leukaemia inhibitory factor (LIF) were identified as essential molecules for this astrocytic differentiation using neutralizing antibodies and recombinant cytokines. Our results suggest that microglia as well as astrocytes contribute to the integrity of the local environment of NSPCs, and at least IL-6 and LIF released by activated microglia promote astrocytic differentiation of NSPCs via the activation of the JAK/STAT and MAPK pathways.

  18. Astrocytic vesicle mobility in health and disease.

    PubMed

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  19. Astrocytic Vesicle Mobility in Health and Disease

    PubMed Central

    Potokar, Maja; Vardjan, Nina; Stenovec, Matjaž; Gabrijel, Mateja; Trkov, Saša; Jorgačevski, Jernej; Kreft, Marko; Zorec, Robert

    2013-01-01

    Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions. PMID:23712361

  20. Astrocyte Aquaporin Dynamics in Health and Disease.

    PubMed

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  1. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    PubMed

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  2. Astrocyte Aquaporin Dynamics in Health and Disease

    PubMed Central

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  3. Peroxisome proliferator-activated receptor γ regulates genes involved in insulin/insulin-like growth factor signaling and lipid metabolism during adipogenesis through functionally distinct enhancer classes.

    PubMed

    Oger, Frédérik; Dubois-Chevalier, Julie; Gheeraert, Céline; Avner, Stéphane; Durand, Emmanuelle; Froguel, Philippe; Salbert, Gilles; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-10

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPAR induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process.

  4. Peroxisome Proliferator-activated Receptor γ Regulates Genes Involved in Insulin/Insulin-like Growth Factor Signaling and Lipid Metabolism during Adipogenesis through Functionally Distinct Enhancer Classes*

    PubMed Central

    Oger, Frédérik; Dubois-Chevalier, Julie; Gheeraert, Céline; Avner, Stéphane; Durand, Emmanuelle; Froguel, Philippe; Salbert, Gilles; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2014-01-01

    The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPARγ induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPARγ also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPARγ utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process. PMID:24288131

  5. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

    PubMed

    Anderova, Miroslava; Benesova, Jana; Mikesova, Michaela; Dzamba, David; Honsa, Pavel; Kriska, Jan; Butenko, Olena; Novosadova, Vendula; Valihrach, Lukas; Kubista, Mikael; Dmytrenko, Lesia; Cicanic, Michal; Vargova, Lydia

    2014-01-01

    Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+) and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+) clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K(+). As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+), α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+). PMID:25426721

  6. Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

    PubMed Central

    Anderova, Miroslava; Benesova, Jana; Mikesova, Michaela; Dzamba, David; Honsa, Pavel; Kriska, Jan; Butenko, Olena; Novosadova, Vendula; Valihrach, Lukas; Kubista, Mikael; Dmytrenko, Lesia; Cicanic, Michal; Vargova, Lydia

    2014-01-01

    Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K+. PMID:25426721

  7. Inhibition of cyclin-dependent kinase 6 suppresses cell proliferation and enhances radiation sensitivity in medulloblastoma cells

    PubMed Central

    Harris, Peter S.; Venkataraman, Sujatha; Alimova, Irina; Birks, Diane K.; Donson, Andrew M.; Foreman, Nicholas K.; Vibhakar, Rajeev

    2015-01-01

    Medulloblastoma accounts for 20 % of all primary pediatric intracranial tumors. Current treatment cures 50–80 % of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase 6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation. PMID:23138228

  8. pH-Sensitive K(+) Currents and Properties of K2P Channels in Murine Hippocampal Astrocytes.

    PubMed

    Weller, Johannes; Steinhäuser, Christian; Seifert, Gerald

    2016-01-01

    Based on their intimate spatial association with synapses and the capillary, astrocytes are critically involved in the control of ion, transmitter, and energy homeostasis as well as regulation of the cerebral blood flow. Under pathophysiological conditions, dysfunctional astrocytes can no longer assure homeostatic control although the underlying mechanisms are poorly understood. Specifically, neurological diseases are often accompanied by acidification of the extracellular space, but the properties of astrocytes in such an acidic environment are still a matter of debate. To meet the homeostatic requirements, astrocytes are equipped with intercellular gap junctions, inwardly rectifying K(+) (Kir) channels, and two-pore domain K(+) (K2P) channels. One goal of the present study was to overview current knowledge about astrocyte K(+) channel function during acidosis. In addition, we combined functional and molecular analyses to clarify how low pH affects K(+) channel function in astrocytes freshly isolated from the developing mouse hippocampus. Extracellular acidification led to a decrease of K(+) currents in astrocytes, probably due to modulation of Kir4.1 channels. After blocking Kir4.1 channels, low pH enhanced K(+) current amplitudes. This current activation was mimicked by modulators of TREK-1 channels, which belong to the K2P channels family. We found no evidence for the presence of acid-sensitive ion channels and transient receptor potential vanilloid receptors in hippocampal astrocytes. In conclusion, the assembly of astrocytic K(+) channels allows tolerating short, transient acidification, and glial Kir4.1 and K2P channels can be considered promising new targets in brain diseases accompanied by pH shifts. PMID:26920692

  9. Nanoscale Properties of Neural Cell Prosthetic and Astrocyte Response

    NASA Astrophysics Data System (ADS)

    Flowers, D. A.; Ayres, V. M.; Delgado-Rivera, R.; Ahmed, I.; Meiners, S. A.

    2009-03-01

    Preliminary data from in-vivo investigations (rat model) suggest that a nanofiber prosthetic device of fibroblast growth factor-2 (FGF-2)-modified nanofibers can correctly guide regenerating axons across an injury gap with aligned functional recovery. Scanning Probe Recognition Microscopy (SPRM) with auto-tracking of individual nanofibers is used for investigation of the key nanoscale properties of the nanofiber prosthetic device for central nervous system tissue engineering and repair. The key properties under SPRM investigation include nanofiber stiffness and surface roughness, nanofiber curvature, nanofiber mesh density and porosity, and growth factor presentation and distribution. Each of these factors has been demonstrated to have global effects on cell morphology, function, proliferation, morphogenesis, migration, and differentiation. The effect of FGF-2 modification on the key nanoscale properties is investigated. Results from the nanofiber prosthetic properties investigations are correlated with astrocyte response to unmodified and FGF-2 modified scaffolds, using 2D planar substrates as a control.

  10. Label-free optical activation of astrocyte in vivo

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Ku, Taeyun; Choi, Kyungsun; Choi, Chulhee

    2011-07-01

    As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

  11. C/EBPβ regulates multiple IL-1β-induced human astrocyte inflammatory genes

    PubMed Central

    2012-01-01

    Background CCAAT enhancer-binding protein (C/EBP)β regulates gene expression in multiple organ systems and cell types, including astrocytes in the central nervous system (CNS). Inflammatory stimuli, interleukin (IL)-1β, tumor necrosis factor-α, human immunodeficiency virus (HIV)-1 and lipopolysaccharide induce astrocyte C/EBPβ expression. C/EBPβ is detectable in brains of Alzheimer’s disease (AD), Parkinson’s disease (PD) and HIV-1-associated dementia (HAD) patients, yet little is known about how C/EBPβ contributes to astrocyte gene regulation during neuroinflammation. Methods The expression of 92 human inflammation genes was compared between IL-1β-treated primary human astrocytes and astrocytes transfected with C/EBPβ-specific small interfering (si)RNA prior to IL-1β treatment for 12 h. Transcripts altered by > two-fold compared to control were subjected to one-way analysis of variance and Newman-Keuls post-test for multiple comparisons. Expression of two genes, cyclooxygenase-2 (COX-2) and bradykinin receptor B2 (BDKRB2) was further confirmed in additional human astrocyte donors. Astrocytes were treated with mitogen-activated protein kinase-selective inhibitors, then with IL-1β for 12 or 24 h followed by COX-2 and BDKRB2, expression analyses. Results IL-1β altered expression of 29 of 92 human inflammation genes by at least two-fold in primary human astrocytes in 12 h. C/EBPβ knockdown affected expression of 17 out of 29 IL-1β-regulated genes by > 25%. Two genes relevant to neuroinflammation, COX-2 and BDKRB2, were robustly decreased and increased, respectively, in response to C/EBPβ knockdown, and expression was confirmed in two additional donors. COX-2 and BDKRB2 mRNA remained altered in siRNA-transfected astrocytes at 12, 24 or 72 h. Inhibiting p38 kinase (p38K) activation blocked IL-1β-induced astrocyte COX-2 mRNA and protein expression, but not IL-1β-induced astrocyte BDKRB2 expression. Inhibiting extracellular

  12. Synergistic enhancement of human bone marrow stromal cell proliferation and osteogenic differentiation on BMP-2-derived and RGD peptide concentration gradients.

    PubMed

    Moore, Nicole M; Lin, Nancy J; Gallant, Nathan D; Becker, Matthew L

    2011-05-01

    Rational design of bioactive tissue engineered scaffolds for directing bone regeneration in vivo requires a comprehensive understanding of cell interactions with the immobilized bioactive molecules. In the current study, substrates possessing gradient concentrations of immobilized peptides were used to measure the concentration-dependent proliferation and osteogenic differentiation of human bone marrow stromal cells. Two bioactive peptides, one derived from extracellular matrix protein (ECM), GRGDS, and one from bone morphogenic protein-2 (BMP-2), KIPKASSVPTELSAISTLYL, were found to synergistically enhance cell proliferation, up-regulate osteogenic mRNA markers bone sialoprotein (BSP) and Runt-related transcription factor 2, and produce mineralization at densities greater than 130 pmol cm(-2) (65 pmol cm(-2) for each peptide). In addition, COOH-terminated self-assembled monolayers alone led to up-regulated BSP mRNA levels at densities above 200 pmol cm(-2) and increased cell proliferation from day 3 to day 14. Taking further advantage of both the synergistic potentials and the concentration-dependent activities of ECM and growth-factor-derived peptides on proliferative activity and osteogenic differentiation, without the need for additional osteogenic supplements, will enable the successful incorporation of the bioactive species into biorelevant tissue engineering scaffolds. PMID:21272672

  13. Estrogen promotes fat mass and obesity-associated protein nuclear localization and enhances endometrial cancer cell proliferation via the mTOR signaling pathway.

    PubMed

    Zhu, Yaping; Shen, Jiaqi; Gao, Liyan; Feng, Youji

    2016-04-01

    Extensive exposure to estrogen is generally acknowledged as a risk factor for endometrial cancer. Given that the accumulation of adipocytes also contributes to the increased production of estrogen, in the present study, we evaluated the expression of the fat mass and obesity-associated (FTO) gene in endometrial tumor tissues and further explored the mechanism of how estrogen facilitates FTO nuclear localization and promotes endometrial cancer cell proliferation. Immunohistochemical (IHC) staining assay was used to detect the FTO expression in endometrial tumor samples. Western blotting was performed to investigate the mechanism of estrogen-induced FTO nuclear localization. siRNA was used to knock down ERα and further explore its role in FTO nuclear localization. MTT assay was carried out to determine cell proliferation. We found that FTO was overexpressed in endometrial carcinoma tissues and served as a poor prognostic marker. Additionally, estrogen induced FTO nuclear accumulation via the mTOR signaling pathway and the nuclear localization was ERα-dependent, which contributed to enhanced proliferative activity. Therefore, the present study provides new insight into the mechanisms of estrogen-induced proliferation, implying the possibility of using FTO as a potential therapeutic target for the treatment of endometrial cancer.

  14. [Voluntary wheel running enhances cell proliferation and expression levels of BDNF, IGF1 and WNT4 in dentate gyrus of adult mice].

    PubMed

    Yu, Jia-Ling; Ma, Li; Ma, Lan; Tao, Ye-Zheng

    2014-10-25

    Adult hippocampal neurogenesis plays important roles in learning, memory and mood regulation. External factors, such as physical exercise, have been found to modulate adult hippocampal neurogenesis. Voluntary running enhances cell proliferation in subgranular zone (SGZ) and increases the number of new born neurons in rodents, but underlying mechanisms are not fully understood. In this study, we used BrdU assay to identify proliferating cells in 2-month-old C57BL/6 mice after 15 days of voluntary wheel running test. mRNA and protein levels for several neural factors in dentate gyrus, Ammon's horn, and cortex were also analyzed by RT-qPCR and Western blot assay after 15 days of voluntary wheel running. Our data show that voluntary wheel running for 15 days elevated the number of proliferation cells in dentate gyrus and significantly up-regulated the mRNA levels of Bdnf, Igf1 and Wnt4. The protein levels of BDNF and IGF1 in dentate gyrus were also increased after voluntary wheel running. These results indicate that the increase of adult hippocampal neurogenesis caused by voluntary wheel running for 15 days might be through up-regulating BDNF, IGF1 and WNT4 in dentate gyrus.

  15. Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling

    PubMed Central

    Filipello, Fabia; Romagnani, Andrea; Mazzitelli, Sonia; Matteoli, Michela; Verderio, Claudia; Grassi, Fabio

    2016-01-01

    Astrocytes play a crucial role in neuroinflammation as part of the glia limitans, which regulates infiltration of the brain parenchyma by leukocytes. The signaling pathways and molecular events, which result from the interaction of activated T cells with astrocytes are poorly defined. Here we show that astrocytes promote the expression and enzymatic activity of CD39 and CD73 ectonucleotidases in recently activated CD4 cells by a contact dependent mechanism that is independent of T cell receptor interaction with class II major histocompatibility complex (MHC). Transforming growth factor-β (TGF-β) is robustly upregulated and sufficient to promote ectonucleotidases expression. T cell adhesion to astrocyte results in differentiation to an immunosuppressive phenotype defined by expression of the transcription factor Rorγt, which characterizes the CD4 T helper 17 subset. CD39 activity in T cells in turn inhibits spontaneous calcium oscillations in astrocytes that correlated with enhanced and reduced transcription of CCL2 chemokine and Sonic hedgehog (Shh), respectively. We hypothesize this TCR-independent interaction promote an immunosuppressive program in T cells to control possible brain injury by deregulated T cell activation during neuroinflammation. On the other hand, the increased secretion of CCL2 with concomitant reduction of Shh might promote leukocytes extravasation into the brain parenchyma. PMID:26784253

  16. Inhibition of astrocyte metabolism is not the primary mechanism for anaesthetic hypnosis.

    PubMed

    Voss, Logan J; Harvey, Martyn G; Sleigh, James W

    2016-01-01

    Astrocytes have been promoted as a possible mechanistic target for anaesthetic hypnosis. The aim of this study was to explore this using the neocortical brain slice preparation. The methods were in two parts. Firstly, multiple general anaesthetic compounds demonstrating varying in vivo hypnotic potency were analysed for their effect on "zero-magnesium" seizure-like event (SLE) activity in mouse neocortical slices. Subsequently, the effect of astrocyte metabolic inhibition was investigated in neocortical slices, and compared with that of the anaesthetic drugs. The rationale was that, if suppression of astrocytes was both necessary and sufficient to cause hypnosis in vivo, then inhibition of astrocytic metabolism in slices should mimic the anaesthetic effect. In vivo anaesthetic potency correlated strongly with the magnitude of reduction in SLE frequency in neocortical slices (R(2) 37.7 %, p = 0.002). Conversely, SLE frequency and length were significantly enhanced during exposure to both fluoroacetate (23 and 20 % increase, respectively, p < 0.01) and aminoadipate (12 and 38 % increase, respectively, p < 0.01 and p < 0.05). The capacity of an anaesthetic agent to reduce SLE frequency in the neocortical slice is a good indicator of its in vivo hypnotic potency. The results do not support the hypothesis that astrocytic metabolic inhibition is a mechanism of anaesthetic hypnosis. PMID:27462489

  17. Astrocytes and pericytes differentially modulate blood–brain barrier characteristics during development and hypoxic insult

    PubMed Central

    Al Ahmad, Abraham; Taboada, Carole Bürgi; Gassmann, Max; Ogunshola, Omolara O

    2011-01-01

    Understanding regulation of blood–brain barrier (BBB) is crucial to reduce/prevent its disruption during injury. As high brain complexity makes interpretation of in vivo data challenging, BBB studies are frequently performed using simplified in vitro models. However, many models fail to address the three-dimensional (3D) cellular interactions that occur in vivo, an important feature that may explain discrepancies in translation of in vitro data to the in vivo situation. We have designed and characterized an innovative 3D model that reproduces morphological and functional characteristics of the BBB in vivo and used it to investigate cellular interactions and contribution of astrocytes and pericytes to BBB development. Our model shows that both astrocytes and pericytes significantly suppress endothelial proliferation. In contrast, differential effects on tubulogenesis were observed with astrocytes reducing the number of tubes formed but increasing diameters and length, whereas pericytes had the opposite effect. Pericytes also induce proper localization of barrier proteins, lumen polarization, and functional activity of ATP-binding cassette (ABC) transporters similar to astrocytes, but the presence of both cells is required to maintain optimal barrier characteristics during hypoxic exposure. This model is simple, dynamic, and convenient to study many aspects of BBB function and represents an exciting new tool to address open questions of BBB regulation. PMID:20827262

  18. Targeted activation of CREB in reactive astrocytes is neuroprotective in focal acute cortical injury.

    PubMed

    Pardo, Luis; Schlüter, Agatha; Valor, Luis M; Barco, Angel; Giralt, Mercedes; Golbano, Arantxa; Hidalgo, Juan; Jia, Peilin; Zhao, Zhongming; Jové, Mariona; Portero-Otin, Manuel; Ruiz, Montserrat; Giménez-Llort, Lydia; Masgrau, Roser; Pujol, Aurora; Galea, Elena

    2016-05-01

    The clinical challenge in acute injury as in traumatic brain injury (TBI) is to halt the delayed neuronal loss that occurs hours and days after the insult. Here we report that the activation of CREB-dependent transcription in reactive astrocytes prevents secondary injury in cerebral cortex after experimental TBI. The study was performed in a novel bitransgenic mouse in which a constitutively active CREB, VP16-CREB, was targeted to astrocytes with the Tet-Off system. Using histochemistry, qPCR, and gene profiling we found less neuronal death and damage, reduced macrophage infiltration, preserved mitochondria, and rescued expression of genes related to mitochondrial metabolism in bitransgenic mice as compared to wild type littermates. Finally, with meta-analyses using publicly available databases we identified a core set of VP16-CREB candidate target genes that may account for the neuroprotective effect. Enhancing CREB activity in astrocytes thus emerges as a novel avenue in acute brain post-injury therapeutics.

  19. Coculture with astrocytes reduces the radiosensitivity of glioblastoma stem-like cells and identifies additional targets for radiosensitization

    PubMed Central

    Rath, Barbara H; Wahba, Amy; Camphausen, Kevin; Tofilon, Philip J

    2015-01-01

    Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization. PMID:26518290

  20. Long interspersed nucleotide acid element-1 ORF-1 protein promotes proliferation and invasion of human colorectal cancer LoVo cells through enhancing ETS-1 activity.

    PubMed

    Li, M Y; Zhu, M; Feng, F; Cai, F Y; Fan, K C; Jiang, H; Wang, Z Q; Linghu, E Q

    2014-04-14

    The human proto-oncogene long interspersed nucleotide acid element-1 (LINE-1) open reading frame-1 protein (ORF-1p) is involved in the progress of several cancers. The transcription factor ETS-1 can mediate the transcription of some downstream genes that play specific roles in the regulation of cancerous cell invasion and metastasis. In this study, the effects of LINE-1 ORF-1p on ETS-1 activity and on the proliferation and invasion of human colorectal cancer LoVo cells were investigated. Results showed that the overexpression of LINE-1 ORF-1p enhanced the transcription of ETS-1 downstream genes and increased their protein levels, and downregulation of the LINE-1 ORF-1p level by small interfering RNA (siRNA) reduced the transcriptional activation of ETS-1. In addition, overexpression of LINE-1 ORF-1p promoted LoVo cell proliferation and anchor-independent growth, and a knockdown of the LINE-1 protein level by siRNA reduced the proliferation and anchor-independent growth ability of LoVo cells. In vivo data revealed that LINE-1 ORF-1p overexpression increased LoVo tumor growth in nude mice, whereas the siRNA knockdown of endogenous LINE-1 ORF-1p expression decreased LoVo cell growth in nude mice. Therefore, LINE- 1 ORF-1p could promote LoVo cell proliferation and invasion both in vitro and in vivo, indicating that it might be a useful molecular target for the treatment of human colorectal cancer.

  1. Specialized contacts of astrocytes with astrocytes and with other cell types in the hypothalamus of the hamster.

    PubMed Central

    Suarez Najera, I; Fernandez Ruiz, B; Garcia Segura, L M

    1980-01-01

    Adult hamsters were used for this electron microscopic study of the hypothalamic region. Specialized contacts between astrocytes and astrocytes, and between astrocytes and other cellular elements, are described and illustrated. The specialized inter-astrocytic junctions occur primarily in perivascular and subpial regions, but also in areas of high synaptic density. The junctions between astrocytic processes are of hemidesmosomal type. Astrocytes are connected to oligodendroglial cells by means of desmosomes, and to neuronal processes by means of zonulae occludens. The functional significance of these arrangements is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7364663

  2. Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival.

    PubMed

    Lataillade, J J; Clay, D; Dupuy, C; Rigal, S; Jasmin, C; Bourin, P; Le Bousse-Kerdilès, M C

    2000-02-01

    The chemokine stromal cell-derived factor-1 (SDF-1), and its receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34(+) cells. We show here that SDF-1 may also be involved in hematopoiesis, promoting the proliferation of human CD34(+) cells purified from normal adult peripheral blood (PB). CXCR-4 was expressed on PB CD34(+) cells. The amount of CXCR-4 on PB CD34(+) cells was 10 times higher when CD34(+) cells were purified following overnight incubation. CXCR-4 overexpression was correlated with a primitive PB CD34(+) cell subset defined by a CD34(high) CD38(low)CD71(low)c-Kit(low)Thy-1(+) antigenic profile. The functional significance of CXCR-4 expression was ascertained by assessing the promoting effect of SDF-1alpha on cell cycle, proliferation, and colony formation. SDF-1 alone increased the percentage of CD34(+) cells in the S+G(2)/M phases and sustained their survival. In synergy with cytokines, SDF-1 increased PB CD34(+) and CD34(high)CD38(low) cell expansion and colony formation. SDF-1 also stimulated the growth of colonies derived from primitive progenitors released from quiescence by anti-TGF-beta treatment. Thus, our results shed new light on the potential role of this chemokine in the stem cell engraftment process, which involves migration, adhesion, and proliferation. Furthermore, both adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity may be of clinical relevance for improving cell therapy settings in stem cell transplantation.

  3. MicroRNA-26b Represses Colon Cancer Cell Proliferation by Inhibiting Lymphoid Enhancer Factor 1 (LEF-1) Expression

    PubMed Central

    Zhang, Zichao; Kim, KyoungHyun; Li, Xiao; Moreno, Myriam; Sharp, Thad; Goodheart, Micheal J.; Safe, Stephen; Dupuy, Adam J.; Amendt, Brad A.

    2014-01-01

    microRNAs (miR) can act as oncogenes and tumor suppressors and several miRs are associated with cancer development and progression through the modulation of multiple cellular processes. miR-26b is down regulated in several cancers and tumors and miR-26b directly targets the Lef-1 3'UTR and inhibits endogenous Lef-1 expression. We report that miR-26b expression is associated with human colon cancer through the regulation of LEF-1 expression in colon cancer cells. Analyses of multiple colon cancer cell lines revealed an inverse correlation between miR-26b and LEF-1 expression. Normal human colon cells express low levels of LEF-1 and high levels of miR-26b, however human colon cancer cells have decreased miR-26b expression and increased LEF-1 expression. We demonstrate that miR-26b expression is a potent inhibitor of colon cancer cell proliferation and significantly decreases LEF-1 expression. The LEF-1 regualted genes Cyclin D1 and c-Myc were indirectly repressed by miR-26b and this was consistent with decreased proliferation. miR-26b overexpression in SW480 colon cancer cells also inhibited tumor growth in nude mice and this was due to decreased tumor growth and not apoptosis. Analyses of human colon cancer databases also demonstrated a link between miR-26b and LEF-1 expression. c-Myc expression is associated with multiple cancers and we propose that miR-26b may act as a potential therapeutic agent in reducing cancer cell proliferation through repressing LEF-1 activation of c-Myc and Cyclin D1 expression. PMID:24785257

  4. Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

    PubMed Central

    Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

    2016-01-01

    Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

  5. Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells.

    PubMed

    Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

    2016-01-01

    Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment.

  6. Hybrid chitosan/β-1,3-glucan matrix of bone scaffold enhances osteoblast adhesion, spreading and proliferation via promotion of serum protein adsorption.

    PubMed

    Przekora, Agata; Benko, Aleksandra; Blazewicz, Marta; Ginalska, Grazyna

    2016-01-01

    Initial protein adsorption to the material surface is crucial for osteoblast adhesion, survival, and rapid proliferation resulting in intensive new bone formation. The aim of this study was to demonstrate that modification of a chitosan matrix of chitosan/hydroxyapatite (chit/HA) biomaterial for bone tissue engineering applications with linear β-1,3-glucan (curdlan) leads to promotion of serum protein adsorption to the resultant scaffold (chit/glu/HA) and thus in enhancement of osteoblast adhesion, spreading and proliferation. Fabricated biomaterials were pre-adsorbed with different protein solutions and then protein adsorption and osteoblast behavior on the scaffolds were compared. Moreover, surface chemical composition, wettability and surface energy of biomaterials were compared. Modification of the chitosan matrix with β-1,3-glucan introduces a greater polarpart in the resultant chitosan/β-1,3-glucan matrix presumably resulting from more OH groups within the curdlan structure. Moreover, FTIR-ATR results suggest that there might be some sort of chemical interaction between the NH group of chitosan and the OH group of β-1,3-glucan. As a consequence, the chit/glu/HA scaffold adsorbs significantly more adhesion proteins that are crucial for osteoblasts compared to the chit/HA material, providing a higher density culture of well-spread osteoblasts on its surface. Obtained results revealed that not only is chit/glu/HA biomaterial a promising scaffold for bone tissue engineering applications, but the specific polysaccharide chit/glu matrix itself is promising for use in the biomedical material field to modify various biomaterials in order to enhance osteoblast adhesion and proliferation on their surfaces. PMID:27388048

  7. Pharmacological administration of the isoflavone daidzein enhances cell proliferation and reduces high fat diet-induced apoptosis and gliosis in the rat hippocampus.

    PubMed

    Rivera, Patricia; Pérez-Martín, Margarita; Pavón, Francisco J; Serrano, Antonia; Crespillo, Ana; Cifuentes, Manuel; López-Ávalos, María-Dolores; Grondona, Jesús M; Vida, Margarita; Fernández-Llebrez, Pedro; de Fonseca, Fernando Rodríguez; Suárez, Juan

    2013-01-01

    Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.

  8. Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.

    PubMed

    Bencze, Maximilien; Negroni, Elisa; Vallese, Denis; Yacoub-Youssef, Houda; Chaouch, Soraya; Wolff, Annie; Aamiri, Ahmed; Di Santo, James P; Chazaud, Bénédicte; Butler-Browne, Gillian; Savino, Wilson; Mouly, Vincent; Riederer, Ingo

    2012-11-01

    Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

  9. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    SciTech Connect

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  10. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    SciTech Connect

    Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

    2013-02-01

    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  11. Astrocytes Protect Against Isoflurane Neurotoxicity by Buffering pro Brain-Derived Neurotrophic Factor

    PubMed Central

    Stary, Creed M.; Sun, Xiaoyun; Giffard, Rona G.

    2015-01-01

    Background Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain derived neurotrophic factor (proBDNF) and activation of post-synaptic p75 neurotrophin receptor (p75NTR). Astrocytes express p75NTR but their role in neuronal p75NTR mediated cell death remains unclear. We investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75NTR neurotoxicity. Methods Cell death was assessed in day-in-vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 hours following 1 hour exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75NTR in co-culture was achieved with small interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used 6–8 replicate cultures/condition, and was repeated at least three times. Results Exposure to isoflurane significantly (p<0.05) increased neuronal cell death in primary neuronal cultures (1.5±0.7 fold, mean±SD) but not in co-culture with DIV 7 (1.0±0.5 fold) or DIV 21 astrocytes (1.2±1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75NTR inhibition. Astrocyte-targeted p75NTR knockdown in co-cultures increased media proBDNF (1.2±0.1 fold) and augmented isoflurane induced neuronal cell death (3.8±3.1 fold). Conclusions The presence of astrocytes provides protection to growing neurons by buffering elevated levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where elevated levels of proBDNF impair neurogenesis. PMID:26270940

  12. Sodium signaling and astrocyte energy metabolism.

    PubMed

    Chatton, Jean-Yves; Magistretti, Pierre J; Barros, L Felipe

    2016-10-01

    The Na(+) gradient across the plasma membrane is constantly exploited by astrocytes as a secondary energy source to regulate the intracellular and extracellular milieu, and discard waste products. One of the most prominent roles of astrocytes in the brain is the Na(+) -dependent clearance of glutamate released by neurons during synaptic transmission. The intracellular Na(+) load collectively generated by these processes converges at the Na,K-ATPase pump, responsible for Na(+) extrusion from the cell, which is achieved at the expense of cellular ATP. These processes represent pivotal mechanisms enabling astrocytes to increase the local availability of metabolic substrates in response to neuronal activity. This review presents basic principles linking the intracellular handling of Na(+) following activity-related transmembrane fluxes in astrocytes and the energy metabolic pathways involved. We propose a role of Na(+) as an energy currency and as a mediator of metabolic signals in the context of neuron-glia interactions. We further discuss the possible impact of the astrocytic syncytium for the distribution and coordination of the metabolic response, and the compartmentation of these processes in cellular microdomains and subcellular organelles. Finally, we illustrate future avenues of investigation into signaling mechanisms aimed at bridging the gap between Na(+) and the metabolic machinery. GLIA 2016;64:1667-1676. PMID:27027636

  13. Spinal astrocytes produce and secrete dynorphin neuropeptides.

    PubMed

    Wahlert, Andrew; Funkelstein, Lydiane; Fitzsimmons, Bethany; Yaksh, Tony; Hook, Vivian

    2013-04-01

    Dynorphin peptide neurotransmitters (neuropeptides) have been implicated in spinal pain processing based on the observations that intrathecal delivery of dynorphin results in proalgesic effects and disruption of extracellular dynorphin activity (by antisera) prevents injury evoked hyperalgesia. However, the cellular source of secreted spinal dynorphin has been unknown. For this reason, this study investigated the expression and secretion of dynorphin-related neuropeptides from spinal astrocytes (rat) in primary culture. Dynorphin A (1-17), dynorphin B, and α-neoendorphin were found to be present in the astrocytes, illustrated by immunofluorescence confocal microscopy, in a discrete punctate pattern of cellular localization. Measurement of astrocyte cellular levels of these dynorphins by radioimmunoassays confirmed the expression of these three dynorphin-related neuropeptides. Notably, BzATP (3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate) and KLA (di[3-deoxy-D-manno-octulosonyl]-lipid A) activation of purinergic and toll-like receptors, respectively, resulted in stimulated secretion of dynorphins A and B. However, α-neoendorphin secretion was not affected by BzATP or KLA. These findings suggest that dynorphins A and B undergo regulated secretion from spinal astrocytes. These findings also suggest that spinal astrocytes may provide secreted dynorphins that participate in spinal pain processing.

  14. Unraveling the complex metabolic nature of astrocytes.

    PubMed

    Bouzier-Sore, Anne-Karine; Pellerin, Luc

    2013-01-01

    Since the initial description of astrocytes by neuroanatomists of the nineteenth century, a critical metabolic role for these cells has been suggested in the central nervous system. Nonetheless, it took several technological and conceptual advances over many years before we could start to understand how they fulfill such a role. One of the important and early recognized metabolic function of astrocytes concerns the reuptake and recycling of the neurotransmitter glutamate. But the description of this initial property will be followed by several others including an implication in the supply of energetic substrates to neurons. Indeed, despite the fact that like most eukaryotic non-proliferative cells, astrocytes rely on oxidative metabolism for energy production, they exhibit a prominent aerobic glycolysis capacity. Moreover, this unusual metabolic feature was found to be modulated by glutamatergic activity constituting the initial step of the neurometabolic coupling mechanism. Several approaches, including biochemical measurements in cultured cells, genetic screening, dynamic cell imaging, nuclear magnetic resonance spectroscopy and mathematical modeling, have provided further insights into the intrinsic characteristics giving rise to these key features of astrocytes. This review will provide an account of the different results obtained over several decades that contributed to unravel the complex metabolic nature of astrocytes that make this cell type unique.

  15. Unraveling the complex metabolic nature of astrocytes

    PubMed Central

    Bouzier-Sore, Anne-Karine; Pellerin, Luc

    2013-01-01

    Since the initial description of astrocytes by neuroanatomists of the nineteenth century, a critical metabolic role for these cells has been suggested in the central nervous system. Nonetheless, it took several technological and conceptual advances over many years before we could start to understand how they fulfill such a role. One of the important and early recognized metabolic function of astrocytes concerns the reuptake and recycling of the neurotransmitter glutamate. But the description of this initial property will be followed by several others including an implication in the supply of energetic substrates to neurons. Indeed, despite the fact that like most eukaryotic non-proliferative cells, astrocytes rely on oxidative metabolism for energy production, they exhibit a prominent aerobic glycolysis capacity. Moreover, this unusual metabolic feature was found to be modulated by glutamatergic activity constituting the initial step of the neurometabolic coupling mechanism. Several approaches, including biochemical measurements in cultured cells, genetic screening, dynamic cell imaging, nuclear magnetic resonance spectroscopy and mathematical modeling, have provided further insights into the intrinsic characteristics giving rise to these key features of astrocytes. This review will provide an account of the different results obtained over several decades that contributed to unravel the complex metabolic nature of astrocytes that make this cell type unique. PMID:24130515

  16. Molecular approaches for manipulating astrocytic signaling in vivo

    PubMed Central

    Xie, Alison X.; Petravicz, Jeremy; McCarthy, Ken D.

    2015-01-01

    Astrocytes are the predominant glial type in the central nervous system and play important roles in assisting neuronal function and network activity. Astrocytes exhibit complex signaling systems that are essential for their normal function and the homeostasis of the neural network. Altered signaling in astrocytes is closely associated with neurological and psychiatric diseases, suggesting tremendous therapeutic potential of these cells. To further understand astrocyte function in health and disease, it is important to study astrocytic signaling in vivo. In this review, we discuss molecular tools that enable the selective manipulation of astrocytic signaling, including the tools to selectively activate and inactivate astrocyte signaling in vivo. Lastly, we highlight a few tools in development that present strong potential for advancing our understanding of the role of astrocytes in physiology, behavior, and pathology. PMID:25941472

  17. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

    PubMed

    Baer, Matthew L; Henderson, Scott C; Colello, Raymond J

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  18. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

    PubMed Central

    Baer, Matthew L.; Henderson, Scott C.; Colello, Raymond J.

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  19. Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

    SciTech Connect

    Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

    2011-06-24

    Highlights: {yields} Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. {yields} Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca{sup 2+} permeant SACs. {yields} The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. {yields} Stretch-induced ET-1 production depends on a calcium influx. {yields} SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 {mu}M GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca{sup 2+} threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

  20. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  1. Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

    PubMed Central

    El‐Jawhari, Jehan J.; Sanjurjo‐Rodríguez, Clara; Jones, Elena

    2015-01-01

    ABSTRACT Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss®. Another collagen‐containing synthetic scaffold, Vitoss® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss® Collagen and Vitoss® was similar but greater than Orthoss® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss® Collagen and Vitoss® after 2‐week culture was also higher compared to Orthoss® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss® collagen and Vitoss® was greater compared to Orthoss® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016. PMID:26466765

  2. Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells.

    PubMed

    El-Jawhari, Jehan J; Sanjurjo-Rodríguez, Clara; Jones, Elena; Giannoudis, Peter V

    2016-04-01

    Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen-containing bovine bone scaffold (Orthoss(®) Collagen) with a non-collagen-containing bovine bone scaffold, Orthoss(®) . Another collagen-containing synthetic scaffold, Vitoss(®) was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit-fibroblast assay and flow-cytometry. The number of BM MSCs initially attached to Orthoss(®) Collagen and Vitoss(®) was similar but greater than Orthoss(®) (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss(®) Collagen and Vitoss(®) after 2-week culture was also higher compared to Orthoss(®) (p = 0.010 and p = 0.023, respectively). Interestingly, collagen-containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture-expanded MSCs on Orthoss(®) collagen and Vitoss(®) was greater compared to Orthoss(®) (p = 0.047 and p = 0.004, respectively). Collectively, collagen-containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.

  3. Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

    PubMed

    Habbas, Samia; Santello, Mirko; Becker, Denise; Stubbe, Hiltrud; Zappia, Giovanna; Liaudet, Nicolas; Klaus, Federica R; Kollias, George; Fontana, Adriano; Pryce, Christopher R; Suter, Tobias; Volterra, Andrea

    2015-12-17

    The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor-α (TNFα). To date, however, no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here, we show that local increase of TNFα in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning-memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS, as well as in other CNS conditions accompanied by inflammatory states or infections. PMID:26686654

  4. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis.

    PubMed

    Muthukumar, Allie K; Stork, Tobias; Freeman, Marc R

    2014-10-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. We found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABA(B)R1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABA(B)R1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.

  5. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia

    PubMed Central

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W.; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-01-01

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. PMID:27739425

  6. Endothelial cells and astrocytes: a concerto en duo in ischemic pathophysiology.

    PubMed

    Berezowski, Vincent; Fukuda, Andrew M; Cecchelli, Roméo; Badaut, Jérôme

    2012-01-01

    The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

  7. Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation.

    PubMed

    Zhou, Shuling; Szczesna, Karolina; Ochalek, Anna; Kobolák, Julianna; Varga, Eszter; Nemes, Csilla; Chandrasekaran, Abinaya; Rasmussen, Mikkel; Cirera, Susanna; Hyttel, Poul; Dinnyés, András; Freude, Kristine K; Avci, Hasan X

    2016-01-01

    Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes. PMID:26798357

  8. Astrocytic adenosine A2A receptors control the amyloid-β peptide-induced decrease of glutamate uptake.

    PubMed

    Matos, Marco; Augusto, Elisabete; Machado, Nuno J; dos Santos-Rodrigues, Alexandre; Cunha, Rodrigo A; Agostinho, Paula

    2012-01-01

    Alzheimer's disease (AD) is characterized by a progressive cognitive impairment tightly correlated with the accumulation of amyloid-β (Aβ) peptides (mainly Aβ(1-42)). There is a precocious disruption of glutamatergic synapses in AD, in line with an ability of Aβ to decrease astrocytic glutamate uptake. Accumulating evidence indicates that caffeine prevents the burden of AD, likely through the antagonism of A(2A) receptors (A(2A)R) which attenuates Aβ-induced memory impairment and synaptotoxicity. Since A(2A)R also modulate astrocytic glutamate uptake, we now tested if A(2A)R blockade could prevent the decrease of astrocytic glutamate uptake caused by Aβ. In cultured astrocytes, Aβ(1-42). (1 μM for 24 hours) triggered an astrogliosis typified by an increased density of GFAP, which was mimicked by the A(2A)R agonist, CGS 26180 (30 nM), and prevented by the A(2A)R antagonist, SCH 58261 (100 nM). Aβ1-42 also decreased D-aspartate uptake by 28 ± 4%, an effect abrogated upon genetic inactivation or pharmacological blockade of A(2A)R. In accordance with the long term control of glutamate transporter expression by A(2A)R, Aβ(1-42). enhanced the expression and density of astrocytic A(2A)R and decreased GLAST and GLT-I expression in astrocytes from wild type, but not from A(2A)R knockout mice. This impact of Aβ(1-42). on glutamate transporters and uptake, dependent on A(2A)R function, was also confirmed in an ex vivo astrocyte preparation (gliosomes) from rats intracerebroventricularly (icv) injected with Aβ(1-42). . These results provide the first demonstration for a direct key role of astrocytic A(2A)R in the ability of Aβ-induced impairment of glutamate uptake, which may underlie glutamatergic synaptic dysfunction and excitotoxicity in AD.

  9. Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

    PubMed Central

    Filous, Angela R.; Miller, Jared H.; Coulson-Thomas, Yvette M.; Horn, Kevin P.; Alilain, Warren J.; Silver, Jerry

    2010-01-01

    Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re-growth across the gradient. The use of selective enzyme inhibitors revealed that MMP-2 enables immature astrocytes to cross the proteoglycan rim. The bridge-building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pre-treatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes co-injected with chondroitinase ABC induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced. PMID:20629049

  10. Redistribution of monocarboxylate transporter 2 on the surface of astrocytes in the human epileptogenic hippocampus

    PubMed Central

    Lauritzen, Fredrik; Heuser, Kjell; de Lanerolle, Nihal C.; Lee, Tih-Shih W.; Spencer, Dennis D.; Kim, Jung H.; Gjedde, Albert; Eid, Tore; Bergersena, Linda H.

    2013-01-01

    Emerging evidence points to monocarboxylates as key players in the pathophysiology of temporal lobe epilepsy (TLE) with hippocampal sclerosis (mesial temporal lobe epilepsy, MTLE). Monocarboxylate transporters (MCT) 1 and 2, which are abundantly present on brain endothelial cells and perivascular astrocyte endfeet, respectively, facilitate the transport of monocarboxylates and protons across cell membranes. Recently, we reported that the density of MCT1 protein is reduced on endothelial cells and increased on astrocyte plasma membranes in the hippocampal formation in patients with MTLE and in several animal models of the disorder. Because the perivascular astrocyte endfeet comprise an important part of the neurovascular unit we now assessed the distribution of the MCT2 in hippocampal formations in TLE patients with (MTLE) or without hippocampal sclerosis (non-MTLE). Light microscopic immunohistochemistry revealed significantly less perivascular MCT2 immunoreactivity in the hippocampal formation in MTLE (n=6) than in non-MTLE (n=6) patients, and to a lesser degree in non-MTLE than in non-epilepsy patients (n=4). Immunogold electron microscopy indicated that the loss of MCT2 protein occurred on perivascular astrocyte endfeet. Interestingly, the loss of MCT2 on astrocyte endfeet in MTLE (n=3) was accompanied by an upregulation of the protein on astrocyte membranes facing synapses in the neuropil, when compared with non-MTLE (n=3). We propose that the altered distribution of MCT1 and MCT2 in TLE (especially MTLE) limits the flux of monocarboxylates across the blood brain barrier and enhances the exchange of monocarboxylates within the brain parenchyma. PMID:22535546

  11. Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia.

    PubMed

    Pang, Y; Cai, Z; Rhodes, P G

    2000-11-15

    Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.

  12. Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

    PubMed

    Zhao, Yi; Yao, Yun-hong; Li, Li; An, Wei-fang; Chen, Hong-zen; Sun, Li-ping; Kang, Hai-xian; Wang, Sen; Hu, Xin-rong

    2014-12-01

    Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC. PMID:25367850

  13. Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

    PubMed

    Zhao, Yi; Yao, Yun-hong; Li, Li; An, Wei-fang; Chen, Hong-zen; Sun, Li-ping; Kang, Hai-xian; Wang, Sen; Hu, Xin-rong

    2014-12-01

    Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC.

  14. High calcium enhances calcium oxalate crystal binding capacity of renal tubular cells via increased surface annexin A1 but impairs their proliferation and healing.

    PubMed

    Chutipongtanate, Somchai; Fong-ngern, Kedsarin; Peerapen, Paleerath; Thongboonkerd, Visith

    2012-07-01

    Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further

  15. Recombinant lentivirus with enhanced expression of caudal-related homeobox protein 2 inhibits human colorectal cancer cell proliferation in vitro.

    PubMed

    He, Sai; Sun, Xue-Jun; Zheng, Jian-Bao; Qi, Jie; Chen, Nan-Zheng; Wang, Wei; Wei, Guang-Bing; Liu, Dong; Yu, Jun-Hui; Lu, Shao-Ying; Wang, Hui

    2015-08-01

    Caudal-related homeobox protein 2 (CDX2), a tumor suppressor in the adult colon, is overexpressed under a non-cancer specific cytomegalovirus promoter in certain tumor cells; furthermore, non-specific expression of CDX2 may result in aberrant side effects in normal cells. The human telomerase reverse transcriptase (hTERT) promoter is active in the majority of cancer cells but not in normal cells. Hypoxia is a key feature of solid tumors, and targeted genes may be significantly upregulated by five copies of hypoxia-response elements (HREs) under hypoxic conditions. However, the effect of CDX2 overexpression, as controlled by five copies of HREs and the hTERT promoter, on human colorectal cancer (CRC) cell proliferation in vitro remains to be fully elucidated. In the current study, a recombinant lentivirus containing the CDX2 gene under the control of five HREs and the hTERT promoter was generated. An immunofluorescence assay was used to detect CDX2 expression by the 5 HhC lentivirus, whereas an MTT assay was used to detect the effects of CoCl2 on the viability of LoVo cells. Western blot analysis was conducted in order to determine the relative ratios of recombinant CDX2 protein to the internal control β-actin, following 5 HhC/LoVo cell culture under normoxic and hypoxic conditions (100, 200, 300, 400 or 500 µmol/l CoCl2) for 24 h, then for 12, 24 or 36 h with the optimal concentration (300 µmol/l) of CoCl2. Reverse transcription polymerase chain reaction analysis was used to determine the transcription of recombinant CDX2 mRNA following culture of 5 HhC/LoVo cells under normoxic or hypoxic conditions. Finally, a cloning assay was used to detect the proliferative ability of 5 HhC/LoVo and 5 Hh cells. High CDX2 expression was observed in hTERT-positive LoVo cells under hypoxic conditions, an effect which was mimicked by treatment with CoCl2 to inhibit LoVo cell proliferation in vitro. High expression of CDX2 therefore provides a promising strategy for the

  16. Increased levels of palmitoylethanolamide and other bioactive lipid mediators and enhanced local mast cell proliferation in canine atopic dermatitis

    PubMed Central

    2014-01-01

    Background Despite the precise pathogenesis of atopic dermatitis (AD) is unknown, an immune dysregulation that causes Th2-predominant inflammation and an intrinsic defect in skin barrier function are currently the two major hypotheses, according to the so-called outside-inside-outside model. Mast cells (MCs) are involved in AD both by releasing Th2 polarizing cytokines and generating pruritus symptoms through release of histamine and tryptase. A link between MCs and skin barrier defects was recently uncovered, with histamine being found to profoundly contribute to the skin barrier defects. Palmitoylethanolamide and related lipid mediators are endogenous bioactive compounds, considered to play a protective homeostatic role in many tissues: evidence collected so far shows that the anti-inflammatory effect of palmitoylethanolamide depends on the down-modulation of MC degranulation. Based on this background, the purpose of the present study was twofold: (a) to determine if the endogenous levels of palmitoylethanolamide and other bioactive lipid mediators are changed in the skin of AD dogs compared to healthy animals; (b) to examine if MC number is increased in the skin of AD dogs and, if so, whether it depends on MC in-situ proliferation. Results The amount of lipid extract expressed as percent of biopsy tissue weight was significantly reduced in AD skin while the levels of all analyzed bioactive lipid mediators were significantly elevated, with palmitoylethanolamide showing the highest increase. In dogs with AD, the number of MCs was significantly increased in both the subepidermal and the perifollicular compartments and their granule content was significantly decreased in the latter. Also, in situ proliferation of MCs was documented. Conclusions The levels of palmitoylethanolamide and other bioactive lipid mediators were shown to increase in AD skin compared to healthy samples, leading to the hypothesis that they may be part of the body’s innate mechanisms to

  17. Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

    PubMed Central

    Trias, Emiliano; Díaz-Amarilla, Pablo; Olivera-Bravo, Silvia; Isasi, Eugenia; Drechsel, Derek A.; Lopez, Nathan; Bradford, C. Samuel; Ireton, Kyle E.; Beckman, Joseph S.; Barbeito, Luis

    2013-01-01

    Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1 G93A mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67 +AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1 G93A rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1 G93A rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS. PMID:24399933

  18. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    SciTech Connect

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  19. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  20. Oxidative damage of copper chloride overload to the cultured rat astrocytes

    PubMed Central

    Hu, Hao-Lu; Ni, Xiu-Shi; Duff-Canning, Sarah; Wang, Xiao-Ping

    2016-01-01

    Disorders of copper metabolism are associated with neurological dysfunction including Wilson’s disease (WD). WD is a autosomal recessive disorder caused by mutations in the ATP7B gene resulting in the inability of the hepatocytes to remove excess copper. Gradual copper accumulation causes damage to liver, brain and other organs manifesting in liver disease, neurological and psychiatric symptoms. Also scond copper-neurometaboic disorder: Menkes disease charaterized with mutated ATP7A gene, is ralated with abnormally neuroal transmission and synaptogenesis. Parkinson’s disease and Alzheimer’s disease both are refered to some degree of copper/iron metabolism changes. The precise mechanisms by which excess copper causes neurological damage remain to be elucidated. In this study, we aimed to investigate the influence of excessive amounts of Cu2+ on the oxidative damage response and survival of primary astrocytes from newborn rats. Primary cultured rat astrocytes were divided into three groups: 30 μmol/L CuCl2, 100 μmol/L CuCl2 and control. At 12, 24, 48, 96 and 120 hours of CuCl2 intervention, cell viability, intracellular reduced glutathione level and glutathion reductase activity, and nitric oxide secretion were determined. It was found that 30 μmol/L CuCl2 might stimulate the exaltation and the compensatory proliferation of astrocytes. The survival rate of astrocytes in the 100 μmol/L CuCl2 group was significantly decreased relative to the 30 μmol/L CuCl2 group. At 24 hours of CuCl2 intervention, intracellular reduced glutathione level and glutathion reductase activity were significantly decreased in the 100 μmol/L CuCl2 group compared to the control group. At 120 hours of CuCl2 intervention, nitric oxide secretion in the 100 μmol/L CuCl2 group was significantly greater than in the control group. Under pathological conditions, excessive amounts of Cu2+ greatly damaged the growth and proliferation of astrocytes, reduced the anti-oxidative capacity of

  1. Oxidative damage of copper chloride overload to the cultured rat astrocytes.

    PubMed

    Hu, Hao-Lu; Ni, Xiu-Shi; Duff-Canning, Sarah; Wang, Xiao-Ping

    2016-01-01

    Disorders of copper metabolism are associated with neurological dysfunction including Wilson's disease (WD). WD is a autosomal recessive disorder caused by mutations in the ATP7B gene resulting in the inability of the hepatocytes to remove excess copper. Gradual copper accumulation causes damage to liver, brain and other organs manifesting in liver disease, neurological and psychiatric symptoms. Also scond copper-neurometaboic disorder: Menkes disease charaterized with mutated ATP7A gene, is ralated with abnormally neuroal transmission and synaptogenesis. Parkinson's disease and Alzheimer's disease both are refered to some degree of copper/iron metabolism changes. The precise mechanisms by which excess copper causes neurological damage remain to be elucidated. In this study, we aimed to investigate the influence of excessive amounts of Cu(2+) on the oxidative damage response and survival of primary astrocytes from newborn rats. Primary cultured rat astrocytes were divided into three groups: 30 μmol/L CuCl2, 100 μmol/L CuCl2 and control. At 12, 24, 48, 96 and 120 hours of CuCl2 intervention, cell viability, intracellular reduced glutathione level and glutathion reductase activity, and nitric oxide secretion were determined. It was found that 30 μmol/L CuCl2 might stimulate the exaltation and the compensatory proliferation of astrocytes. The survival rate of astrocytes in the 100 μmol/L CuCl2 group was significantly decreased relative to the 30 μmol/L CuCl2 group. At 24 hours of CuCl2 intervention, intracellular reduced glutathione level and glutathion reductase activity were significantly decreased in the 100 μmol/L CuCl2 group compared to the control group. At 120 hours of CuCl2 intervention, nitric oxide secretion in the 100 μmol/L CuCl2 group was significantly greater than in the control group. Under pathological conditions, excessive amounts of Cu(2+) greatly damaged the growth and proliferation of astrocytes, reduced the anti-oxidative capacity of

  2. Self-repair in a bidirectionally coupled astrocyte-neuron (AN) system based on retrograde signaling

    PubMed Central

    Wade, John; McDaid, Liam; Harkin, Jim; Crunelli, Vincenzo; Kelso, Scott

    2012-01-01

    In this paper we demonstrate that retrograde signaling via astrocytes may underpin self-repair in the brain. Faults manifest themselves in silent or near silent neurons caused by low transmission probability (PR) synapses; the enhancement of the transmission PR of a healthy neighboring synapse by retrograde signaling can enhance the transmission PR of the “faulty” synapse (repair). Our model of self-repair is based on recent research showing that retrograde signaling via astrocytes can increase the PR of neurotransmitter release at damaged or low transmission PR synapses. The model demonstrates that astrocytes are capable of bidirectional communication with neurons which leads to modulation of synaptic activity, and that indirect signaling through retrograde messengers such as endocannabinoids leads to modulation of synaptic transmission PR. Although our model operates at the level of cells, it provides a new research direction on brain-like self-repair which can be extended to networks of astrocytes and neurons. It also provides a biologically inspired basis for developing highly adaptive, distributed computing systems that can, at fine levels of granularity, fault detect, diagnose and self-repair autonomously, without the traditional constraint of a central fault detect/repair unit. PMID:23055965

  3. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-01

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types. PMID:27422823

  4. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-01

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

  5. Astrocyte-Specific Overexpression of Nrf2 Protects Striatal Neurons from Mitochondrial Complex II Inhibition

    PubMed Central

    Calkins, Marcus J.; Vargas, Marcelo R.; Johnson, Delinda A.; Johnson, Jeffrey A.

    2010-01-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is known to regulate a variety of cytoprotective genes through the antioxidant response element (ARE). This endogenous response is one of the major pathways by which cells are protected from xenobiotic or innate oxidative insults. Furthermore, in neural systems, astrocyte-specific activation of Nrf2 is known to protect neurons. In previous work, our laboratory found that Nrf2 protects from intrastriatal injections of the mitochondrial complex II inhibitor malonate. Here, we extend these results to show that multiple methods of astrocyte-specific Nrf2 overexpression provide protection from neurotoxicity in vivo. GFAP-Nrf2 transgenic mice are significantly more resistant to malonate lesioning. This outcome is associated with an increased basal resistance, but more so, an enhanced Nrf2 response to lesioning that attenuated the ensuing neurotoxicity. Furthermore, striatal transplantation of neuroprogenitor cells overexpressing Nrf2 that differentiate into astrocytes after grafting also significantly reduced malonate toxicity. Overall, these data establish that enhanced astrocytic Nrf2 response and Nrf2 preconditioning are both sufficient to protect from acute lesions from mitochondrial complex II inhibition. PMID:20211941

  6. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  7. Light attenuates lipid accumulation while enhancing cell proliferation and starch synthesis in the glucose-fed oleaginous microalga Chlorella zofingiensis.

    PubMed

    Chen, Tianpeng; Liu, Jin; Guo, Bingbing; Ma, Xiaonian; Sun, Peipei; Liu, Bin; Chen, Feng

    2015-10-07

    The objective of this study was to investigate the effect of light on lipid and starch accumulation in the oleaginous green algae Chlorella zofingiensis supplemented with glucose. C. zofingiensis, when fed with 30 g/L glucose, synthesized lipids up to 0.531 g/g dry weight; while in the presence of light, the lipid content dropped down to 0.352 g/g dry weight. Lipid yield on glucose was 0.184 g/g glucose, 14% higher than that cultured with light. The light-mediated lipid reduction was accompanied by the down-regulation of fatty acid biosynthetic genes at the transcriptional level. Furthermore, light promoted cell proliferation, starch accumulation, and the starch yield based on glucose. Taken together, light may attenuate lipid accumulation, possibly through the inhibition of lipid biosynthetic pathway, leading to more carbon flux from glucose to starch. This study reveals the dual effects of light on the sugar-fed C. zofingiensis and provides valuable insights into the possible optimization of algal biomass and lipid production by manipulation of culture conditions.

  8. Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: Enhanced antitumor immunity and impaired cell proliferation

    PubMed Central

    Perez-Villaroel, P.; Lee, C.; Cheng, F.; Knox, T.; Woods, D.M.; Barrios, K.; Powers, J.; Sahakian, E.; Wang, H.W.; Canales, J.; Marante, D.; Smalley, K.S.M.; Bergman, J.; Seto, E.; Kozikowski, A.; Pinilla-Ibarz, J.; Sarnaik, A.; Celis, E.; Weber, J.; Sotomayor, E.M.; Villagra, A.

    2015-01-01

    The median survival for metastatic melanoma is in the realm of 8–16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan-HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti-melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti-melanoma agents. PMID:25957812

  9. MiR-100 Inhibits Osteosarcoma Cell Proliferation, Migration, and Invasion and Enhances Chemosensitivity by Targeting IGFIR.

    PubMed

    Liu, Yang; Zhu, Shu-Tao; Wang, Xiao; Deng, Jun; Li, Wei-Hua; Zhang, Peng; Liu, Bing-Shan

    2016-10-01

    MicroRNAs are highly conserved noncoding RNA that negatively modulate protein expression at a posttranscriptional and/or translational level. MicroRNAs play an important role in the development and progression of human cancers, including osteosarcoma. Recent studies have shown that miR-100 was downregulated in many cancers; however, the role of miR-100 in human osteosarcoma has not been totally elucidated. In this study, we demonstrate that the expression of miR-100 was significantly downregulated in human osteosarcoma tissues compared to the adjacent tissues. Enforced expression of miR-100 inhibited cell proliferation, migration, and invasion abilities of osteosarcoma cells, U-2OS, and MG-63. Additionally, miR-100 also sensitized osteosarcoma cells to cisplatin and promoted apoptosis. Furthermore, overexpression of miR-100 decreased the expression of insulin-like growth factor I receptor and inhibited PI3K/AKT and MAPK/ERK signaling. In human clinical specimens, insulin-like growth factor I receptor was inversely correlated with miR-100 in osteosarcoma tissues. Collectively, our results demonstrate that miR-100 is a tumor suppressor microRNA and indicate its potential application for the treatment of osteosarcoma in future.

  10. Light attenuates lipid accumulation while enhancing cell proliferation and starch synthesis in the glucose-fed oleaginous microalga Chlorella zofingiensis

    PubMed Central

    Chen, Tianpeng; Liu, Jin; Guo, Bingbing; Ma, Xiaonian; Sun, Peipei; Liu, Bin; Chen, Feng

    2015-01-01

    The objective of this study was to investigate the effect of light on lipid and starch accumulation in the oleaginous green algae Chlorella zofingiensis supplemented with glucose. C. zofingiensis, when fed with 30 g/L glucose, synthesized lipids up to 0.531 g/g dry weight; while in the presence of light, the lipid content dropped down to 0.352 g/g dry weight. Lipid yield on glucose was 0.184 g/g glucose, 14% higher than that cultured with light. The light-mediated lipid reduction was accompanied by the down-regulation of fatty acid biosynthetic genes at the transcriptional level. Furthermore, light promoted cell proliferation, starch accumulation, and the starch yield based on glucose. Taken together, light may attenuate lipid accumulation, possibly through the inhibition of lipid biosynthetic pathway, leading to more carbon flux from glucose to starch. This study reveals the dual effects of light on the sugar-fed C. zofingiensis and provides valuable insights into the possible optimization of algal biomass and lipid production by manipulation of culture conditions. PMID:26442783

  11. Artesunate inhibits proliferation of naïve CD4(+) T cells but enhances function of effector T cells.

    PubMed

    Lee, Sung Ho; Cho, Young-Chang; Kim, Kyung Hee; Lee, Ik-Soo; Choi, Hyun Jin; Kang, Bok Yun

    2015-06-01

    Artesunate is an artemisinin derivative from Artemisia annua and is being applied as a first-line drug for malaria treatment. In addition to anti-malarial effects, anti-cancer, anti-viral, and anti-inflammatory activities have been reported for artemisinin derivatives. In this study, we investigated the effects of artesunate on naïve T cell activation and Th1/Th2 differentiation. Artesunate inhibited the proliferation of CD4(+) T cells and the production of IL-2, T cell growth factor. Moreover, artesunate reduced the expression of cell surface protein CD25 (IL-2 receptor alpha chain) and CD69 on CD4(+) T cells. Artesunate showed inhibitory effects on naïve T cell activation but artesunate increased the production of IFN-γ and IL-4 under Th1 and Th2 skewed condition respectively. Therefore, these results suggest that artesunate has a negative mitogenic effect on CD4(+) T cells but reinforces the function of effector T cells. PMID:25370606

  12. An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.

    PubMed

    Yan, Yang; Cui, Huachun; Guo, Chuanyu; Li, Jun; Huang, Xiaohong; Wei, Jingguang; Qin, Qiwei

    2013-12-01

    Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.

  13. Enhanced proliferation and activation of peripheral blood mononuclear cells in patients with psoriasis vulgaris mediated by streptococcal antigen with bacterial DNA.

    PubMed

    Cai, Yi-Hua; Lu, Zhi-Yong; Shi, Ruo-Fei; Xue, Feng; Chen, Xiao-Ying; Pan, Meng; Yuan, Wei-Ru; Xu, Han; Li, Wei-Ping; Zheng, Jie

    2009-11-01

    Streptococcal infection is believed to have an intimate relationship with psoriasis, although the pathogenic role of streptococcal DNA is not fully understood. To gain a clearer understanding of these dynamics, we investigated the effect of streptococcal DNA on lymphocyte proliferation and activation as well as cytokine secretion in psoriasis. Peripheral blood mononuclear cells (PBMCs) from psoriatic patients had higher proliferative responses upon stimulation by streptococcal antigen (SA) when compared with those from healthy individuals. Strikingly, this enhanced proliferation of PBMCs was attenuated after administration of SA treated with DNase-I. In addition, CD69 expression levels on T cells, including skin-homing lymphocyte cutaneous lymphocyte-associated antigen positive T cells, and IFN-alpha secretion by PBMCs were also attenuated in patients after stimulation with SA without nucleic acid (non-nucleic acid SA, non-NASA) compared with stimulation with untreated SA. However, activation marker CD86 expression levels on B cells as well as the secretion of IFN-gamma and tumor necrosis factor (TNF)-alpha following stimulation with SA or non-NASA were not significantly altered. Interestingly, the attenuated T-cell activation and IFN-alpha secretion in psoriatic patients could be reconstituted when stimulated by non-NASA combined with synthetic CpG-A, but not when combined with synthetic CpG-B. This study demonstrates the integral function of SA, particularly streptococcal DNA, in the pathogenesis of psoriasis.

  14. IGF-1R and ErbB3/HER3 contribute to enhanced proliferation and carcinogenesis in trastuzumab-resistant ovarian cancer model

    SciTech Connect

    Jia, Yanhan; Zhang, Yan; Qiao, Chunxia; Liu, Guijun; Zhao, Qing; Zhou, Tingting; Chen, Guojiang; Li, Yali; Feng, Jiannan; Li, Yan; Zhang, Qiuping; Peng, Hui

    2013-07-12

    Highlights: •We established trastuzumab-resistant cell line SKOV3/T. •SKOV3/T enhances proliferation and in vivo carcinogenesis. •IGF-1R and HER3 genes were up-regulated in SKOV3/T based on microarray analysis. •Targeting IGF-1R and/or HER3 inhibited the proliferation of SKOV3/T. •Therapies targeting IGF-1R and HER3 might be effective in ovarian cancer. -- Abstract: Trastuzumab (Herceptin®) has demonstrated clinical potential in several types of HER2-overexpressing human cancers. However, primary and acquired resistance occurs in many HER2-positive patients with regimens. To investigate the possible mechanism of acquired therapeutic resistance to trastuzumab, we have developed a preclinical model of human ovarian cancer cells, SKOV3/T, with the distinctive feature of stronger carcinogenesis. The differences in gene expression between parental and the resistant cells were explored by microarray analysis, of which IGF-1R and HER3 were detected to be key molecules in action. Their correctness was validated by follow-up experiments of RT-PCR, shRNA-mediated knockdown, downstream signal activation, cell cycle distribution and survival. These results suggest that IGF-1R and HER3 differentially regulate trastuzumab resistance and could be promising targets for trastuzumab therapy in ovarian cancer.

  15. Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo.

    PubMed

    Liu, Nei-Chi; Hsieh, Pei-Fang; Hsieh, Ming-Kun; Zeng, Zih-Ming; Cheng, Hsiao-Ling; Liao, Jiunn-Wang; Chueh, Pin Ju

    2012-03-14

    Cancer chemoprevention is employed to block or reverse the progression of malignancies. To date, several thousands of agents have been found to possess chemopreventative activity, one of which is capsaicin, a component of chili peppers that exhibits antigrowth activity against various cancer cell lines. However, the role of capsaicin in tumorigenesis remains controversial because both cancer prevention and promotion have been proposed. Here, we made the unexpected discovery that treatment with low concentrations of capsaicin up-regulates tNOX (tumor-associated NADH oxidase) expression in HCT116 human colon carcinoma cells in association with enhanced cell proliferation and migration, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX-knockdown in HCT116 cells by RNA interference reversed capsaicin-induced cell proliferation and migration in vitro and decreased tumor growth in vivo. Collectively, these findings provide a basis for explaining the tumor-promoting effect of capsaicin and might imply that caution should be taken when using capsaicin as a chemopreventive agent. PMID:22353011

  16. Target cell-specific modulation of neuronal activity by astrocytes

    NASA Astrophysics Data System (ADS)

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S.

    2006-06-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network. glutamate | GABA | inhibition | olfactory bulb | synchronization

  17. Role of astrocytes in brain function and disease.

    PubMed

    Sidoryk-Wegrzynowicz, Marta; Wegrzynowicz, Michal; Lee, Eunsook; Bowman, Aaron B; Aschner, Michael

    2011-01-01

    Astrocytes assume multiple roles in maintaining an optimally suited milieu for neuronal function. Select astrocytic functions include the maintenance of redox potential, the production of trophic factors, the regulation of neurotransmitter and ion concentrations, and the removal of toxins and debris from the cerebrospinal fluid (CSF). Impairments in these and other functions, as well as physiological reactions of astrocytes to injury, can trigger or exacerbate neuronal dysfunction. This review addresses select metabolic interactions between neurons and astrocytes and emphasizes the role of astrocytes in mediating and amplifying the progression of several neurodegenerative disorders, such as Parkinson's disease (PD), hepatic encephalopathy (HE), hyperammonemia (HA), Alzheimer's disease (AD), and ischemia.

  18. Regional astrocyte allocation regulates CNS synaptogenesis and repair.

    PubMed

    Tsai, Hui-Hsin; Li, Huiliang; Fuentealba, Luis C; Molofsky, Anna V; Taveira-Marques, Raquel; Zhuang, Helin; Tenney, April; Murnen, Alice T; Fancy, Stephen P J; Merkle, Florian; Kessaris, Nicoletta; Alvarez-Buylla, Arturo; Richardson, William D; Rowitch, David H

    2012-07-20

    Astrocytes, the most abundant cell population in the central nervous system (CNS), are essential for normal neurological function. We show that astrocytes are allocated to spatial domains in mouse spinal cord and brain in accordance with their embryonic sites of origin in the ventricular zone. These domains remain stable throughout life without evidence of secondary tangential migration, even after acute CNS injury. Domain-specific depletion of astrocytes in ventral spinal cord resulted in abnormal motor neuron synaptogenesis, which was not rescued by immigration of astrocytes from adjoining regions. Our findings demonstrate that region-restricted astrocyte allocation is a general CNS phenomenon and reveal intrinsic limitations of the astroglial response to injury.

  19. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis*

    PubMed Central

    Robertson, Chadia L.; Srivastava, Jyoti; Siddiq, Ayesha; Gredler, Rachel; Emdad, Luni; Rajasekaran, Devaraja; Akiel, Maaged; Shen, Xue-Ning; Corwin, Frank; Sundaresan, Gobalakrishnan; Zweit, Jamal; Croniger, Colleen; Gao, Xiaoli; Ghosh, Shobha; Hylemon, Philip B.; Subler, Mark A.; Windle, Jolene J.; Fisher, Paul B.; Sarkar, Devanand

    2015-01-01

    Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity. PMID:26070567

  20. Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

    SciTech Connect

    Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter; Wells, Peter G.

    2013-07-01

    Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity in the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic

  1. New tools for investigating astrocyte-to-neuron communication

    PubMed Central

    Li, Dongdong; Agulhon, Cendra; Schmidt, Elke; Oheim, Martin; Ropert, Nicole

    2013-01-01

    Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca2+ signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in astrocytes. PMID:24194698

  2. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

    PubMed Central

    Muthukumar, Allie K.; Stork, Tobias; Freeman, Marc R.

    2014-01-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system, however mechanisms by which astrocytes regulate GAT levels are undefined. Here we show at mid-pupal stages the Drosophila CNS neuropil is devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrate the neuropil coordinate with synaptogenesis and a strocyte ablation reduces synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses form in earnest, the GABA transporter, GAT, is up-regulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor (GABABR1/2) signaling in astrocytes leads to decreased astrocytic GAT levels. Interestingly, developmental depletion of astrocytic GABABR1/2 signaling suppresses mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling, and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity. PMID:25151265

  3. Enhanced effects by 4-phenylbutyrate in combination with RTK inhibitors on proliferation in brain tumor cell models

    SciTech Connect

    Marino, Ana-Maria; Sofiadis, Anastasios; Baryawno, Ninib; Johnsen, John Inge; Larsson, Catharina; Vukojevic, Vladana; Ekstroem, Tomas J.

    2011-07-22

    Highlights: {yields} The histone deacetylase inhibitor 4-phenylbutyrate substantially enhance efficacy of the receptor tyrosine kinase inhibitors gefitinib or vandetanib in glioma and medulloblastoma cell lines. {yields} Cell death increases and clonogenic survival is reduced in the combination treatments, over mono-therapy. {yields} Combination treatments with these drugs may improve clinical outcome for cancer therapy. -- Abstract: We have investigated in vitro effects of anticancer therapy with the histone deacetylase inhibitor (HDACi) 4-phenylbutyrate (4-PB) combined with receptor tyrosine kinase inhibitors (RTKi) gefitinib or vandetanib on the survival of glioblastoma (U343MGa) and medulloblastoma (D324Med) cells. In comparison with individual effects of these drugs, combined treatment with gefitinib/4-PB or vandetanib/4-PB resulted in enhanced cell killing and reduced clonogenic survival in both cell lines. Our results suggest that combined treatment using HDACi and RTKi may beneficially affect the outcome of cancer therapy.

  4. Recent molecular approaches to understanding astrocyte function in vivo

    PubMed Central

    Davila, David; Thibault, Karine; Fiacco, Todd A.; Agulhon, Cendra

    2013-01-01

    Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms in vivo is just beginning. In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes – with an emphasis on astrocyte signaling – in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions. PMID:24399932

  5. Metabolic pathways for glucose in astrocytes.

    PubMed

    Wiesinger, H; Hamprecht, B; Dringen, R

    1997-09-01

    Cultured astroglial cells are able to utilize the monosaccharides glucose, mannose, or fructose as well as the sugar alcohol sorbitol as energy fuel. Astroglial uptake of the aldoses is carrier-mediated, whereas a non-saturable transport mechanism is operating for fructose and sorbitol. The first metabolic step for all sugars, including fructose being generated by enzymatic oxidation of sorbitol, is phosphorylation by hexokinase. Besides glucose only mannose may serve as substrate for build-up of astroglial glycogen. Whereas glycogen synthase appears to be present in astrocytes as well as neurons, the exclusive localization of glycogen phosphorylase in astrocytes and ependymal cells of central nervous tissue correlates well with the occurrence of glycogen in these cells. The identification of lactic acid rather than glucose as degradation product of astroglial glycogen appears to render the presence of glucose-6-phosphatase in cultured astrocytes an enigma. The colocalization of pyruvate carboxylase, phosphenolpyruvate carboxykinase and fructose-1,6-bisphosphatase points to astrocytes as being the gluconeogenic cell type of the CNS. PMID:9298844

  6. The use of biomaterials for cell function enhancement: acceleration of fibroblast migration and promotion of stem cell proliferation

    NASA Astrophysics Data System (ADS)

    Qin, Sisi

    , while remained constant for the cells on the flat surfaces. The increased speed on the 8microm fiber surfaces could be correlated with a 20% increase in the nuclear deformation, and a decrease around 30% in the number of focal adhesion during the same observation period. RNA expression of Myosin IIA, a protein which complexes to the actin and is responsible for exertion of traction forces during migration was not upregulated during this process. On the other hand, histochemical staining of Myosin IIA showed that the protein had re-organized into large fibers which spanned the length of the cells. Observation of the cell morphology indicated that a new mode of motion had been established. Rather than the classical retraction of the cytoplasm followed by center of mass translation, which was observed on the flat surfaces, the cells were now moving by a contractile motion around the nucleus similar to that found in muscular motion. This mode was found to be more efficient when undergoing oriented motion. In addition to orientation, surface mechanics are also important in the tissue regeneration process. This study demonstrated that mechanical factors are important for the maintenance of pluripotency and control of proliferation rates. CD34+ hematopoietic stem cells (HSCs) were transduced with ICD (intracellular domain)-Notch and plated on gelatin hydrogels, whose moduli were controlled by the crosslinking ratio. On the softer hydrogel, a synergy was achieved which resulted in more than a five-fold increase in proliferation and a four-fold increase in the preservation of stemness, while HSCs maintained their ability to differentiate into multiple blood cell lineages. These results point the way for achieving clinically significant expansion of human HSCs.

  7. RKIP suppresses gastric cancer cell proliferation and invasion and enhances apoptosis regulated by microRNA-224.

    PubMed

    Liu, Hongyi; Li, Peng; Li, Bing; Sun, Peng; Zhang, Jiajin; Wang, Baishi; Jia, Baoqing

    2014-10-01

    The purposes of this study were to determine the expression profile of Raf kinase inhibitor protein (RKIP) in human gastric cancer cells and its effect on the biological characteristics of SGC-7901 cell lines, to examine the modulatory effect of microRNA-224 (miR-224) on RKIP. The research will provide novel strategies for gastric cancer treatment in the future. Quantitative real-time reverse transcription PCR (qRT-PCR) was employed to determine the expression profile of RKIP in gastric cancer cell lines (SGC-7901, MGC80-3, and MKN45). A eukaryotic expression vector, pcDNA3.1-RKIP, was constructed and transfected into SGC-7901 cells. Changes in RKIP protein expression were examined by Western blot assays, and the effect of RKIP overexpression on SCG-7901 cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assays. The effect of RKIP overexpression on SGC-7901 cell proliferation and apoptosis was analyzed by flow cytometry and that on the migration of SGC-7901 cells was investigated by Transwell migration assays. RKIP was identified to be a regulatory target gene of miR-224 using a luciferase reporter gene system, and the effect of miR-224 on intracellular RKIP protein expression was examined by Western blot assays. The regulatory effect of miR-224 on the biological characteristics of RKIP was investigated by MTT, flow cytometry, and Transwell invasion chamber assays. The expression of RKIP in gastric cancer cells was decreased significantly in comparison to that of normal gastric mucosal epithelial cells (GES-1) (p < 0.01), as demonstrated by qRT-PCR assays. Compared with the control group, the up-regulation of RKIP intracellular expression was observed in SGC-7901 cells after transfection of pcDNA3.1-RKIP for 48 h (p < 0.01). There were significant decreases in cell viability and the S-phase fraction (p < 0.05), concomitant with a significant increase in apoptosis (p < 0.01), as well as a significant

  8. The poplar basic helix-loop-helix transcription factor BEE3 - Like gene affects biomass production by enhancing proliferation of xylem cells in poplar.

    PubMed

    Noh, Seol Ah; Choi, Young-Im; Cho, Jin-Seong; Lee, Hyoshin

    2015-06-19

    Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

  9. Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

    PubMed

    Kaneda, Atsushi; Wang, Chiaochun J; Cheong, Raymond; Timp, Winston; Onyango, Patrick; Wen, Bo; Iacobuzio-Donahue, Christine A; Ohlsson, Rolf; Andraos, Rita; Pearson, Mark A; Sharov, Alexei A; Longo, Dan L; Ko, Minoru S H; Levchenko, Andre; Feinberg, Andrew P

    2007-12-26

    Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade. PMID:18087038

  10. The poplar basic helix-loop-helix transcription factor BEE3 – Like gene affects biomass production by enhancing proliferation of xylem cells in poplar

    SciTech Connect

    Noh, Seol Ah Choi, Young-Im Cho, Jin-Seong Lee, Hyoshin

    2015-06-19

    Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems. - Highlights: • We identify the BEE3-like gene form hybrid poplar (Populus alba × Populus glandulosa). • We examine effects of overexpression of PagBEE3L on growth in poplar. • We found that 35S:BEE3L transgenic plants showed more rapid growth than wild-type plants. • BEE3L protein plays an important role in the development of plant stem.

  11. Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

    PubMed

    Kaneda, Atsushi; Wang, Chiaochun J; Cheong, Raymond; Timp, Winston; Onyango, Patrick; Wen, Bo; Iacobuzio-Donahue, Christine A; Ohlsson, Rolf; Andraos, Rita; Pearson, Mark A; Sharov, Alexei A; Longo, Dan L; Ko, Minoru S H; Levchenko, Andre; Feinberg, Andrew P

    2007-12-26

    Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

  12. MicroRNA-148b enhances proliferation and apoptosis in human renal cancer cells via directly targeting MAP3K9.

    PubMed

    Nie, Fang; Liu, Tianming; Zhong, Liang; Yang, Xianggui; Liu, Yunhong; Xia, Hongwei; Liu, Xiaoqiang; Wang, Xiaoyan; Liu, Zhicheng; Zhou, Li; Mao, Zhaomin; Zhou, Qin; Chen, Tingmei

    2016-01-01

    Increasing evidence revealed that miRNAs, the vital regulators of gene expression, are involved in various cellular processes, including cell growth, differentiation, apoptosis and progression. In addition, miRNAs act as oncogenes and/or tumor suppressors. The present study aimed to verify the potential roles of miR148b in human renal cancer cells. miR‑148b was found to be downregulated in human renal cancel tissues and human renal cancer cell lines. Functional studies demonstrated that plasmid‑mediated overexpression of miR‑148b promoted cell proliferation, increased the S‑phase population of the cell cycle and enhanced apoptosis in the 786‑O and OS‑RC‑2 renal cancer cell lines, while it did not appear to affect the total number of viable cells according to a Cell Counting Kit‑8 assay. Subsequently, a luciferase reporter assay verified that miR148b directly targeted mitogen‑activated protein kinase (MAPK) kinase kinase 9 (MAP3K9), an upstream activator of MAPK kinase/c‑Jun N‑terminal kinase (JNK) signaling, suppressing the protein but not the mRNA levels. Furthermore, western blot analysis indicated that overexpression of miR148b in renal cancer cells inhibited MAPK/JNK signaling by decreasing the expression of phosphorylated (p)JNK. In addition, overexpression of MAP3K9 and pJNK was detected in clinical renal cell carcinoma specimens compared with that in their normal adjacent tissues. The present study therefore suggested that miR‑148b exerts an oncogenic function by enhancing the proliferation and apoptosis of renal cancer cells by inhibiting the MAPK/JNK pathway.

  13. Prostate cancer stem-like cells proliferate slowly and resist etoposide-induced cytotoxicity via enhancing DNA damage response

    SciTech Connect

    Yan, Judy; Tang, Damu

    2014-10-15

    Despite the development of chemoresistance as a major concern in prostate cancer therapy, the underlying mechanisms remain elusive. In this report, we demonstrate that DU145-derived prostate cancer stem cells (PCSCs) progress slowly with more cells accumulating in the G1 phase in comparison to DU145 non-PCSCs. Consistent with the important role of the AKT pathway in promoting G1 progression, DU145 PCSCs were less sensitive to growth factor-induced activation of AKT in comparison to non-PCSCs. In response to etoposide (one of the most commonly used chemotherapeutic drugs), DU145 PCSCs survived significantly better than non-PCSCs. In addition to etoposide, PCSCs demonstrated increased resistance to docetaxel, a taxane drug that is commonly used to treat castration-resistant prostate cancer. Etoposide produced elevated levels of γH2AX and triggered a robust G2/M arrest along with a coordinated reduction of the G1 population in PCSCs compared to non-PCSCs, suggesting that elevated γH2AX plays a role in the resistance of PCSCs to etoposide-induced cytotoxicity. We have generated xenograft tumors from DU145 PCSCs and non-PCSCs. Consistent with the knowledge that PCSCs produce xenograft tumors with more advanced features, we were able to demonstrate that PCSC-derived xenograft tumors displayed higher levels of γH2AX and p-CHK1 compared to non-PCSC-produced xenograft tumors. Collectively, our research suggests that the elevation of DNA damage response contributes to PCSC-associated resistance to genotoxic reagents. - Highlights: • Increased survival in DU145 PCSCs following etoposide-induced cytotoxicity. • PCSCs exhibit increased sensitivity to etoposide-induced DDR. • Resistance to cytotoxicity may be due to slower proliferation in PCSCs. • Reduced kinetics to growth factor induced activation of AKT in PCSCs.

  14. Nano-curcumin inhibits proliferation of esophageal adenocarcinoma cells and enhances the T cell mediated immune response.

    PubMed

    Milano, Francesca; Mari, Luigi; van de Luijtgaarden, Wendy; Parikh, Kaushal; Calpe, Silvia; Krishnadath, Kausilia K

    2013-01-01

    In Western countries the incidence of the esophageal adenocarcinoma (EAC) has risen at a more rapid rate than that of any other malignancy. Despite intensive therapies this cancer is associated with extreme high morbidity and mortality. For this reason, novel effective therapeutic strategies are urgently required. Dendritic Cell (DC)-based immunotherapy is a promising novel treatment strategy, which combined with other anti-cancer strategies has been proven to be beneficial for cancer patients. Curcumin (diferuloylmethane), is a natural polyphenol that is known for its anti-cancer effects however, in it's free form, curcumin has poor bioavailability. The aim of this study was to investigate whether using a highly absorptive form of curcumin, dispersed with colloidal nano-particles, named Theracurmin would be more effective against EAC cells and to analyze if this new compound affects DC-induced T cell response. As a result, we show efficient uptake of nano-curcumin by the EAC cell lines, OE33, and OE19. Moreover, nano-curcumin significantly decreased the proliferation of the EAC cells, while did not affect the normal esophageal cell line HET-1A. We also found that nano-curcumin significantly up-regulated the expression of the co-stimulatory molecule CD86 in DCs and significantly decreased the secretion of pro-inflammatory cytokines from in vitro activated T cells. When we combined T cells with nano-curcumin treatment in OE19 and OE33, we found that the basic levels of T cell induced cytotoxicity of 6.4 and 4.1%, increased to 15 and 13%, respectively. In conclusion, we found that nano-curcumin is effective against EAC, sensitizes EAC cells to T cell induced cytotoxicity and decreases the pro-inflammatory signals from T cells. Combining DC immunotherapy with nano-curcumin is potentially a promising approach for future treatment of EAC. PMID:23755374

  15. Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains

    PubMed Central

    1991-01-01

    There is evidence that the cytokine tumor necrosis factor alpha (TNF- alpha) contributes to the pathogenesis of neurological autoimmune diseases such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). TNF-alpha exerts damaging effects on oligodendrocytes, the myelin-producing cell of the central nervous system (CNS), and myelin itself. We have recently demonstrated TNF- alpha expression from astrocytes induced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), and interleukin 1 beta (IL-1 beta). Astrocytes secrete TNF-alpha in response to LPS alone, and can be primed by IFN-gamma to enhance LPS-induced TNF-alpha production. IFN- gamma and IL-1 beta, cytokines known to be present in the CNS during neurological disease states, do not induce TNF-alpha production alone, but act synergistically to stimulate astrocyte TNF-alpha expression. Inbred Lewis and Brown-Norway (BN) rats differ in genetic susceptibility to EAE, which is controlled in part by major histocompatibility complex (MHC) genes. We examined TNF-alpha gene expression by astrocytes derived from BN rats (resistant to EAE) and Lewis rats (highly susceptible). Astrocytes from BN rats express TNF- alpha mRNA and protein in response to LPS alone, yet IFN-gamma does not significantly enhance LPS-induced TNF-alpha expression, nor do they express appreciable TNF-alpha in response to the combined stimuli of IFN-gamma/IL-1 beta. In contrast, astrocytes from Lewis rats express low levels of TNF-alpha mRNA and protein in response to LPS, and are extremely responsive to the priming effect of IFN-gamma for subsequent TNF-alpha gene expression. Also, Lewis astrocytes produce TNF-alpha in response to IFN-gamma/IL-1 beta. The differential TNF-alpha production by astrocytes from BN and Lewis strains is not due to the suppressive effect of prostaglandins, because the addition of indomethacin does not alter the differential pattern of TNF-alpha expression. Furthermore, Lewis and BN astrocytes

  16. Gemfibrozil, a Lipid-lowering Drug, Inhibits the Induction of Nitric-oxide Synthase in Human Astrocytes*

    PubMed Central

    Pahan, Kalipada; Jana, Malabendu; Liu, Xiaojuan; Taylor, Bradley S.; Wood, Charles; Fischer, Susan M.

    2007-01-01

    Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by

  17. Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

    PubMed Central

    Noh, Y H; Kim, K-Y; Shim, M S; Choi, S-H; Choi, S; Ellisman, M H; Weinreb, R N; Perkins, G A; Ju, W-K

    2013-01-01

    Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important

  18. Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

    SciTech Connect

    Jensen, Pia; Gramsbergen, Jan-Bert; Zimmer, Jens; Widmer, Hans R.; Meyer, Morten

    2011-07-15

    Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neurob